University of Southern California Dissertations and Theses

Crosslink Precursors In Fetal Calf Skin Soluble Collagens

(USC Thesis Other)

Crosslink Precursors In Fetal Calf Skin Soluble Collagens

PDF

Download

Open document

Flip pages

Download a page range

Download transcript

Copy asset link

Request this asset

Transcript (if available)

Content CROSSLINK PRECURSORS IN PETAL CALF SKIN SOLUBLE COLLAGENS by D avid P a u l Norby A D i s s e r t a t i o n P re s e n te d t o th e FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA I n P a r t i a l F u l f i l l m e n t o f th e R e q u ire m e n ts f o r t h e D egree DOCTOR OF PHILOSOPHY ( B io c h e m is tr y ) F e b r u a ry 1973 INFORMATION TO USERS This dissertation was produced from a microfilm copy of the original docum ent. While the most advanced technological means to photograph and reproduce this docum ent have been used, the quality is heavily dependent upon the quality of the original submitted. The following explanation of techniques is provided to help you understand markings or patterns which may appear on this reproduction. 1. The sign or "target" for pages apparently lacking from the docum ent photographed is "Missing Page(s)". If it was possible to obtain the missing page(s) or section, they are spliced into the film along with adjacent pages. This may have necessitated cutting thru an image and duplicating adjacent pages to insure you com plete continuity. 2. When an image on the film is obliterated w ith a large round black mark, it is an indication that th e photographer suspected th a t the copy may have moved during exposure and thus cause a blurred image. You will find a good image of the page in the adjacent frame. 3. When a map, drawing or chart, etc., was part of the material being p h o to g rap h e d the photographer followed a definite m ethod in "sectioning" the material. It is custom ary to begin photoing a t the upper left hand corner of a large sheet and to continue photoing from left to right in equal sections w ith a small overlap. If necessary, sectioning is continued again — beqinning below the first row and continuing on until complete. 4. The majority of users indicate th a t the textual content is of greatest value, however, a somewhat higher quality reproduction could be made from "photographs" if essential to th e understanding o f the dissertation. Silver prints of "photographs" may be ordered at additional charge by writing the O rder Department, giving the catalog number, title, author and specific pages you wish reproduced. University Microfilms 300 North Zeeb Road Ann Arbor, Michigan 48106 A Xerox Education Company 73-9318 NORBY, David Paul, 1944- CROSSLINK PRECURSORS IN FETAL CALF SKIN SOLUBLE COLLAGENS. U niversity o f Southern C a lifo rn ia , Ph.D., 1973 Biochemistry University Microfilms, A X ER O X Com pany, Ann Arbor, Michigan THIS DISSERTATION HAS BEEN MICROFILMED EXACTLY AS RECEIVED. UNIVERSITY O F SO U T H E R N CALIFORNIA TH E GRADUATE SCHOO L UNIVERSITY PARK LOS A N G ELES. CA LIFO RN IA 9 0 0 0 7 This dissertation, written by under the direction of fiLS.... Dissertation Com mittee, and approved by all its members, has been presented to and accepted by The Graduate School, in partial fulfillment of requirements of the degree of D O C T O R O F P H I L O S O P H Y DAVID PAUL NORBY Dtan Date. February 1973 DISSERTATION COMMITTEE Chairman PLEASE NOTE: Some p ag es may have indistinct print. Filmed a s r e c e iv e d . University Microfilms, A Xerox Education Company ACKNOWLEDGMENTS I w ish t o th a n k D r. M arcel Nimni f o r h i s g u id a n c e and s p o n s o r s h i p . I would a l s o l i k e to th a n k D r. K a lin d i Deshmukh f o r many h e l p f u l d i s c u s s i o n s and h e r d i r e c t i o n i n d o in g amino a c id a n a l y s e s . I am g r a t e f u l t o Mary J u s t and S y l v i a P a d i l l a f o r t h e i r t e c h n i c a l a s s i s t a n c e . I w ould e s p e c i a l l y l i k e t o th a n k my w if e , L in d a . H er e n c o u ra g e m e n t, p a t i e n c e , and t e c h n i c a l a s s i s t a n c e have c o n t r i b u t e d i n g r e a t m easu re t o c o m p le tio n o f t h i s d i s s e r t a t i o n . ii TABLE OP CONTENTS Page ACKNOWLEDGMENTS...................................................................................... i i LIST OP TABLES ...................................................................................... v LIST OP F I G U R E S ...................................................................................... v i C h a p te r I . INTRODUCTION ............................................................................ 1 G e n e ra l Review o f C o lla g e n A ld eh y d es and C r o s s l i n k i n g L a th y ris m and P e n i c i l l a m i n e E f f e c t s S o l u b i l i t y C h a r a c t e r i s t i c s o f T is s u e C o lla g e n F e a t u r e s L y sy l O x id a se I I . STATEMENT OP THE PR O B L E M .............................................. 30 I I I . MATERIALS AND METHODS........................................................ 32 E x t r a c t i o n o f C o lla g e n s P u r i f i c a t i o n o f E x t r a c t s I s o l a t i o n o f S u b u n its L im ite d C le a v a g e w ith C yanogen Brom ide C om plete C le a v a g e w ith C yanogen Brom ide S e p a r a t i o n o f C yanogen Brom ide P e p tid e s S ephadex G-50 C hrom atography B io -G e l P-2 C hrom atography P e p s in D i g e s t i o n A c ry la m id e G el E l e c t r o p h o r e s i s o f CNBr P e p t i d e s NaBH4 (^H ; R e d u c tio n MBTH A ldehyde E s tim a tio n s H y d r o ly s is f o r Amino Acid A n a ly s is S o u rc e s o f I m p o r ta n t R e a g e n ts iii C h a p te r Page IV. RESULTS .................................................................................. 56 V. D IS C U S S IO N ............................................................................. 118 V I. SUMMARY....................................................................................... 136 REFERENCES ........................................................................................... 138 iv LIST OP TABLES Table page I . C r o s s l i n k P r e c u r s o r s Pound i n V e r t e b r a t e Co H a g e n s ................................................................... 18 I I . C r o s s l i n k s Pound I n V e r te b r a t e C o lla g e n s . . 21 I I I . E x t r a c t a b i l i t y o f C o lla g e n from P e t a l C a lf S k i n ................................................................... 57 IV . A ldehyde C o n te n t o f S e le c te d E x t r a c t s . . . 59 V. Amino Acid C o m p o sitio n o f E x t r a c t s and S e l e c te d S u b u n its .............................. . . . . . 69 V I . A lp h a S u b u n it C o m p o sitio n o f Acid S o lu b le C o lla g e n s o f P e t a l C a lf S k in .......................... 74 V I I . A ldehyde C o n te n t o f S u b u n its from C i t r a t e S o lu b le C o lla g e n ......................................... 78 V I I I . Amino A cid C o m p o sitio n o f P r a c t i o n Vc fro m F i g u r e 8 ......................................................... 87 v LIST OP FIGURES Figure Page 1 . S u b u n it A g g re g a te s I s o l a b l e from C o lla g e n o f B one, S k in , o r Tendon o f V e r t e b r a t e s . . 6 2 . C o lla g e n F i b e r Viewed I n P e r s p e c t i v e P a r a l l e l t o i t s Long A x i s ......................................... 9 Z>, Flow S h e e t f o r P r e p a r a t i o n o f E x t r a c t s from F e t a l C a lf S k i n ........................................................ 33 4 . Flow S h e e t f o r P u r i f i c a t i o n o f E x t r a c t s from F e t a l C a lf S k i n ........................................................ 38 5 . A b s o r p tio n S p e c t r a fro m R e a c tio n o f S e l e c t e d E x t r a c t s w ith M B T H .................................... 6 l 6 . DNPH S p e c t r a o f E x t r a c t s o f F e t a l C a lf S k i n ................................................................................................. 62 7 . C ro to n a ld e h y d e 2 , 4 - D i n i tr o p h e n y lh y d r a z i n e . . 65 8 . G lu ta r a ld e h y d e 2 ,4 - D i n i t r o p h e p y l h y d r a z i n e . . 66 9 . A c ry la m id e G el E l e c t r o p h o r e s i s o f S e l e c te d E x t r a c t s .................................................................................. 67 1 0 . E l u t i o n P r o f i l e o f C arb o x y m eth y l C e l l u l o s e C h ro m ato g rap h y o f A cid S o lu b le C o lla g e n . . 71 1 1 . A b s o r p tio n S p e c t r a fro m R e a c tio n o f S u b u n its o f A cid S o lu b le C o lla g e n w ith M BTH..................... 76 1 2 . C a rb o x y m e th y l C e l l u l o s e C hrom atography o f CNBr D i g e s t o f A cid S o lu b le C o lla g e n o^| - S u b u n i t s .................................... 8 l 13 . C arb o x y m e th y l C e l l u l o s e C hrom atography ( a c e t a t e p r o c e d u r e ) o f F r a c t i o n V from C a rb o x y m e th y l C e l l u l o s e C hrom ato graph y ( c i t r a t e ) o f CNBr D ig e s t o f &3 vi Figure Page 1 4 . A c ry la m id e Gel E l e c t r o p h o r e s i s o f F r a c t i o n V from F ig u r e 1 2 ....................................................................... 84 1 5 . MBTH A ldehyde S p e c tru m o f F r a c t i o n Vc . . . . 8 6 1 6 . C arb oxym ethyl C e l l u l o s e C hrom atography o f CNBr D ig e s t o f <^,3. S u b u n its o f F e t a l C a lf S k in , A cid S o lu b le C o l l a g e n .................................... 88 1 7 . P h o s p h o c e llu lo s e C hrom atography 2 ,4 - D i n i t r o p h e n y l h y d r a z i n e - r e a c t e d 2 - M e r c a p to - e th y la m in e - s o lu b le F e t a l C a lf S k in C o l l a g e n ....................................................................... 89 1 8 . CysSC DNPH CNBr - P h o s p h o c e llu lo s e F r a c t i o n I , B io -G e l P-2 F r a c t i o n I ..................... 91 1 9 . F o rm ald eh y d e 2 - 4 - D itr o p h e n y lh y d r a z in e . . . . 92 2 0 . CysSC DNPH CNBr B io -G e l P -2 F r a c t i o n I P h o s p h o c e llu lo s e F r a c t i o n I I .................................... 93 2 1 . A ceto n e 2 ,4 - D i n i tr o p h e n y lh y d r a z i n e ........................... 95 2 2 . CysSC DNPH CNBr - P h o s p h o c e llu lo s e F r a c t i o n I I I B io -G e l P -2 F r a c t i o n I ......................................... 9 6 2 2 . CysSC DNPH P h o s p h o c e llu lo s e F r a c t i o n IV B io -G e l P -2 F r a c t i o n I ................................................... 97 2 4 . CysSC DNPH CNBr P h o s p h o c e llu lo s e F r a c t i o n V B io -G e l P-2 F r a c t i o n I ................................................... 99 25* CysSC DNPH CNBr P h o s p h o c e llu lo s e F r a c t i o n IV B io -G e l P -2 F r a c t i o n I ................................................... 100 2 6 . B io -G e l P -2 F i l t r a t i o n C hrom atography o f CNBr D ig e s t o f C y s S C ........................................................ 102 2 7 . CysSC DNPH CNBr D ig e s t B io -G e l P-2 F r a c t i o n I ................................................................................. 102 2 8 . CysSC DNPH CNBr D ig e s t B io -G e l P -2 ■ # F r a c t i o n I I ............................................................................ 104 vii Pigure Page 2 9 . CysSC DNPH CNBr D ig e s t B io -G e l P -2 F r a c t i o n I I I .............................................. 105 3 0 . CysSC DNPH CNBr D ig e s t B io -G e l P -2 F r a c t i o n IV .............................................. 106 31• C arb o x y m eth y l C e l l u l o s e C hrom atography o f Void Volume ( F r a c t i o n I ) fro m B io -G e l P -2 C hrom ato graph y o f CNBr D ig e s t o f 2 ,4 D i n l t r o p h e n y l h y d r a z i n e - r e a c t e d 2 -M e rc a p to e th y la m in e S o lu b le C o lla g e n . . . 109 3 2 . CysSC DNPH CNBr D i g e s t , N o n d ia ly z a b le C arb o x y m eth y l C e l l u l o s e F r a c t i o n I , B io -G e l P -2 F r a c t i o n l a ................................ I l l 33* CysSC DNPH B io -G e l P -2 C arbo xym eth yl C e l l u l o s e F r a c t i o n l b ................................................... 112 3 4 . CysSC DNPH CNBr D ig e s t B io -G e l P -2 F r a c t i o n I C arb o x y m eth y l C e l l u l o s e F r a c t i o n I c . . . . 113 3 5 . C arb o x y m eth y l C e l l u l o s e C hrom atography o f CNBr D ig e s t o f N aB T ^-reduced CysSC C o l l a g e n ........................................................................ 113 3 6 . F r a c t i o n V from C arb o x y m eth y l C e l l u l o s e C h rom atograp hy o f CNBr D ig e s t o f NaB^H^- re d u c e d CysSC. T r it i u m R a d i o a c t i v i t y P a t t e r n from Amino Acid A a n ly s is o f B ase H y d r o l y s a t e .............................................................. 117 viii CHAPTER I INTRODUCTION G e n e ra l Review o f C o lla g e n Among t h e d e te r m i n a n ts o f th e m e c h a n ic a l p r o p e r t i e s o f v e r t e b r a t e c o n n e c tiv e t i s s u e s , c o l l a g e n i s th e m ost i m p o r ta n t o r g a n i c c o n s t i t u e n t . T h is im p o rta n c e comes from t h e a b i l i t y o f c o l l a g e n t o s e l f - a g g r e g a t e i n t o h ig h ly o r d e r e d t h r e e d im e n s io n a l m o le c u la r a r r a y s w hich a r e v e ry r i c h i n c o v a l e n t i n t e r m o l e c u l a r c r o s s l i n k s . Such c r o s s - lin k e d a r r a y s p r o v i d e f o r m ost o f th e t e n s i l e s t r e n g t h o f c o n n e c ti v e t i s s u e s ( l ) . The t e n s i l e s t r e n g t h p ro v id e d t o c o n n e c ti v e t i s s u e s by c o l l a g e n i s d i r e c t l y p r o p o r t i o n a l t o t h e num ber o f i n t e r m o l e c u l a r c r o s s l i n k s p e r c o l l a g e n m o l e c u l e ( 2 ) . The m e c h a n ic a l f u n c t i o n o f c o l l a g e n i n c o n n e c tiv e t i s s u e s i s t h e p rim a ry s o u r c e o f i t s b i o l o g i c a l im p o r ta n c e , a lth o u g h o t h e r r o l e s have b e e n s u g g e s te d . Some o f t h e s e r o l e s i n c l u d e th e f o l lo w in g : a ) i n i t i a t i o n o f t h e m i n e r a l i z a t i o n o f bone(35,*0 b ) i n d u c t i o n o f t h e p r o l i f e r a t i o n o f c o n n e c tiv e t i s s u e c o m p o n e n ts( 5 *6 ) c ) p a r t i c i p a t i o n i n t h e o r g a n i z a t i o n o f m ucop oly - 1 2 s a c c h a r i d e s (g ly c o s a m in o g ly c a n s ) i n e x t r a c e l l u l a r s p a c e s ( 7 , 8 ) . C o lla g e n h a s b e e n shown t o be c a p a b le o f f u n c t i o n i n g i n eac h o f t h e s e r o l e s . However, t h e b i o l o g i c a l s i g n i f i c a n c e o f e ac h o f t h e s e c a p a b i l i t i e s re m a in s t o be e s t a b l i s h e d . R e c e n tly s y n t h e s i z e d c o l l a g e n c a n be e x t r a c t e d from th e e x t r a c e l l u l a r m a tr ix o f m ost c o n n e c tiv e t i s s u e s w ith s o l v e n t s w hich do n o t d e n a tu r e m ost p r o t e i n s . V a rio u s p u r i f i c a t i o n p r o c e d u r e s a r e a v a i l a b l e w hich rem ove m ost o f t h o s e g l y c o p r o t e i n s and g ly c o s a m in o g ly c a n s w hich a r e c o - e x t r a c t e d w ith c o lla g e n ( 9 ) * Such p r o c e d u r e s g e n e r a l l y i n v o lv e d i f f e r e n t i a l p r e c i p i t a t i o n r e s u l t i n g from c h an g es i n i o n i c s t r e n g t h , ch an g e s i n pH, o r b o t h . A d d itio n a l p u r i f i c a t i o n i s g e n e r a l l y a c h ie v e d w ith e th a n o l c o a c e r v a - t i o n a n d / o r d i f f e r e n t i a l c e n t r i f u g a t i o n . The f i n a l m a t e r i a l i s o p e r a t i o n a l l y d e f in e d a s ''pure*' o r " p u r i f i e d ” c o l l a g e n . The b u lk o f m a t e r i a l i n p u r i f i e d c o ll a g e n s o l u t i o n s c o n s i s t s o f r o d - l i k e monomers, c a l l e d " t r o p o - c o l l a g e n . " S o lu b le c o l l a g e n m o le c u le s h av e a m o le c u la r w e ig h t o f a b o u t 3 0 0 , 0 0 0 . I f t r o p o c o l l a g e n s o l u t i o n s a r e in c u b a te d a t 4 0 ° C, s u b u n i t s a r e r e l e a s e d . The s m a l l e s t s u b u n i t s t h a t a r e o b ta in e d from t r o p o c o l l a g e n s o l u t i o n s w ith o u t p e p t i d e bond c le a v a g e a r e o f a b o u t 1 0 0 ,0 0 0 m o le c u l a r w e ig h t, and a r e c a l l e d a lp h a c h a i n s . C o lla g e n d e r iv e d from m ost t i s s u e s i s c o m p rise d o f two t y p e s o f s u b u n i t s : a lp h a one ( f ) and a lp h a 3 two ( ^ v ) * su ch t i s s u e s e a c h t r o p o c o l l a g e n m o le c u le c o n s i s t s o f two c^, s u b u n i t s and one s u b u n i t ( 1 0 ) . T h ese s u b u n i t s , th o u g h s i m i l a r i n s e v e r a l ways have d i s t i n c t amino a c id c o m p o s itio n s and s e q u e n c e s . F o r most ' v e r t e b r a t e s p e c i e s , t h e c i , and c h a in s o f t r o p o c o l l a g e n e x t r a c t e d from b o n e, s k i n , and te n d o n have th e same i n i t i a l amino a c id s e q u e n c e s ( 1 1 ) . The d e g re e o f h y d r o x y l a t i o n o f p r o l y l and l y s y l r e s i d u e s may v a ry , and t h e e x t e n t o f g l y c o s y l a t i o n o f t h e h y d r o x y la te d l y s y l r e s i d u e s may v a r y ( l 2 ) . T hese l a t t e r m o d i f i c a t i o n s o c c u r s u b s e q u e n t t o p e p t i d e bond f o r m a t io n , so t h e s ta te m e n t t h a t t h e i n i t i a l amino a c id s e q u e n c e s o f o{, and a r e c o n s t a n t among t h e s e t i s s u e s i s a p p a r e n t l y v a l i d . R e c e n tly , h ow ev er, v a r i a n t form s o f th e a ip n a s u b u n i t s n av e b een fo u n d t o o c c u r i n c a r t i l a g e , i n c a s e m ent m embrane, ana i n e m b ry o n ic t i s s u e s . The v a r i a n t s u b u n i t s from c a r t i l a g e d i f f e r i n amino a c id c o m p o s itio n and se q u e n c e from e i t h e r ^ , o r ^ ^ o f b o n e , s k i n , and t e n d o n ( l 3 )- Due t o t h e c h ro m a to g ra p h ic s i m i l a r i t y o f t h i s v a r i a n t a lp h a s u b u n i t t o of / from o t h e r t i s s u e s , i t h a s b e en named a lp h a one ty p e two ( c J j ( I I ) ) . The off s u b u n i t found i n b o n e, s k i n , and te n d o n i s renam ed a lp h a on e ty p e one ( , ( I ) ) t o d i f f e r e n t i a t e i t from th e new s u b u n i t . T r o p o c o lla g e n m o le c u le s w hich c o n t a i n &{f ( I I ) a r e c o m p rise d s o l e l y o f t h i s s u b u n i t ( l 4 ) . T h e re a re a p p a r e n t l y no t r o p o c o l l a g e n s wriicn c o n s i s t o f two ^ ( ,( 1 1 ) 4 s u b u n i t s and one > s u b u n i t . Nor a r e t h e r e any t r o p o - c o H a g e n s w hich c o n s i s t o f on e q ^ ( l ) , one e ^ ( H ) > and one I n t h e p a s t , a c o n s i d e r a b l e amount o f i n v e s t i g a t i o n was done t o d e te r m in e w h e th e r t h e two s u b u n i t s i n e a c h t r o p o c o l l a g e n w ere i n f a c t i n i t i a l l y o f i d e n t i c a l am ino a c id c o m p o s itio n and s e q u e n c e ( l 5 ) • I t was found t h a t t r o p o c o l l a g e n m o le c u le s from c a rp swim b l a d d e r e a c h c o n s i s t e d o f t h r e e d i s t i n c t a lp h a s u b u n i t s c a l l e d a lp h a o n e , a lp h a tw o, and a lp h a t h r e e ( l 6 ) . T h is h a s n o t been fo u n d e x c e p t among f i s h e s . More r e c e n t l y a n o th e r v a r i a n t o f a lp h a s u b u n i t s h a s b e e n fou n d i n t r o p o c o l l a g e n p r e p a r a t i o n s from e m b ry o n ic c h ic k s k i n ( l 7 ) * T h is s u b u n i t i s d i s t i n c t i n amino a c id c o m p o s itio n and s e q u e n c e from ^ i , ( I ) , and ef^. Due t o i t s c h ro m a to g ra p h ic s i m i l a r i t y to of; ( I ) and ^ j ( l l ) , t h i s new s u b u n i t h a s b e en named a lp h a one t y p e t h r e e ( Off( II I ) ) . As w ith q ) , ( I I ) , < 3 ^ ( I I I ) a p p e a rs t o o c c u r o n ly i n t r o p o c o l l a g e n m o le c u le s w hich c o n s i s t s o l e l y o f t h i s v a r i a n t s u b u n i t . R e c e n t s t u d i e s w ith c o l l a g e n e x t r a c t e d from b asem en t membrane o f human k id n e y , from D e s c e m e n t's m em brane, and fro m basem en t membrane o r t h e l e n s c a p s u le o f t h e eye hav e d e m o n s tra te d t h a t t h e r e i s an a d d i t i o n a l ty p e o f a lp h a c h a i n i n t h e s e t i s s u e s ( 1 8 ,1 9 ) . U n lik e e x t r a c e l l u l a r c o l l a g e n from any o t h e r n orm al v e r t e b r a t e 5 t i s s u e t h a t h a s b een so f a r e x am in e d , t h i s a lp h a c h a in h a s b een shown t o c o n t a i n 4 -8 c y s t e i n e r e s i d u e s p e r 1 ,0 0 0 amino a c id r e s i d u e s . S e v e r a l o t h e r s u b s t a n t i a l d i f f e r e n c e s i n amino a c id c o m p o s itio n w ere r e p o r t e d . As w ith o t h e r v a r i a n t a lp h a c h a i n s , i t was fo u n d t h a t t r o p o c o l l a g e n w hich c o n t a i n s t h i s new a lp h a c h a i n i B co m p rised s o l e l y o f su c h s u b u n i t s . Due t o th e c h ro m a to g ra p h ic s i m i l a r i t y o f t h i s a lp h a c h a i n t o M ( from s k i n , b o n e, and te n d o n , th e new s u b u n i t was a ls o r e f e r r e d to a s I t h a s n o t y e t b een r e f e r r e d t o as a lp h a one ty p e f o u r ( etyiv)). I n t r o p o c o l l a g e n s w hich a r e composed o f two f( l ) and one ^ , o t h e r s u b u n i t s a r e found w hich hav e m o le c u la r w e ig h ts e q u a l t o a b o u t 2 0 0 ,0 0 0 . T hese a re c a l l e d b e t a s u b u n i t s and a r i s e by c o v a le n t c r o s s l i n k a g e o f two a lp h a s u b u n i t s . B e ta s u b u n i t s a r i s i n g from i n t r a m o le c u la r c o v a l e n t c r o s s l i n k a g e a r e o f two t y p e s : b e t a o n e -tw o d e r iv e d from one one and b e t a o n e -o n e ( ^ ^ ) , d e r iv e d from two F ig u r e 1 . C o lla g e n p r e p a r a t i o n s w hich c o n t a i n i n t e r m o l e - c u l a r l y c r o s s l i n k e d d im e r s , o lig o m e r s , and h i g h e r n -m ers o f t r o p o c o l l a g e n hav e b e e n shown t o p ro d u c e a d d i t i o n a l ty p e s o f s u b u n i t s . B e ta tw o -tw o d e r iv e d from i n t e r m o l e c u l a r c o v a l e n t c r o s s l i n k i n g o f two ^ s u b u n i t s , h a s b e en r e p o r t e d ( 2 0 ) . O th e r s t u d i e s hav e r e p o r t e d c o v a l e n t l y c r o s s l i n k e d s u b u n i t s i n i n s o l u b l e c o l l a g e n w hich h a v e m o le c u la r w e ig h ts o f a b o u t 3 0 0 ,0 0 0 . T h ese w ere FIGURE 1 IX. IIJL /* \ ✓ V \ / V £ S u b u n it a g g r e g a t e s i s o l a b l e from c o ll a g e n o f b o n e , s k i n , o r te n d o n o f v e r t e b r a t e s 7 named gamma ( y O s u b u n i t s . T h ese s t u d i e s r e p o r t e d v a r i o u s ✓ ty p e s u b u n i t s w hich c o n ta in e d a l l o f t h e p o s s i b l e c o m b in a tio n s o f ^ j , and ^ s u b u n i t s ta k e n t h r e e a t a tim e : gamma o n e -o n e -o n e ( gamma o n e -o n e -tw o ( gamma o n e -tw o -tw o arK* Ean™a tw o -tw o -tw o ( 2 1 ) . O th e r h i g h e r m o le c u la r w e ig h t s u b u n i t s o f i n s o l u b l e p o ly m e ric c o l l a g e n w ere a l s o r e p o r t e d and w ere c a l l e d ^ , to r e f e r t o t h e w hole c o l l e c t i o n o f s u b u n i t s o f m o le c u la r w e ig h t g r e a t e r t h a n 3 0 0 ,0 0 0 . The c o m p o s itio n s o f t h e s e ^ s u b u n i t s w ere n o t e s t a b l i s h e d , b u t d id a p p e a r t o have a ra n g e o f c o m b in a tio n s o f o r i g i n a l of, and ©( ^ s u b u n i t s . The a lp h a s u b u n i t s a r e a rra n g e d i n t h e t r o p o c o l l a g e n m o le c u le i n a p a r a l l e l f a s h i o n w ith a l l t h r e e a lp h a s u b u n i t s r u n n in g t h e f u l l l e n g t h o f th e m o le c u le ( 2 2 ) . The a lp h a c h a in s a l l h a v e t h e same amino t e r m i n a l to c a rb o x y l t e r m i n a l o r i e n t a t i o n ( 2 3 )• Each a lp h a c h a i n i s a h e l i x o f t h e p o l y p r o l i n e ty p e and t h e t h r e e h e l i c e s a r e c o i l e d t o form a l a r g e r t r i p l e c h a in h e l i x ( 2 4 ) . The t r i p l e h e l i c a l s t r u c t u r e e x te n d s o v e r n e a r l y a l l o f t h e l e n g t h o f t h e t r o p o c o l l a g e n . The n o t a b l e e x c e p t i o n t o th e t r i p l e h e l i x i s a s e c t i o n c o m p r is in g a b o u t one p e r c e n t o f t h e l e n g t h o f t h e m o le c u le , w hich o c c u rs on t h e amino t e r m i n a l end o f e a c h a lp h a c h a i n . T h is n o n - h e l i c a l r e g i o n i s w e l l e s t a b l i s h e d a s t h e s i t e o f t h e i n i t i a l f o r m a tio n o f c o v a le n t c r o s s l i n k a g e s ( 2 3 ) . 8 C o lla g e n from s k i n , bone, and te n d o n o f v e r t e b r a t e s s p o n ta n e o u s ly s e l f a s s e m b le s i n t o f i b e r s u n d e r p h y s i o l o g i c a l c o n d it i o n s o f pH and i o n i c s t r e n g t h . The lo n g a x es o f th e f i b e r s a r e o r i e n t e d p a r a l l e l t o t h e lo n g a x es o f t h e i r c o n s t i t u e n t m o le c u le s ( 2 6 ,2 7 ) . F ig u r e 2 . I n a p e r s p e c t i v e p a r a l l e l t o t h e lo n g a x i s o f a f i b e r , t h e c o l l a g e n monomers a re o r g a n iz e d i n a s ta g g e r e d a r r a y . The s t a g g e r i n g o f th e m o le c u le s i s a t i n t e r v a l s o f a p p ro x im a te ly o n e - q u a r t e r t h e l e n g t h o f th e c o l l a g e n m onom ers: 640 A ngstrom u n i t s . T h is s ta g g e r e d a r r a y i s r e f e r r e d t o as " q u a r t e r s t a g g e r e d ." I n th e p e r s p e c t i v e o f a p e r p e n d i c u l a r c r o s s - s e c t i o n o f t h e lo n g a x is o f th e f i b e r , t h e c o l l a g e n monomers a r e asse m b le d i n a h e x a g o n a l c lo s e - p a c k i n g a r r a y . A ld eh y d es and C r o s s l i n k i n g S e v e r a l ty p e s o f b o nds have b e e n p ro p o se d f o r t h e c r o s s l i n k i n g o f c o l l a g e n . Among them a r e £ -am ino l y s i n e p e p t i d e b o n d s, y ^ - g ly ta m y l b o n d s, / ^ - a s p a r t y l b o n d s, c a r b o h y d r a te b r i d g e s , p h o s p h a te b r i d g e s , p e r o x i d e s , a r g i n i n e c r o s s l i n k s , and i n t e r m e d i a r y m e t a b o l i t e b r i d g e c r o s s l i n k s . An £ -am ino l y s i n e p e p t i d e bond was r e p o r t e d i n an a c id p a r t i a l h y d r o l y s a t e o f c o l l a g e n ( 2 8 ) . V ^ g l u t a m y l bonds have b e e n r e p o r t e d i n an e n z y m a tic h y d r o l y s a t e o f c o l l a g e n ( 2 9 ) . P h o s p h a te -m e d ia te d c r o s s - l i n k a g e s hav e b e en r e p o r t e d t o o c c u r i n h a rd t i s s u e FIGURE 2 C o lla g e n f i b e r view ed i n p e r s p e c t i v e p a r r a l l e l t o i t s lo n g a x i s C C o lla g e n f i b e r view ed i n p e r p e c t i v e p e r p e n d i c u l a r t o i t s lo n g a x is 10 c o H a g e n s ( 3 0 ,2 1 ) • I n te r m e d ia r y m e t a b o l i t e s w hich c o n ta in c a r b o n y l f u n c t i o n s ( p a r t i c u l a r l y a ld e h y d e s ) hav e b e en shown t o be c a p a b le o f c r o s s l i n k i n g c o ll a g e n by i n v i t r o s t u d i e s ( 3 5 2 ) . C o lla g e n h a s a l s o b e e n shown to be c r o s s - l in k e d i n v i t r o i n t h e p r e s e n c e o f h o r s e r a d i s h p e r o x i d a s e , w hich c a t a l y z e d t h e f o r m a tio n o f d i t y r o s i n e ( 3 3 ) • S id e c h a i n e s t e r and am ide c r o s s l i n k a g e s w ere s u g g e s te d by i n v i t r o s t u d i e s w hich showed a l o s s o f s o l u b i l i t y i n c o l l a g e n a f t e r a c e t y l a t i o n ( 3 4 ) . ©(-amino a ld e h y d e s were s u g g e s te d by s t u d i e s on h y d r o l y s a t e s o f N aBH^-reduced c o l l a g e n s ( 3 5 ) • T h ese s t u d i e s hav e n o t b e en fo llo w e d by c o n f ir m a to r y s t u d i e s . S p e c i f i c a l l y , no d o u b le c h a in p e p t i d e h a s b e en i s o l a t e d w hich c o n ta in e d any o f t h e s e bonds and was s m a ll enough t o be c o m p le te ly c h a r a c t e r i z e d a s s u c h . The s u b j e c t o f i n t r o c h a i n " e s t e r l i k e " bonds h a s r e c e i v e d a s u b s t a n t i a l amount o f a t t e n t i o n s i n c e i t was fo u n d t n a t © (-c h ain s c o u ld be c le a v e d i n an a p p a r e n t l y s p e c i f i c m anner by h y d ro x y la m in e (3 6 ) . R ecen t se q u e n c e s t u d i e s on a p e p ti d e d e r iv e d iro m t h e cyanogen orom ide d i g e s t i o n o f th e © (^-chain o f r a t s k i n c o l l a g e n have i d e n t i f i e d two s i t e s w hich a r e c le a v e d by h y d ro x y la m in e ( 3 7 j 3 8 ) . T h ese s i t e s a p p e a r t o be a s p a r t y l - g l y c y l o r a s p a r a g i n y l - g l y c y l p e p t i d e bonds w hich have b e e n c o n v e rte d t o c y c l i c im id e s i n v o lv i n g t h e ^ ( - a n d ^ - c a r b o x y l g ro u p s o f t h e a s p a r t y l r e s i d u e and t h e am ino g ro u p o f th e g y l c y l r e s i d u e (3 9 )• 11 P a s t s t u d i e s hav e shown t h a t c o l l a g e n w i l l r e a c t w ith c a r b o n y l r e a g e n t s g i v i n g p r o d u c ts w ith s p e c t r a l c h a r a c t e r i s t i c s o f a ld e h y d e d e r i v a t i v e s ( 4 0 , 4 l ) . An a ld e h y d e was found i n a p e p ti d e from a c o ll a g e n a s e d i g e s t o f e o l l a g e n ( 4 2 ) . I t a p p e a re d t o be a /4 -h y d ro x y a ld e h y d e , and c o u ld e a s i l y d e h y d r a te t o form an o| ,^ 5 - u n s a t u r a t e d a ld e h y d e d e r i v a t i v e . T h is p e p t i d e h a s s i n c e b e e n shown t o be from t h e amino t e r m i n a l end o f t h e m o le c u le and t o c o n t a i n th e i n t r a m o l e c u l a r c r o s s l i n k (4.3). The c r o s s l i n k i s d e r iv e d from an a l d o l c o n d e n s a tio n b etw een two a ld e h y d e s , one from th e amino t e r m i n a l r e g i o n o f e ac h o f two c h a i n s . D i r e c t e v id e n c e f o r t h i s p e p t i d e b e in g an i n t e r c h a i n c r o s s l i n k was g a in e d from t h e c o m p a riso n o f p e p t i d e s d e r iv e d from t h e cy an o g en b ro m id e (CNBr) d i g e s t i o n o f o<, and c h a in s w ith t h e a n a lo g o u s p e p t i d e s from t h e c r o s s l i n k e d d im e r o f and © ^ c a l l e d / S /-> j ( 4 4 ) . A s i n g l e c r o s s l i n k e d p e p t i d e was i s o l a t e d and s e q u e n c e d , and th e c r o s s l i n k was i d e n t i f i e d a s t h e a l d o l c o n d e n s a tio n p r o d u c t (ACP) o f th e r e a c t i o n o f two r e s i d u e s o f ^(-am ino a d i p i c se m ia ld e h y d e (AASA). S i m i l a r r e s u l t s w ere o b ta in e d from s t u d y in g d i g e s t s o f , t h e d im e r from two c h a i n s . C om parison o f t h e c r o s s l i n k r e g i o n p e p t i d e s from new ly s y n th e s iz e d o r l a t h y r i t i c c o ll a g e n s w ith t h o s e fro m more m a tu re p r e p a r a t i o n s o f c o l l a g e n have shown t h a t AASA i s d e r iv e d from t h e o x i d a t i v e d e a m in a tio n o f l y s y l r e s i d u e s ( 4 5 ) . The i n t r a m o l e c u l a r c r o s s l i n k r e g i o n o f t h e 12 m o le c u le c o m p rise s a b o u t one p e r c e n t o f i t s l e n g t h ( 4 6 , 1 5 ) . T h is r e g i o n c a n be s e l e c t i v e l y c le a v e d from t h e b a la n c e o f th e m o le c u le w ith v a r i o u s p r o t e a s e s o r CNBr u n d e r c o n d i t i o n s w hich do n o t p e r t u r b t h e h e l i c a l s t r u c t u r e o f th e m o le c u le ( 4 7 , 4 8 , 4 9 ) . Such c le a v a g e d o e s n o t s i g n i f i c a n t l y change t h e m o le c u la r w e ig h t o f t h e l a r g e r d i g e s t i o n p r o d u c t , i n d i c a t i n g t h a t t h e c r o s s l i n k r e g i o n i s on t h e end o f t h e m o le c u le . S tudy o f th e se q u e n c e o f e a c h end o f t h e m o le c u le b e f o r e and a f t e r rem oval o f t h e c r o s s l i n k r e g i o n h a s shown th e c r o s s l i n k to be i n i t s amino t e r m i n a l r e g i o n ( 2 2 ) . T h ese s t u d i e s h av e b e e n c a r r i e d o u t on c o l l a g e n e x t r a c t s i n s o l u t i o n . I t h a s b e e n found t h a t l e s s a l d o l c o n d e n s a tio n p r o d u c t i s d e m o n s tra b le i n r e c o n s t i t u t e d c o l l a g e n f i b e r s t h a n i n c o l l a g e n i n s o l u t i o n ( 5 0 ) . T h is s u g g e s t s t h a t t h e f o r m a tio n o f a l d o l con d e n s a t i o n p r o d u c t s may be t h e r e s u l t o f s o l u b i l i z i n g t h e c o l l a g e n , r a t h e r th a n b e in g a n a t u r a l l y o c c u r r i n g c r o s s l i n k i n v iv o . A l t e r n a t i v e l y , th e a l d o l c o n d e n s a tio n p r o d u c t may be an i n t e r m e d i a t e , w hich r e a c t s f u r t h e r when r e c o n s t i t u t e d i n t o n a t i v e - t y p e f i b r i l s , s i n c e i t a ls o i s an a ld e h y d e . The above r e s u l t s r e l a t e p r i m a r i l y t o i n t r a m o le c u la r c r o s s l i n k i n g , b u t a r e o f im p o rta n c e i n u n d e r s t a n d i n g c u r r e n t know ledge o f i n t e r m o l e c u l a r c r o s s l i n k i n g . I t i s t h e i n t e r m o l e c u l a r c r o s s l i n k s w hich g i v e c o l l a g e n f i b e r s t h e i r g r e a t s t r e n g t h and s t a b i l i t y . B ut 13 i n t e r m o l e c u l a r c r o s s l i n k s a l s o make c o l l a g e n i n s o l u b l e . The c lo s e r e l a t i o n b e tw e e n i n t r a m o l e c u l a r c r o s s l i n k i n g and i n t e r m o l e c u l a r c r o s s l i n k i n g i s i l l u s t r a t e d i n l a t h y r i s m and a f t e r p e n i c i l l a m i n e a d m i n i s t r a t i o n . I n e a c h o f t h e s e c o n d i t i o n s , b o th i n t r a m o l e c u l a r and i n t e r m o l e c u l a r c r o s s l i n k i n g a r e p r e v e n t e d . I n l a t h y r i s m t h e f o r m a tio n o f a ld e h y d e s i s p r e v e n t e d , a p p a r e n t l y by i n h i b i t i o n o f th e am ine e x id a s e w h ich c o n v e r t s p e p tid e -b o u n d l y s i n e (and h y d r o x y ly s in e ) t o th e a l d e h y d e ( 5 l ) • I n p e n c i ll a m i n e i n t o x i c a t i o n , a ld e h y d e s a re form ed b u t th e y a r e c h e m ic a lly b lo c k e d by p e n i c i l l a m i n e and a r e u n a v a i l a b l e f o r p a r t i c i p a t i o n i n c r o s s l i n k f o r m a t i o n ( 5 2 ) . Removal o f t h e p e n c i ll a m i n e by d i a l y s i s fo llo w e d by r e c o n s t i t u t i o n o f n a t i v e - t y p e f i b e r s l e a d s t o r a p id f o r m a tio n o f b o th i n t r a - and i n t e r m o l e c u l a r c r o s s l i n k s . The e x i s t e n c e o f i n t e r m o l e c u l a r c r o s s l i n k s h a s b e e n c l e a r l y d e m o n s tr a te d by t h e d e n a t u r a t i o n o f i n s o l u b l e c o l l a g e n f o llo w e d by d e m o n s tr a tio n o f i n t e r m o l e c u l a r e n t i t i e s i n t h e e x t r a c t ( 2 0 ) . S in c e e ach c o l l a g e n m o le c u le from s k i n , b o n e , o r te n d o n c o n t a i n s two f c h a in s and one Ji v c h a i n , d e m o n s tr a ti o n o f a co m p o n en t( 5 3 ) ( c r o s s - l in k e d d im e r o f two ^ ^ c h a i n s ) shows t h e e x i s t e n c e o f i n t e r m o l e c u l a r c r o s s l i n k s . S i m i l a r l y , c r o s s l i n k e d t r i m e r s , c a l l e d ■y/com ponents h a v e b e en found w h ich a r e composed o f t h r e e ^ c h a i n s (Vj n ) ; one ^ c h a in , and two ^ v c h a in s and t h r e e tA ^ c h a i n s ( ( 2 1 ) . The com ponents 14 and have b e en d e m o n s tra te d and c o u ld be i n t e r m o l e c u l a r c o m p o n en ts, b u t th e y may a l s o be i n t r a m o le c u la r i n o r i g i n . E a r ly s t u d i e s on s o l u b l e c o ll a g e n s i n d i c a t e d t h a t c o l l a g e n had a ld e h y d e s o n ly i n t h e amino t e r m i n a l r e g i o n o f th e m o le c u l e ( 4 5 ) . How ever, some o f t h e c o l l a g e n f o rm e rly th o u g h t t o be i n s o l u b l e c a n be s o l u b i l i z e d by t r e a tm e n t w ith s o l u t i o n s o f ^f-am ino t h i o l com pounds a t n e u t r a l pH ( 2 1 ) . T h is f r a c t i o n h a s b e e n shown t o c o n t a i n a b o u t t h r e e r e s i d u e s o f a ld e h y d e s p e r a lp h a c h a in (5 ^ )* T h ese a ld e h y d e s have b e en l o c a l i z e d t o s p e c i f i c p e p t i d e s i n a cy an o g en brom ide d i g e s t o f t h e m a t e r i a l ( 2 ) . I n a d d i t i o n t o t h e a ld e h y d e s c o n ta in e d o n C 6 I ’ and t w h ich a r e d e r iv e d from th e amino t e r m i n a l , n o n - h e l i c a l r e g i o n w here i n t r a m o l e c u l a r c r o s s l i n k s a r e fo rm ed , t h e h e l i c a l r e g i o n p e p t i d e s , and d e m o n s tra te d s i g n i f i c a n t am ounts o f r e a c t i v e a l d e h y d e s . I t h as b e e n shown t h a t u n d e n a tu re d p r e p a r a t i o n s o f t h i s ty p e o f c o l l a g e n c an be r e c o n s t i t u t e d i n t o f i b r i l s w ith n a t i v e - t y p e s p a c in g and c a n form i n t e r m o l e c u l a r c r o s s l i n k s e v e n i f t h e amino t e r m i n a l r e g i o n ox’ t h e m o le c u le h a s b e e n s e l e c t i v e l y rem oved(5 5)* T h ese c r o s s l i n k s w ere fo u n d t o be p r e d o m in a n tly o f th e S c h i f f ’ s b a s e t y p e . The l o c a t i o n o f t h e s e c r o s s l i n k s re m a in s t o be d e te r m in e d . To l o c a t e t h e s e , i t w i l l be n e c e s s a r y t o i s o l a t e a c r o s s l i n k e d p e p t i d e w hich i s o f s u f f i c i e n t s i z e so t h a t th e d e t e r m i n a t i o n o f t h e amino a c id c o m p o s itio n and se q u e n c e o f e a c h o f th e com ponent c h a in s w i l l be s u f f i c i e n t l y s p e c i f i c t o a llo w a s s ig n m e n t t o s p e c i f i c s i t e s on th e m o le c u le . Knowledge o f t h e n a t u r e and l o c a t i o n o f th e i n t e r m o l e c u l a r c r o s s l i n k s w i l l a id i n u n d e r s ta n d in g how c o l l a g e n m o le c u le s a r e o r g a n iz e d i n t o f i b r i l s . Such u n d e r s ta n d in g w i l l h e lp t o e x p l a i n how c o l l a g e n sy s te m s a r e o r g a n iz e d i n t o v a r i a n t n e tw o rk s found i n c a r t i l a g e , b asem en t mem brane, c o rn e a , and l e n s c a p s u l e . I t h a s b e e n s u g g e s te d t h a t c o l l a g e n m o le c u le s a r e o r g a n iz e d i n a h ig h ly s p e c i f i c m anner i n t o t e t r a m e r s b e f o r e o r g a n i z a t i o n i n t o f i b r i l s . T h is was b ased on t h e f i n d i n g t h a t m a t e r i a l e x t r a c t e d from d e n a tu r e d b o v in e c o riu m g a v e a much h i g h e r c o n te n t o f , n , and co m p o n en ts, r e l a t i v e t o t h e amount o f , y/ \ , and v f vV. th a n would be e x p e c te d by assu m in g t h a t s e l e c t i o n was on a random b a s i s . S t u d i e s have a ls o b e e n c a r r i e d o u t t r y i n g t o i s o l a t e c r o s s l i n k e d p e p t i d e s from a cy an o g en b ro m id e d i g e s t o f i n s o l u b l e c o l l a g e n . Some r e c e n t s u c c e s s h a s b e e n made u s in g c a t i l a g e c o l l a g e n ( 5 6 ) . T h is work found a d o u b le c h a in p e p t i d e composed o f n o n - i d e n t i c a l c h a i n s . The c o v a le n t c r o s s l i n k was shown to a r i s e from two r e s i d u e s w hich w ere o r i g i n a l l y h y d r o x y ly s y l r e s i d u e s . I t was n o t d e te rm in e d w h e th e r t h e c r o s s l i n k was a S c h i f f b a s e o r an a l d o l c o n d e n s a tio n p r o d u c t . The c r o s s l i n k i s p ro b a b ly o f i n t e r m o l e c u l a r 16 o r i g i n , due to t h e u n e q u a l c o n s t i t u e n t p e p ti d e c h a i n s . C a r t i l a g e c o l l a g e n h a s b e e n shown t o be composed o f t h r e e i d e n t i c a l a lp h a s u b u n i t s . The t h r e e a lp h a c h a in s a r e i n r e g i s t e r and h av e th e same amino t o c a rb o x y l t e r m in a l o r i e n t a t i o n . I n t r a m o l e c u l a r c r o s s l i n k s c o u ld t h e r e f o r e o n ly be in v o lv e d i f th e c o n s t i t u e n t p e p ti d e c h a in s o f a c r o s s l i n k e d r e g i o n w ere i d e n t i c a l . R e c e n tly , s t u d i e s on c o l l a g e n s o l u b i l i z e d w ith yS -am ino t h i o l compounds i n d i c a t e d t h a t t h e r e a r e com p o n e n ts i n su ch e x t r a c t s w h ich a r e i n t e r m o l e c u l a r l y c r o s s l i n k e d ( 5 7 ) . Such e x t r a c t s g iv e a much h i g h e r i n t r i n s i c v i s c o s i t y t h a n w ould be e x p e c te d on th e b a s i s o f a m o n o d is p e rs e s o l u t i o n o f t r o p o c o l l a g e n . U l t r a c e n t r i f u g a l a n a l y s i s r e v e a le d a m in o r com ponent w hich se d im e n te d v e ry r a p i d l y , a f t e r w hich th e n orm al s e d im e n ta tio n p a t t e r n o f o t h e r d e n a tu r e d s o l u b l e c o ll a g e n s was s e e n . When such a m i n o - th i o l s o l u b l e c o l l a g e n i s d i s s o l v e d i n 1 M CaCl2 and p u t on a g e l f i l t r a t i o n colum n, u s in g B io -G e l A-5M o r A-15M a s th e s e p a r a t i o n medium, a s m a ll f r a c t i o n c o n s i s t e n t l y e l u t e s w ith t h e v o id volum e. The m o le c u la r w e ig h t o f t h i s f r a c t i o n i s p ro b a b ly g r e a t e r t h a n 3 0 0 , 0 0 0 , s u g g e s t i n g th e e x i s t e n c e o f a n a t u r a l l y o c c u r r i n g s o l u b l e s p e c i e s o f c o l l a g e n w hich i s i n t e r m o l e c u l a r l y c r o s s l i n k e d ( 5 7 ) . A ld eh y d es w ere f i r s t d e m o n s tra te d i n c o l l a g e n a f t e r r e a c t i o n w ith 2 , 4 - d i n i t r o p h e n y l h y d r a z i n e (DNPH) ( 3 8 ,4 2 ) . The D N PH -reacted c o lla g e n was d i g e s t e d w ith b a c t e r i a l c o l l a g e n a s e . A DN PH -peptide was fo u n d i n th e v o id volume f r a c t i o n upon c h ro m ato g rap h y o f t h e d i g e s t on a colum n o f S ephadex G-2 5 . T h is p e p t i d e was s u b s e q u e n tly shown t o a r i s e from a s h o r t n o n - h e l i c a l r e g i o n a t t h e amino t e r m i n a l end o f th e m o le c u le . The a ld e h y d e - c o n t a i n i n g r e s i d u e h a s s i n c e been shown t o be a lp h a amino a d i p i c - 5 - s e m ia ld e h y d e . T ab le I . I t i s form ed by t h e o x i d a t i v e d e a m in a tio n o f t h e £ -am ino g ro u p o f a l y s y l r e s i d u e . The e x i s t e n c e o f such a ld e h y d e f u n c t i o n s h a s a l s o b e en d e m o n s tr a te d by o x i d a t i o n o f t h e a ld e h y d e t o a c a rb o x y l f u n c t i o n ( 5 9 * 6 0 ) . The p ro d u c e c o -c h ro m a to g ra p h s w ith a u t h e n t i c a lp h a amino a d ip i c a c id on th e amino a c id a n a l y z e r . T h ese s t u d i e s d id n o t r e p o r t lo o k in g f o r a lp h a am ino, ^ - h y d r o x y a d i p i c a c i d . T h is would be t h e a n a lo g o u s p r o d u c t d e r iv e d from o x i d a t i o n o f t h e a lp h a am ino <§-h y d ro x y a d i p i c se m ia ld e h y d e w hich would a r i s e from o x i d a t i v e d e a m in a tio n o f h y d r o x y ly s in e ( 6l ) . The p r e s e n c e o f t h e h y d ro x y l g ro u p on a c arb o n atom a d j a c e n t to t h e c a r b o x y l f u n c t i o n w ould make th e r e s i d u e s u s c e p t i b l e t o o x i d a t i v e d e c a r b o x y l a t i o n u n d e r t h e o x i d a t i o n c o n d i t i o n s , fo rm in g g lu ta m ic a c id and c a rb o n d i o x i d e . The i n c r e a s e o f one r e s i d u e p e r th o u s a n d o f g lu ta m ic a c id i n t h e amino a c id sp e c tru m o f c o l l a g e n would be v e ry d i f f i c u l t t o d i s c e r n among t h e 7 0 -8 0 g lu ta m ic a c id r e s i d u e s p e r th o u s a n d w h ich n o rm a lly o c c u r . Such a d i f f e r e n c e c o u ld TABLE I CROSSLINK PRECURSORS FOUND IN VERTEBRATE COLLAGENS ( p * p o ly p e p tid e b ackb one o f p r o t e i n ) Cu++, (0 « ?) ^ 0 p-CH9-CH9-CH9-CH9-NH9 ------- * * p-CH9-CH9-CH9-C ^ 2 2 2 2 2 am ine o x id a s e 2 2 2 N r l y s y l s i d e c h a in o f c o lla g e n a l l y s y l s i d e c h a in ? H Cu++t ( 0 2? ) ?H . 0 p-CHp-CHp-CH-CHp-NHp -----> * p-CH9-CH9-CH-C^ am ine o x id a s e h y d r o x y ly s y l s i d e c h a in 5 -h y d ro x y a l l y s y l s i d e c h a in 19 e a s i l y a r i s e from th e v a r i a t i o n s w hich n o rm a lly o c c u r from one chrom atogram t o t h e n e x t i n amino a c id a n a l y s i s . A n o th e r m ethod w hich h a s b e e n used f o r d e t e c t i o n o f a ld e h y d e r e s i d u e s i n c o l l a g e n i s r e d u c t i o n w ith 3 H -sodium b o r o h y d r id e , w hich g i v e s r a d i o a c t i v e l y l a b e l l e d a lc o h o l a s t h e p r o d u c t ( 6 2 ) . The r e d u c t i o n p r o d u c t re m a in s i n t a c t th r o u g h h y d r o l y s i s f o r amino a c id a n a l y s i s i f t h e p o l y - p e p t i d e i s h y d ro ly s e d w ith b a s e . The s i t e s o f e l u t i o n o f t h e r e d u c t i o n p r o d u c ts o f l y s y l - and h y d ro - x y l y s y l - d e r i v e d a ld e h y d e s a r e w e ll e s t a b l i s h e d . The a ld e h y d e r e s i d u e s i n c o l l a g e n c an r e a c t w ith o t h e r a ld e h y d e f u n c t i o n s o r w ith amino f u n c t i o n s i n t h e same o r a d j a c e n t m o le c u le . The r e a c t i o n o f two a l l y s y l r e s i d u e s would p ro c e e d th ro u g h an a l d o l c o n d e n s a tio n , fo rm in g an £ - u n s a t u r a t e d a ld e h y d e . R e c e n tly , some i n v e s t i g a t o r s hav e q u e s tio n e d w h e th e r a n a l d o l c o n d e n s a tio n c r o s s l i n k i s a c t u a l l y b i o l o g i c a l l y s i g n i f i c a n t (6 2 ) • T h ese i n v e s t i g a t o r s h av e fo u n d t h a t r e d u c t i o n o f s o l u b l e c o l l a g e n i n t h e d i s s o l v e d s t a t e r e a d i l y a llo w s t h e d e m o n s tr a tio n o f a l d o l c r o s s l i n k s . But r e c o n s t i t u t i o n o f t h e same p r e p a r a t i o n i n t o t h e n a t i v e - t y p e f i b r i l s b e f o r e r e d u c t i o n c a u s e s t h e amount o f d e m o n s tr a b le a l d o l s t o become v a n i s h i n g l y s m a l l . C o n c u rre n t w ith t h e d is a p p e a r a n c e o f a l d o l s was t h e a p p e a ra n c e o f i n c r e a s e d am ounts o f S c h i f f 's b a s e - t y p e c r o s s l i n k s . 20 S c h i f f 's b a s e s a re form ed i n c o l l a g e n by t h e r e a c t i o n o f a l y s i n e o r h y d r o x y l y s i n e - d e r iv e d a ld e h y d e w ith th e £ -a m in o g ro u p o f a l y s y l o r h y d r o x y ly s y l r e s i d u e ( 6 3 ) . They may a l s o be form ed by a s u b s e q u e n t r e a c t i o n o f t h e a ld e h y d ic a l d o l c o n d e n s a tio n p r o d u c t w ith an £ -am ino g ro u p from a l y s y l o r h y d r o x y ly s y l r e s i d u e ( 6 4 ) . P o u r ty p e s o f S c h i f f ' s b a s e s c o u ld be form ed by t h e r e a c t i o n o f an a ld e h y d e from e i t h e r a h y d r o x y ly s y l o r l y s y l r e s i d u e w ith th e € -am in o g ro u p o f a l y s y l o r h y d ro x y l y s y l r e s i d u e ( s e e T a b le I I ) ; d e h y d r o l y s in o n o r l e u c i n e ( l y s i n e p l u s AASA), d e h y d ro h y d r o x y ly s in o n o r le u c in e ( h y d r o x y ly s in e p l u s ASSA), d e h y d ro ly s in o h y d r o x y n o r le u c in e ( l y s i n e p l u s ^ -h y d ro x y -A A S A ), and d e h y d ro h y d r o x y ly s in o - h y d r o x y n o r le u c in e ( a l s o c a l l e d d e h y d ro d ih y d r o x y ly s in o n o r l e u c i n e - from h y d r o x y ly s in e and ^ -h y d ro x y -A A S A )(6 5 )• T h ese c r o s s l i n k e d u n i t s a r e u n s t a b l e u n d e r t h e c o n d i t i o n s w hich h y d ro ly z e p e p t i d e bonds n o n - e n z y m a tic a lly , a s a r e a l d o l c o n d e n s a tio n p r o d u c t s . They a r e a ls o l a b i l e u n d e r t h e c o n d i t i o n s used f o r r e s o l u t i o n o f th e com ponents o f su c h h y d r o l y s a t e s (am ino a c id a n a l y s i s ) . So, c r o s s l i n k i n g e n t i t i e s a r e o r d i n a r i l y red u c ed w ith sodium b o r o h y d r id e (NaBH^) p r i o r t o a c id o r b a se h y d r o l y s i s , f r e q u e n t l y u s in g H ^ - la b e lle d m a t e r i a l ( 6 6 , 6 7 )• Such t r e a t m e n t , h o w e v er, c o n v e r ts two ty p e s o f c r o s s l i n k s ( d e h y d r o h y d r o x y ly s in o n o r le u c in e and d e h y d ro ly s in o h y d r o x y n o r l e u c i n e ) i n t o t h e same p r o d u c t , h y d r o x y ly s in o n o r le u c in e TABLE II CROSSLINKS POUND IN VERTEBRATE COLLAGENS I . A ld o l c o n d e n s a tio n p r o d u c t p - c h 2- c h 2- c h 2- c ' + ^ c - c h 2- c h 2- c h 2- p ^ p - c h 2- c h 2- c h - c h - c h 2- c h 2- c h 2-p H H OH OH 8 ♦t p-CH2-CH2-y=CH-CH2-CH2-CH2-p CH 8 I I . S c h l f f b a s e s A. d e h y d r o ly s in o n o r le u c in e p-CH2-CH2-CH2-C ' + H2N-CH2-CH2-CH2-CH2-p ^ p-CH2-CH2-CH2-CH*N-CH2-CH2-CH2-CH2-p B. d e h y d ro h y d r o x y ly s in o n o r le u c in e P-CH2” CH2 "CH2’ C\ + H2N-CH2-CH-CH2-CH2- p p-CH2-CH2-CH2-CH=N-CH2-CH-CH2-CH2-p H OH OH ro TABLE II continued d e h y d r o ly s in o h y d ro x y n o r le u c in e 2-CH2-CH-C ' + H2N-CH2-CH2-CH2-CH2- p ^ p-CH2-CH2-CH-GH=N-CH2- OH H OH d e h y d ro h y d ro x y ly s in o h y d ro x y n o rle u c in e 23 ( b b ) . The n a t u r e o f t h e o r i g i n a l c r o s s l i n k c a n s t i l l be d e te r m in e d . I t r e q u i r e s t h e c a r e f u l c le a v a g e o f th e red u c ed c r o s s l i n k i n g m a t e r i a l w ith p e r i o d a t e and s u b s e q u e n t d e t e r m i n a t i o n o f w h e th e r th e t r i t i u m o f r e d u c t i o n was ta k e n up on t h e h y d r o x y la te d o r u n h y d ro x y la te d s i d e o f t h e c r o s s l i n k ( 5 0 ) . Each o f th e t y p e s o f p o s s i b l e re d u c e d S c h i f f 's b a s e s h as now b een i d e n t i f i e d i n c o l l a g e n ( 6 3 , 6 9 , 7 0 , / i ) The r e l a t i v e ab u n d an ce o f e a c h ty p e o f c r o s s l i n k v a r i e s i n d i f f e r e n t a n im a ls , i n d i f f e r e n t t i s s u e s i n t h e same a n im a l, and i n th e same t i s s u e i n t h e same a n im a l a t d i f f e r e n t a g e s ( 6 9 ,7 2 ,7 3 , 7 4 , 7 5 ) • A ld o l c o n d e n s a tio n c r o s s l i n k s , i f th e y do o c c u r n a t u r a l l y , a r e c o n s id e r e d t o be e x c l u s i v e l y o f i n t r a m o l e c u l a r o r i g i n . No one h as y e t p r e s e n te d e v id e n c e f o r S c h i f f ' s b a se c r o s s l i n k s b e in g o f i n t r a m o l e c u l a r o r i g i n . S e v e r a l i n v e s t i g a t o r s h av e p r e s e n te d e v id e n c e t h a t S c h i f f ' s b a s e s a re in v o lv e d i n i n t e r m o l e c u l a r c r o s s l i n k i n g ( 7 6 ) . S in c e i n t e r m o l e c u l a r c r o s s l i n k s a r e w hat g iv e c o l l a g e n f i b e r s y s te m s t h e i r b i o l o g i c a l l y im p o r ta n t m e c h a n ic a l p r o p e r t i e s , i t i s im p o r ta n t t o i d e n t i f y t h e n a t u r e and l o c a t i o n o f a l l t h e i r p o t e n t i a l c r o s s l i n k i n g s i t e s . The c r o s s l i n k s t h a t h a v e b e en shown t o o c c u r i n c o ll a g e n a re a l l b a se d upon t h e re q u ire m e n t f o r an a ld e h y d e d e riv e d from e i t h e r p e p t i d y l l y s i n e o r h y d r o x y ly s in e . T h is p o i n t s t o th e p rim a ry im p o rta n c e o f e l u c i d a t i n g t h e 24 l o c a t i o n and n a tu r e ( a l l y s y l o r h y d r o x y a l l y s y l ?) and num ber o f a ld e h y d e s i n c o l l a g e n . E a rly s t u d i e s o f t h e a ld e h y d e s i n c o l l a g e n fo c u s e d upon t h e amino te r m in a l end o f s a l t - and a c id s o l u b l e c o l l a g e n s . T hese s t u d i e s showed t h a t e a c h c h a in o r c h a in h a s a l y s y l r e s i d u e i n t h e n o n - h e l i c a l r e g i o n a t i t s amino t e r m in a l e n d (7 7 )> A ld o l c o n d e n s a tio n s o f t h e s e a ld e h y d e s form i n t r a m o l e c u l a r d im e rs c a l l e d b e t a c h a i n s . L a th y ris m and P e n i c i l l a m i n e E f f e c t s The b i o l o g i c a l s i g n i f i c a n c e o f t h e c r o s s l i n k i n g o f c o l l a g e n i n c o n n e c tiv e t i s s u e s h a s b e e n w e ll e s t a b l i s h e d . The m ost c l e a r d e m o n s tr a tio n o f t h i s s i g n i f i c a n c e i s p r o v id e d by t h e p a t h o l o g i c a l s t a t e c a l l e d l a t h y r i s m . Young e x p e r im e n ta l a n im a ls w hich a r e s u b j e c t e d t o a c u te s u b l e t h a l d o e se o f b e ta -a m in o p r o p i o n i t r i l e f u m a r a te (BAPN) i n t h e d i e t d e v e lo p v e ry f r a g i l e c o n n e c tiv e t i s s u e s and num erous s k e l e t a l d e f o r m i t i e s . The c o l l a g e n i n th e s e a n im a ls shows a marked d e c r e a s e i n c r o s s l i n k i n g . C o lla g e n w h ich i s s y n th e s iz e d a f t e r t h e i n s u l t o f BAPN i s s u b s e q u e n tl y u n a b le to form c r o s s l i n k s ( 7 8 , 7 9 )• T h is i n a b i l i t y t o form c r o s s l i n k s h a s b e e n shown t o be d i r e c t l y r e l a t e d t o t h e i n a b i l i t y to form a ld e h y d e s on th e c o l l a g e n m o le c u le . The a ld e h y d e s a r e o r d i n a r i l y form ed by th e a c t i o n o f l y s y l o x id a s e on t h e ( -am in o g ro u p s o f s e l e c t e d 2 5 l y s y l and h y d r o x y ly s y l m o i e t i e s . BAPN h a s b e en shown t o be an i n h i b i t o r o f l y s y l o x i d a s e ( 8 0 ) . The p r o g r e s s i v e l o s s o f s o l u b i l i t y t h a t n o rm a lly acc o m p a n ie s t h e p r o c e s s o f c r o s s l i n k f o r m a tio n i s p r e v e n te d by BAPN. As a r e s u l t , s o l u b l e c o l l a g e n a c c u m u la te s i n th e c o n n e c tiv e t i s s u e s o f l a t h y r i t i c e x p e r im e n ta l a n im a ls . A d d i t io n a l e v id e n c e f o r th e s i g n i f i c a n c e o f th e c o n t r i b u t i o n o f c o l l a g e n c r o s s l i n k i n g to t h e t e n s i l e s t r e n g t h o f c o n n e c tiv e t i s s u e s i s p ro v id e d by s u b j e c t i n g young e x p e r im e n ta l a n im a ls t o p e n ic illa m in e e i t h e r a s a d i e t a r y com ponent ( 0 .5 p e r c e n t by w e ig h t) o r by i n j e c t i o n i n t o t h e p e rito n e u m . The r e s u l t i n g e x p e r im e n ta l p a th o lo g y re s e m b le s l a t h y r i s m i n a s u p e r f i c i a l s e n s e ( 8 l , 8 2 ) . Such a n im a ls hav e v e ry low t e n s i l e s t r e n g t h i n t h e i r c o n n e c tiv e t i s s u e s . How ever, c o l l a g e n w hich i s s y n t h e s i z e d a f t e r th e i n s u l t o f p e n i c i l l a m i n e r e g a i n s t h e a b i l i t y t o c r o s s l i n k i f t h e a d m i n i s t r a t i o n o f p e n i c i l l a m i n e c e a s e s ( 8 2 ) . T h is i s d u e t o th e f a c t t h a t a ld e h y d e s a r e a b le t o form n o rm a lly i n t h e new ly s y n t h e s i z e d c o l l a g e n . B ut p e n i c i l l a m i n e r e a c t s r e v e r s i b l y w ith t h e a ld e h y d e s t o form a t h i a z o l i d i n e r i n g com pound: R = c o lla g e n p e p t i d e c h a in a t a l l y s y l r e s i d u e C r o s s li n k s a re form ed by r e a c t i o n o f c o l l a g e n a ld e h y d e s w ith o t h e r a p p r o p r i a t e m o ie t ie s i n th e same o r a d ja c e n t m o le c u l e ( 8 4 ) . R e a c tio n w ith p e n i c i l l a m i n e i n h i b i t s t h i s p r o c e s s , p r e v e n t i n g t h e l o s s o f s o l u b i l i t y t h a t acc o m p a n ie s c r o s s l i n i n g . As a r e s u l t , a n im a ls r e c e i v i n g p e n i c i l l a m i n e a c c u m u la te s o l u b l e c o l l a g e n i n t h e i r c o n n e c tiv e t i s s u e s . I f p e n i c i l l a m i n e a d m i n i s t r a t i o n i s d i s c o n t i n u e d , t h e s o l u b l e c o l l a g e n t h a t h a s a c c u m u la te d l o s e s s o l u b i l i t y and becom es i n d i s t i n g u i s h a b l e from o r d in a r y i n s o l u b l e c o l l a g e n . T h ese phenom ena c a n be d e m o n s tra te d i n th e i s o l a t e d c o l l a g e n from a n im a ls w hich have b e e n t r e a t e d w ith p e n i c i l l a m i n e o r BAPN. S o lu b le c o ll a g e n from s k i n , b o n e , and te n d o n c an be r e c o n s t i t u t e d i n t o n a t i v e - t y p e f i b r i l s by i n c u b a t i o n a t 3 7 ° C a t n e u t r a l pH and p h y s i o l o g i c a l 27 i o n i c s t r e n g t h ( 8 5 ) • W ith s o l u b l e c o l l a g e n i s o l a t e d from norm al a n im a ls , r e c o n s t i t u t i o n i n t o n a t i v e - t y p e f i b r i l s i s a t l e a s t p a r t i a l l y r e v e r s i b l e by c o o li n g t o 4 ° C. The p r o p o r t i o n t h a t c a n be r e d i s s o l v e d g r a d u a l l y d e c r e a s e s w ith i n c r e a s e i n tim e o f i n c u b a t i o n a t 3 7 ° C ( 8 6 ) . W ith s o l u b l e c o l l a g e n from l a t h y r i t i c r a t s , r e c o n s t i t u t i o n i n t o n a t i v e - t y p e f i b r i l s i s e s s e n t i a l l y t o t a l l y r e v e r s i b l e , and t h e r e v e r s i b i l i t y d o e s n o t v a ry d e m o n s tra b ly w ith tim e o f i n c u b a t i o n . C o n v e r s e ly , i f p e n i c i l l a m i n e i s d i a l y s e d o u t o f s o l u b l e c o l l a g e n p r e p a r a t i o n s from a n im a ls t r e a t e d w ith p e n i c i l l a m i n e , r e c o n s t i t u t i o n o f su c h p r e p a r a t i o n s i n t o n a t i v e - t y p e f i b r i l s r e s u l t s i n a r a p i d l o s s o f s o l u b i l i t y w ith tim e o f i n c u b a t i o n . T h is l o s s o f s o l u b i l i t y p r o c e e d s a t a g r e a t e r r a t e t h a n t h a t o b s e rv e d w ith c o l l a g e n from n o rm al a n i m a l s . B u t t h e s o l u b i l i t y l o s s c a n be p r e v e n te d by t h e i n t r o d u c t i o n o f p e n i c i l l a m i n e i n t o t h e r e c o n s t i t u t i o n medium. E ven w ith n o rm al c o l l a g e n p r e p a r a t i o n s , t h e i n t r o d u c t i o n o f p e n i c i l l a m i n e i n t o t h e r e c o n s t i t u t i o n medium p r e v e n t s t h e n o rm al s t e a d y l o s s o f s o l u b i l i t y . O th e r /3 - am ino t h i o l com pounds a r e s i m i l a r l y e f f e c t i v e i n v i t r o s t u d i e s . S o l u b i l i t y C h a r a c t e r i s t i c s o f T is s u e C o lla g e n F e a t u r e s I t s h o u ld b e n o te d t h a t t h e te rm s " s o l u b l e c o lla g e r i' and " i n s o l u b l e c o l l a g e n " a r e o p e r a t i o n a l i n o r i g i n and do n o t r e f e r t o a s i n g l e m o le c u la r e n t i t y . I t h a s b e e n shown t h a t f o r any e x t r a c t e d c o l l a g e n from any i n d i v i d u a l i n any t i s s u e , p o l y d i s p e r s i t y e x i s t s w ith r e s o e c t t o h y d r o x y l a t i o n o f p r o l y l and l y s y l r e s i d u e s ( 8 7 , 8 8 , 8 9 ) , g l y c o s y l a t i o n o f h y d r o x y ly s y l r e s i d u e s ( 9 0 , 9 l) > amount o f c r o s s l i n k i n g , and m o le c u la r s i z e . The n a t u r e o f t h e e x t r a c t i n g medium h a s a s u b s t a n t i a l e f f e c t on th e ty p e o f c o l l a g e n e x t r a c t e d . N e u t r a l s a l t s o l u t i o n s o f m o d e ra te i o n i c s t r e n g t h ( 0 .5 M) e x t r a c t a s m a ll amount o f c o l l a g e n from n o rm al t i s s u e s . T h is h a s b e e n shown by i s o t o p e l a b e l l i n g s t u d i e s t o be t h e p r e c u r s o r o f b o th i n s o l u b l e c o l l a g e n and a c id s o l u b l e c o l l a g e n ( 9 2 , 9 3 )• Such n e u t r a l s a l t s o l u b l e c o l l a g e n (NSS) h a s b e en shown to be t h e m ost r e c e n t l y s y n t h e s i z e d s o l u b l e c o l l a g e n i n a n im a ls o f a l l a g e s . A d d i t io n a l am ounts o f c o ll a g e n c a n u s u a l l y be e x t r a c t e d from th e t i s s u e r e s i d u e a f t e r e x t r a c t i o n o f NSS. L e ss r e c e n t l y s y n t h e s i z e d c o ll a g e n s c a n be e x t r a c t e d e i t h e r by i n c r e a s i n g th e i o n i c s t r e n g t h a t n e u t r a l pH o r by d e c r e a s i n g th e pH a t p h y s i o l o g i c a l i o n i c s t r e n g t h . N e u t r a l s a l t s o l u t i o n s up t o 2 M h av e b e e n used s u c c e s s f u l l y i n t h e e x t r a c t i o n o f c o l l a g e n . C i t r a t e o r a c e t a t e b u f f e r s , pH 1 . 5 - ^ . 0 , and 0 .1 M - 0 .5 M, a r e t y p i c a l l y u se d f o r e x t r a c t i o n o f a c id s o l u b l e c o l l a g e n (ASC). 29 L y sy l O x id ase I n l a t h y r i s m , th e c o n te n t o f a ld e h y d e s i n c o ll a g e n i s d im in is h e d , and t h e c o n te n t o f l y s y l r e s i d u e s i s e l e v a t e d . T h is was t h e f i r s t i n d i c a t i o n t h a t some l y s y l r e s i d u e s w ere t h e p r e c u r s o r s o f a ld e h y d e s i n c o l l a g e n . S u b se q u e n t s t u d i e s showed t h a t c o n n e c tiv e t i s s u e s c o n t a i n a s p e c i f i c enzym e, l y s y l o x id a s e , w hich c o n v e r t s s p e c i f i c l y s y l and h y d r o x y ly s y l r e s i d u e s to a ld e h y d e f u n c t i o n s by an o x i d a t i v e d e a m in a tio n p r o c e s s (9*0 • T h is enzyme h a s a m o le c u la r w e ig h t o f a b o u t 1 7 0 ,0 0 0 . The enzyme a p p a r e n t l y r e q u i r e s Cu> m f o r a c t i v i t y ( 9 5 ,5 1 ) . The a c t i v i t y o f t h e enzyme i n c r e a s e s w ith i n c r e a s i n g oxygen t e n s i o n . T h is a p p a r e n t l in k a g e o f t h e enzyme w ith m ole c u l a r oxygen h a s n o t y e t b e en f i r m l y e s t a b l i s h e d . BAPN i n h i b i t s t h e enzyme i r r e v e r s i b l y . CHAPTER II STATEMENT OP THE PROBLEM T h is s tu d y was u n d e rta k e n to i n v e s t i g a t e th e num ber and d i s t r i b u t i o n o f a ld e h y d e r e s i d u e s p r e s e n t on c o l l a g e n from t h e s k i n s o f f e t a l c a l v e s . S p e c i f i c a l l y , t h e p r e s e n c e o f a ld e h y d e s i n a d d i t i o n t o t h o s e i n th e amino t e r m i n a l , n o n - h e l i c a l p o r t i o n o f th e m o le c u le was d e t e r m i n e d . Such m easu rem en ts w ere made upon c o ll a g e n p r e p a r a t i o n s u s in g v a r i o u s e x t r a c t i o n m ed ia, and upon g e l a t i n d e r iv e d from t h e i n s o l u b l e r e s i d u e o b t a i n e d a f t e r e x h a u s t i v e e x t r a c t i o n w ith n o n - d e n a tu r in g m e d ia . T hese m easu rem en ts w ere made t o e v a l u a t e t h e g e n e r a l s i g n i f i c a n c e o f t h e p r e v i o u s l y r e p o r t e d p r e s e n c e o f a ld e h y d e m o ie t ie s i n c o l l a g e n d e r iv e d from r a t s k in s w ith 0 .2 M yd - m e rc a p to e th y la m in e ( 1 , 2 ) . The g e n e r a l a p p e a ra n c e o f a ld e h y d e m o ie t ie s i n t h e h e l i c a l r e g i o n o f c o l l a g e n w i l l have c o n s i d e r a b l e s i g n i f i c a n c e p a r t i c u l a r l y a s r e l a t e d t o t h e t o t a l c r o s s l i n k i n g c a p a c i ty o f c o l l a g e n . The num ber o f i n t e r m o l e c u l a r c r o s s l i n k s p e r m o le c u le o f c o l l a g e n d e te r m in e s t h e t e n s i l e s t r e n g t h o f t h e t i s s u e s o u r c e . 30 31 The g e n e r a l a p p ro a c h o f t h i s s tu d y h a s b e en tw o f o ld . F i r s t , th e u n m o d ifie d c o l l a g e n p r e p a r a t i o n s w ere exam ined f o r th e p r e s e n c e o f a ld e h y d e m o i e t i e s i n t h e t o t a l m a t e r i a l and i n t h e u ndeg rad ed and d e g ra d e d s u b u n i t s . S eco n d , c o ll a g e n s m o d ifie d w ith 2 , 4 - d i n i t r o p h e n y l h y d r a z in e o r -^H-sodium b o r o h y d rid e w ere exam ined f o r th e p r e s e n c e o f c o v a l e n t l y bound m o d if i e r i n t h e t o t a l c o lla g e n , i n th e d e r iv e d s u b u n i t s , and i n com ponents d e r iv e d from v a r i o u s d i g e s t i o n p r o c e d u r e s . CHAPTER III MATERIALS AND METHODS E x t r a c t i o n o f C o lla g e n s (9 6 ) ( f i g u r e 3 ) S k in s o f f e t a l c a l v e s (1 0 -1 5 Kg) w ere removed im m e d ia te ly a f t e r s l a u g h t e r and packed i n i c e . S u b se q u e n t s t e p s w ere a l l c a r r i e d o u t a t 4° C o r l e s s . E p id e rm is and su b d erm al f a s c i a w ere removed by s c r a p i n g . The d e rm is w ere m inced and w ashed o v e r n i g h t w ith 0 .1 5 M NaCl (4 0 m l/5 gtn m in c e a t e ) . I n a l l c a s e s , e x t r a c t s w ere s e p a r a t e d from r e s i d u e s by c e n t r i f u g a t i o n a t 1 5 ,0 0 0 rpm f o r 40 m in u te s i n th e SS 34 r o t o r o f a S o r v a l l RC2B c e n t r i f u g e . The washed d e rm is r e s i d u e was hom ogenized i n 0 .4 5 M NaCl ♦ .02 M Na-PO^ b u f f e r , pH 7 - 0 (40 m l/5 gm s t a r t i n g m a t e r i a l ) w ith a V i r t i s "45" h o m o g en ize r a t h ig h sp eed t o two m i n u t e s . I c e was p ack ed aro und th e homoge n i z e r f l a s k s t o p r e v e n t h e a t b u i ld u p . O v e rn ig h t e x t r a c t i o n was done on a m e c h a n ic a l s h a k e r i n a r e f r i g e r a t e d room. The e x t r a c t was s e p a r a t e d fro m t h e r e s i d u e and l a b e l l e d a s "NSS" c o l l a g e n ( f o r n e u t r a l s a l t s o l u b l e c o l l a g e n ) . E x t r a c t was p u r i f i e d by t h e p r o c e d u r e d e s c r i b e d below . The r e s i d u e was t h e n t r e a t e d i n e i t h e r o f two w ays: 32 FIGURE 3 Flow C h a rt f o r P r e p a r a t i o n o f E x t r a c t s o f F e t a l C a lf S k in remove s k i n from f e t u s a t s l a u g h t e r i c h i l l on i c e to 4° C ( a l l su b s e q u e n t s t e p s a t s S 4 ° C) 1 . s c r a p e o f f e p id e rm is and su b d erm al f a s c i a w ith s c a l p e l 2 . m ince s k i n t o p i e c e s 2 x 3 m m I e x t r a c t w ith 0 .1 5 NaCl (40 m l/5 gra m inced d e rm is ) 24 h o u rs I c e n t r i f u g e 4 0 ,0 0 0 x g f o r 45 m in u te s s u p e r n a ta n t 4-------------------------------- c ru d e 0 .1 5 NSS p r e c i p i t a t e : hom ogenize i n V i r t i s ”45" hom o g en izer 2 m in u te s a t to p speed e x t r a c t w ith 0 .4 5 M N aC l+.02 M Na-PO^ b u f f e r . pH 7 .0 (40 ml/ 5 gm minced d e rm is ) 24 h o u rs FIGURE 3 (continued) 1 c e n t r i f u g e 4 0 ,0 0 0 x g f o r 45 m in u te s s u p e r n a t a n t c ru d e 0 .4 5 NSS p r e c i p i t a t e Homogenize w ith V i r t i s "45" f o r 2 m in u te s a t h ig h s p e e d . E x tr a c t h o n o g e n a te ( i n 0 . 5 H a c e t i c a c id - 4 o m l/5 gm m inced d e rm is ) 24 h o u rs i C e n tr if u g e 4 0 ,0 0 0 x g f o r 45 m in u te s Homogenize w ith V i r t i s "45" 2 m in u te s a t h ig h speed E x tr a c t w ith 0 .5 H Na- c i t r a t e b u f f e r , pH 3 - 6 (40 ral/5 gm minced d e rm is ) 24 h o u rs ^ C e n tr if u g e 4 0 ,0 0 0 x g f o r 45 m in u te s s u p e r n a ta n t c ru d e ASC s u p e r n a t a n t ^ c ru d e CSC p r e c i p i t a t e : i n s o l u b l e r e s i d u e p r e c i p i t a t e : wash t o pH 7 w ith r e p e a te d s u s p e n s io n i n H_0 and t i t r a t i o n as needed C e n tr if u g e 4 0 ,0 0 0 x g f o r 45 m in u te s s u p e r n a ta n t d i s c a r d * FIGURE J > (continued) ^ P r e c i p i t a t e : su sp en d i n 0 .2 M c y ste a m in e i n 0 .4 5 M N aC l+.02 M NaPO^ b u f f e r , pH 7 . 0 . Homogenize 2 m in u te s i n V i r t i s '*45” a t h ig h speed 24 h o u rs C e n tr if u g e 4 0 ,0 0 0 x g f o r 45 m in u te s s u p e r n a t a n t c ru d e CysSC I P r e c i p i t a t e : i n s o l u b l e c o ll a g e n ^ R e flu x 1 00-1 05° C 2 gm i n s o l u b l e c o lla g e n /4 0 ml H20 f o r 1 h o u r C e n tr ifu g e 1 ,0 0 0 x g 10 m in u te s s u p e r n a t a n t i n s o l u b l e g e l a t i n P r e c i p i t a t e : d i s c a r d R e sid u e w h ich was t o be used s u b s e q u e n tly f o r e x t r a c t i o n w ith 0 .2 M 2 -m e rc a p to e th y la m in e was f i r s t e x t r a c t e d w ith 0 .5 M sodium c i t r a t e b u f f e r , pH 3 . 6 f o r 48 h o u r s . The r e s i d u e was t h e n r e - e x t r a c t e d w ith t h e same c o n d it i o n s f o r an a d d i t i o n a l 48 h o u r s . B oth e x t r a c t s w ere com bined and l a b e l l e d as CSC ( f o r c i t r a t e s o l u b l e c o l l a g e n ) . P u r i f i c a t i o n p r o c e d u r e s a r e d e s c r i b e d b elo w . The r e s i d u e was t h e n d i s p e r s e d i n d i s t i l l e d w a te r and w ashed by m e c h a n ic a l s h a k in g i n t h e c o ld f o r a p p r o x i m a te ly one h o u r , c e n t r i f u g e d and t h e r e s i d u e was s u b j e c t e d to r e p e t i t i o n s o f t h i s p ro c e d u re u n t i l t h e s u p e r n a t a n t was n e a r l y n e u t r a l pH ( 6 - 7 ) . The r e s i d u e was t h e n d iv id e d i n h a l f and reh o m o g en ized i n 0 .2 M 2 -m e rc a p to e th y la m in e i n 0 .4 5 M NaCl ♦ .02 H p h o s p h a te b u f f e r , pH 7 - 0 . The pH was checked and a d ju s te d a s n e c e s s a r y to 7 . 0 . E x t r a c t i o n was t h e n c a r r i e d o u t f o r 48 h o u r s w ith m e c h a n ic a l s h a k in g i n t h e c o ld . G e n e r a lly , t h e e x t r a c t i o n p ro c e d u re was r e p e a te d o n c e . The e x t r a c t s w ere com bined and l a b e l l e d "CysSC" ( f o r c y s te a m in e s o l u b l e c o l l a g e n ) . The r e s i d u e was washed w ith w a te r s e v e r a l tim e s t o rem ove any r e s i d u a l m e rc a p to - yi e th y la m in e . C o lla g e n i n t h e r e s i d u e was o p e r a t i o n a l l y r e f e r r e d t o a s " I n s o l u b l e C o l la g e n ." When p r e p a r a t i o n s o f t h i s r e s i d u e w ere needed f o r a ld e h y d e m e a su re m e n ts, 2 gm o f r e s i d u e w ere p la c e d w ith 40 ml o f d i s t i l l e d w a te r i n a 100 ml round b o tto m f l a s k and a t t a c h e d t o a v e r t i c a l l y o r i e n t e d c o n d e n s e r and r e f l u x e d f o r one h o u r s , t h e n c e n t r i f u g e d w h ile s t i l l h o t . The s u p e r n a t a n t was l a b e l l e d as " I n s o l u b l e G e l a t i n . " 2 . R e sid u e from e x t r a c t i o n w ith 0 .4 5 M NaCl ♦ .02 M N a-P0^ b u f f e r , pH 7 - 0 , was i n some c a s e s hom ogenized i n 0 .5 M a c e t i c a c id and e x t r a c t e d f o r 48 h o u rs i n t h i s medium. The r e s i d u e was u s u a l l y r e - e x t r a c t e d w ith t h e same medium once o r t w i c e , d e p e n d in g upon how much m a t e r i a l was s o l u b i l i z e d i n t h e p r e v io u s e x t r a c t i o n . The e x t r a c t s w ere com bined and l a b e l l e d "ASC" ( f o r a c e t a t e s o l u b l e c o l l a g e n ) . The f i n a l r e s i d u e was a ls o l a b e l l e d o p e r a t i o n a l l y as " I n s o l u b l e R e s id u e ." P u r i f i c a t i o n o f E x t r a c t s (5 5 ) ( F i g u r e 4 ) The p u r i f i c a t i o n p r o c e d u r e f o r a l l e x t r a c t s was th e sam e, e x c e p t a s n o te d b e lo w . E x t r a c t s w ere d i a l y z e d o v e r n i g h t a g a i n s t d e io n iz e d w a t e r , fo llo w e d by .0 2 M 38 FIGURE 4 Flow S h e e t f o r P u r i f i c a t i o n o f E x t r a c t s from F e t a l C a lf S k in 1 . Crude e x t r a c t ( 0 .1 5 NSS, 0 .4 5 NSS, ASC, CysSC, o r CSC) 1 2 . D ia ly z e v s . d i s t i l l e d H O , 4 h o u rs 1 5 . D ia ly z e v s . .02 M NagHPO^, o v e r n ig h t I 4 . C e n tr i f u g e 1 0 ,0 0 0 rpm, 5 m in u te s -> s u p e r n a t a n t - d i s c a r d 5 . P r e c i p i t a t e : d i s s o l v e i n 0 .1 M a c e t i c a c id (40 m l/5 gm o r i g i n a l m inced d e rm is ) i 6 . And s o l i d NaCl t o 5 p e r c e n t (w /v ) 1 7 - C e n tr i f u g e 1 0 ,0 0 0 rpm, 10 m in u te s ^ ----------------------► s u p e r n a ta n t- d is c a r d 8 . - 1 0 . P r e c i p i t a t e : r e p e a t s t e p s 5 -7 i 1 1 . D is s o lv e s t e p 10 p r e c i p i t a t e i n : a . 0 .4 5 M N aCl4 .0 2 M Na-P04 b u f f e r , pH 7 -0 ( f o r 0 .1 5 NSS, 0 .4 5 NSS, o r CysSC) o r b . 0 .1 M a c e t i c a c id ( f o r ASC o r CSC) 40 m l/5 gm o r i g i n a l m inced d e rm is i 1 2 . D i a l y s i s vs 11a o r l i b f o r 24 h o u rs i 1 ^ . C e n tr i f u g e 1 5 ,0 0 0 rpm f o r 45 m in u te s ir - ► p r e c i p i ta t e - d i s c a r d 39 FIGURE 4 ( c o n tin u e d ) 1 1 4 .- 2 2 . S u p e r n a t a n t : r e p e a t s t e p s 2 -1 0 i 2.5• -2 5 • S te p 22 p r e c i p i t a t e : r e p e a t s t e p s 1 1 -1 5 i 2 6 . S u p e r n a t a n t: c e n t r i f u g e 1 0 0 ,0 0 0 x g f o r 90 m in u te s - ► p r e c ip ita te - d is c a r d 27* S u p e r n a t a n t: p u r i f i e d c o ll a g e n 40 NagHPO^ f o r f o u r t o s i x h o u r s . The p r e c i p i t a t e d c o l l a g e n was c o l l e c t e d by c e n t r i f u g a t i o n f o r f i v e m in u te s a t 1 0 ,0 0 0 rpm i n t h e SS 3 4 r o t o r o f a S o r v a l l RC2B r e f r i g e r a te d c e n t r i f u g e . The p r e c i p i t a t e was d i s s o l v e d i n 0 .1 M a c e t i c a c i d . The s o l u t i o n p r o c e s s was a c c e l e r a t e d by h o m o g e n iz a tio n i n a g l a s s - t e f l o n t i s s u e h o m o g e n iz e r w ith a m e c h a n ic a lly d r i v e n p e s t l e . Dry NaCl was added t o b r i n g th e c o n c e n t r a t i o n t o 5 p e r c e n t . M e c h a n ic a l a g i t a t i o n was used t o a c c e l e r a t e th e p r e c i p i t a t i o n p r o c e s s . The p r e c i p i t a t e d c o l l a g e n was c o l l e c t e d by c e n t r i f u g a t i o n f o r t e n m in u te s a t 1 0 ,0 0 0 rpm i n t h e SS 34 r o t o r o f th e S o r v a l l RC2B r e f r i g e r a t e d c e n t r i f u g e . The p r e c i p i t a t e was r e d i s s o l v e d i n 0 .1 M a c e t i c a c id and r e p r e c i p i t a t e d w ith 5 p e r c e n t N a C l. The p r e c i p i t a t e from t h e seco n d 5 p e r c e n t NaCl p r e c i p i t a t i o n was r e d i s s o l v e d i n 0 .4 5 M NaCl ♦ .02 Na-PO^ b u f f e r , pH 7 * 0 , f o r NSS and CysSC, and i n 0 .1 M a c e t i c a c id f o r CSC and ASC. The e x t r a c t s w ere d i a l y z e d o v e r n i g h t a g a i n s t t h e s o l u t i o n medium, p . 45 M NaCl ♦ .02 M Na-PO^ b u f f e r , pH 7 . 0 , f o r NSS and CysSC; and 0 .1 M a c e t i c a c id f o r CSC and ASC. The d i a l y z e d e x t r a c t s w ere t h e n c e n t r i f u g e d f o r 45 m in u te s a t 1 5 )0 0 0 rpm i n t h e SS 34 r o t o r o f t h e S o r v a l l RC2B r e f r i g e r a t e d c e n t r i f u g e . The p r e c i p i t a t e was d i s c a r d e d . The s u p e r n a t a n t was d i a l y z e d o v e r n i g h t a g a i n s t d e io n i z e d w a t e r , t h e n f o r f o u r t o s i x h o u r s a g a i n s t .02 M Na2HP0^. The p r e c i p i t a t e was c o l l e c t e d by c e n t r i f u g a t i o n a s b e f o r e , and d i s s o l v e d i n 0 .4 5 M NaCl + .02 M Na2HP0^, pH 7 .0 , f o r NSS and CysSC, o r 0 .1 M a c e t i c a c id f o r ASC and CSC. D i a l y s i s was c a r r i e d o u t o v e r n ig h t a g a i n s t th e medium used f o r d i s s o l v i n g t h e p r e c i p i t a t e . The s o l u t i o n s w ere t h e n c e n t r i f u g e d f o r 45 m in u te s a t 1 5 ,0 0 0 rpm i n t h e SS 24 r o t o r i n a S o r v a l l RC2B r e f r i g e r a t e d c e n t r i f u g e . The s u p e r n a t a n t from t h i s was c e n t r i f u g e d a t 2 0 ,0 0 0 rpm f o r 90 m in u te s i n t h e num ber 20 r o t o r o f a Beckman Model L -50 u l t r a c e n t r i f u g e . The s u p e r n a t a n t from t h i s was p a s s e d th ro u g h g l a s s w ool t o rem ove l i p i d m a t e r i a l , and t h e f i l t r a t e was o p e r a t i o n a l l y ta k e n a s p u r i f i e d c o ll a g e n . P u r i f i e d c o l l a g e n was f r o z e n f o r s t o r a g e . I s o l a t i o n o f S u b u n i t s ( 9 7 ,4 5 ) ASC o r CSC was s e p a r a t e d i n t o i t s and /S s u b u n its by c h ro m a to g ra p h y on c a rb o x y m e th y l c e l l u l o s e colum ns 2 . 5 cm x 15 cm. R e s o l u ti o n was o b ta in e d by u s in g a l i n e a r g r a d i e n t made from 600 ml o f s t a r t i n g b u f f e r ( . 0 6 M sodium a c e t a t e , pH 4 .8 ) and th e same volum e o f l i m i t b u f f e r ( . 0 6 M sodium a c e t a t e , pH 4.8 p l u s 0 .1 M N a C l). E l u t i o n was c a r r i e d o u t a t 100-120 m l/ h r w ith p e r i s t a l t i c pum ping. E lu a n t was m o n ito re d a t 220 mu i n a U vicon flo w m o n ito r p ro d u c e d by Toyo I n s t r u m e n t s . 10 ml f r a c t i o n s w ere c o l l e c t e d . F r a c t i o n s r e p r e s e n t i n g m a jo r p e a k s w ere p o o le d and d e s a l t e d by d i a l y s i s a g a i n s t r e p e a te d c h a n g e s o f d e io n iz e d w a t e r . The d i a l y z e d p e a k s w ere lyophilized for storage. L im ite d C le a v a g e w ith Cyanogen B ro m id e( 4 4 ,4 5 ) C o lla g e n s w ere d i s s o l v e d i n 0 .1 M NCI. N itr o g e n g a s was b u b b le d th ro u g h th e s o l u t i o n . A p p ro x im a te ly 2 0 0 - t o 5 0 0 - f o l d m o la r e x c e s s o f cy an o g en bro m id e was added r e l a t i v e t o m e th io n in e and r e a c t i o n was a llo w e d to p ro c e e d o v e r n i g h t a t room te m p e r a tu re w ith t h e r e a c t i o n f l a s k im m ersed i n a b e a k e r o f w a te r t o a c t a s a te m p e r a tu r e b u f f e r . At t h e end o f th e r e a c t i o n , an e q u a l volum e o f w a t e r was added and t h e r e a c t i o n m ix tu re was th e n f r o z e n and l y o p h i l i z e d . I n m ost c a s e s , t h e d r i e d m a t e r i a l was r e d i s s o l v e d i n 0 .1 M a c e t i c a c id and r e l y o p h i l i z e d to rem ove any r e s i d u a l cyano gen b ro m id e . The p r o d u c t was s t o r e d i n t h e d ry fo rm . C om plete C lea v ag e w ith Cyanogen B ro m id e ( 9 8 ,9 9 ) The m ethods f o r c o m p lete c le a v a g e w ith cy anogen b ro m id e w ere t h e same a s th o s e used f o r l i m i t e d c le a v a g e , w ith t h e f o l l o w i n g e x c e p t i o n s : 1 . C o lla g e n s o l u t i o n i n 0 .1 N NCI was d e n a tu r e d a t 60° C f o r 15 m in u te s a f t e r f l u s h i n g r e a c t i o n v e s s e l w ith n i t r o g e n g a s , b u t p r i o r t o a d d i t i o n o f cy ano gen b ro m id e . 2 . R e a c tio n sy ste m was m a in ta in e d a t 40° C th r o u g h o u t r e a c t i o n . 43 S e p a r a t i o n o f C yanogen Brom ide P e p t id e s Carb o x y m e th y l c e l l u l o s e c h ro m a to g ra p h y o f CNBr d i g e s t(l6o) D rie d p r o d u c t from c o m p le te d i g e s t i o n o f c o ll a g e n o r i t s s u b u n i t s w ith cy an o g en bro m id e was d i s s o l v e d i n .02 M sodium c i t r a t e b u f f e r , pH 3 * 6 , and t h e pH was r e a d j u s t e d t o 3 - 6 w ith 5 N sodium h y d ro x id e a s n e e d e d . T h is was a p p lie d t o a colum n, (P h a rm a c ia 2 .5 x 20 cm w ith flo w a d a p t e r s ) f o c a rb o x y m e th y l c e l l u l o s e (Whatman m lc r o g r a n u la r CM 3 2 ) w hich had b e e n e q u i l i b r a t e d w ith .02 M sodium c i t r a t e b u f f e r , pH 3 - 6 . The colum n was th e n washed w ith 100-150 ml o f s t a r t i n g b u f f e r t o rem ove u n a b so rb ed p e p t i d e s . A l i n e a r g r a d i e n t was c o n s t r u c t e d c o n s i s t i n g o f 1 ,0 0 0 ml e ac h o f s t a r t i n g b u f f e r v e r s u s s t a r t i n g b u f f e r p l u s 0 .1 M N aCl. Plow was m a in ta in e d a t 100-1 5 0 c c / h r . One h u n d red d ro p f r a c t i o n s w ere c o l l e c t e d u n t i l t h e i n i t i a l wash w ith s t a r t i n g b u f f e r was c o m p le te . Two h u n d red d ro p f r a c t i o n s w ere c o l l e c t e d d u r i n g t h e g r a d i e n t . At t h e end o f th e g r a d i e n t , th e colum n was w ashed w ith a t l e a s t 100 ml o f s t a r t i n g b u f f e r p l u s 0 . 5 M N aC l, t h e n w ith .01 m NaOH * .5 M N aC l. E f f l u e n t from t h e colum n was c o n tin u o u s ly m o n ito re d a t 2 3 4 -2 3 6 mji. T ubes c o m p r is in g v a r i o u s p e a k s w ere p o o le d and l y o p h i l i z e d . 44 P h o s p h o c e llu lo s e c h ro m a to g ra p h y (97>44) P e p tid e m a t e r i a l was d i s s o l v e d i n .001 M sodium fo rm a te b u f f e r , pH 3 . 6 , and a p p lie d t o a 2 .5 x 20 cm colum n o f p h o s p h o c e l l u lo s e (Whatman) w hich had been e q u i l i b r a t e d w ith t h e same b u f f e r . A f t e r w a sh in g th e colum n w ith 100-150 ml o f s t a r t i n g b u f f e r , a l i n e a r g r a d i e n t was a p p li e d w hich was made up o f 400 ml e ach o f s t a r t i n g b u f f e r v e r s u s s t a r t i n g b u f f e r p l u s 0 . 6 M N aC l. The j a c k e t e d colum n was m a in ta in e d a t 40° C th ro u g h o u t t h e r u n . At t h e end o f t h e ru n , 0 .0 1 M NaOH ♦ 0 .5 M NaCl was a p p lie d t o t h e colum n. Tubes c o m p ris in g 230 mji a b s o r b in g p e a k s w ere p o o le d and l y o p h i l i z e d . S ephadex G-50 C hrom atography A P h a rm a c ia colum n, 1 .5 x 90 cm was p ack ed w ith Sephadex G -50, 2 0 0 -4 0 0 m esh, and e q u i l i b r a t e d w ith 0 .1 M a c e t i c a c i d . S am ples w ere a p p lie d d i r e c t l y t o t h e colum n bed and a llo w e d t o s e e p i n t o t h e colum n. The to p o f t h e colum n bed was t h e n r i n s e d w ith two 0 .2 ml a l i q u o t s o f 0 .1 M a c e t i c a c i d . Each wash was a llo w e d t o se e p i n t o t h e g e l b e f o r e n e x t a l i q u o t was a p p l i e d . Then th e s p a c e above t h e to p o f t h e colum n bed was f i l l e d w ith t h e e q u i l i b r a t i o n b u f f e r , and th e to p o f t h e colum n was c lo s e d and a t t a c h e d to t h e r e s e r v l o r and e l u t i o n was a llo w e d t o p ro c e e d a t a b o u t 15 cc p e r h o u r . E lu a n t was m o n ito re d c o n tin u o u s ly a t 230 mji th ro u g h a flo w c e l l i n 45 a Beckman D U -2 -K ln tra c VTI s p e c tr o p h o to m e te r . Twenty d ro p f r a c t i o n s w ere c o l l e c t e d i n an LKB f r a c t i o n c o l l e c t o r w ith a d ro p c o u n t a tta c h m e n t. B io -G e l P-2 C hrom atography C h ro m a tro n ix L C -^-43 colum ns e q u ip p e d w ith two flo w a d a p t e r s , w ere p acked w ith B io -G e l P -2 and e q u i l i b r a t e d w ith 0 .1 M a c e t i c a c i d . A c c o rd in g t o t h e volum e o f sam p le t o be c h ro m a to g ra p h e d , up t o f o u r colum ns w ere c o n n e c te d i n s e r i e s . T hese colum ns w ere u sed p r i m a r i l y t o d e s a l t f r a c t i o n s from p h o sp h o - and c a rb o x y m e th y l c e l l u l o s e r u n s . O c c a s io n a lly , h o w ever, th e y w ere em ployed t o r e s o l v e s m a ll p e p t i d e s . P e p s in D i g e s t io n Sam ples o f u n d e n a tu re d c o l l a g e n w ere d i s s o l v e d i n 0 .1 M HC1 and p e p s i n was added i n a 1 :1 0 0 r a t i o r e l a t i v e t o s u b s t r a t e . The d i g e s t i o n was a llo w e d to p ro c e e d i n t h e c o ld o v e r n i g h t . R e a c tio n was te r m in a te d by f r e e z i n g and l y o p h i l o z a t i o n . When s u b s e q u e n t s t u d i e s w ere t o be done w hich in v o lv e d d e n a t u r a t i o n o f t h e d i g e s t e d c o l l a g e n , t h e p e p s i n was removed by g e l f i l t r a t i o n on Sephadex G -50. T h is was done t o a v o id s u b s e q u e n t d i g e s t i o n o f t h e c o l l a g e n by r e s i d u a l p e p s in a c t i v i t y . A c ry la m id e Gel E l e c t r o p h o r e s i s o f CNBr P e p tid e s TO T) 46 S to c k s o l u t i o n s : I . G e ls A. 48 ml IN KOH 2 1 .1 g l a c i a l a c e t i c a c id 4 . 6 ml N ,N ,N * ,N '- te tr a m e th y le th y le n e d ia m in e b r i n g t o f i n a l volume 100 ml w ith H20 B. J > § gm a c ry la m id e 0 .8 gm N ,N * -m e th y le n e b is a c ry la m id e b r i n g t o f i n a l volum e 100 ml w ith H20 C. 0 .1 5 gm ammonium p e r s u l f a t e b r i n g t o 100 ml w ith HgO I I . B u f f e r - 17*3 gm g l y c i n e (a m m o n ia -fre e ) 2 .2 6 ml g l a c i a l a c e t i c a c id b r i n g t o pH 4 .0 and f i n a l volume 1 l i t e r w ith H20 - f o r u se d i l u t e l / l O I I I . S t a i n - 0 .5 gm B u f f a lo B lac k 45 ml w a te r 50 ml m eth an o l 7 ml g l a c i a l a c e t i c a c id b r i n g t o f i n a l volume 100 ml w ith HgO P r e p a r a t i o n o f g e l s G els c o n t a i n i n g 12 p e r c e n t a c ry la m id e w ere p r e p a r e d by m ix in g A : B : C : : 0 .9 : ^ .2 : 3 « 9 - 1 .0 ml o f t h i s m ix tu re was p la c e d i n e a c h 0 .5 x 7 cm g l a s s g e l t u b e . 0 .2 ml o f d i s t i l l e d w a te r was la y e r e d o n to t h e to p o f e ac h g e l and p o l y m e r iz a ti o n was a llo w e d t o p ro c e e d f o r one h o u r . The g e l s w ere t h e n a tt a c h e d t o e l e c t r o p h o r e s i s a p p a r a tu s made by C an alco ( R o c k v il l e , M a ry la n d ). The u p p e r and lo w e r r e s e r v o i r s w ere f i l l e d w ith d i l u t e d g l y c i n e b u f f e r . P r i o r t o a p p l i c a t i o n o f s a m p le s , t h e g e l s w ere e q u i l i b r a t e d w ith r e s e r v i o r b u f f e r by a p p l i c a t i o n o f an e l e c t r o p h o r e s i s c u r r e n t e q u a l t o 5 m illia m p e r e s p e r tu b e f o r 30 m i n u t e s . P r o t e i n sa m p le s had b e e n p r e p a r e d p r e v i o u s l y i n d i l u t e d b u f f e r and t h e n mixed 1:1 w ith 60 p e r c e n t s u c r o s e . S am ples w ere d e n a tu r e d a t 40° C f o r 30 m in u te s . D e n a tu re d sa m p le s w ere a p p lie d to t h e to p s o f t h e g e l s by l a y e r i n g u n d e r th e e l e c t r o p h o r e s i s b u f f e r w ith a m ic ro s y r i n g e . E l e c t r o p h o r e s i s was done u s in g a c u r r e n t o f 5 m illia m p e r e s p e r t u b e . I n o r d e r t o i n s u r e t h a t t h e s m a l l e s t p e p t i d e s w ere n o t l o s t by p a s s i n g th ro u g h t h e g e l s to o q u i c k l y , e ach sam p le was s u b j e c t e d t o e l e c t r o p h o r e s i s f o r 15 m i n u t e s . To i n s u r e t h a t th e l a r g e r p e p t i d e s a r e r e s o l v e d f u l l y , e a c h p e p t i d e was s e p a r a t e l y s u b j e c t e d t o e l e c t r o p h o r e s i s f o r 30 m i n u t e s . At t h e end o f e a c h e l e c t r o p h o r e s i s p e r i o d , th e g e l s w ere removed from t h e g e l tu b e s and s t a i n e d by im m e rsio n i n th e dye d e s c r i b e d a b o v e . S t a i n i n g was a llo w e d t o p ro c e e d f o r 20 t o 30 m i n u t e s . The g e l s w ere t h e n removed from t h e s t a i n s o l u t i o n and r i n s e d b r i e f l y w ith t a p w a te r 48 and p la c e d i n i n d i v i d u a l b e a k e r s o f t a p w a t e r . The g e l s w ere so ak ed i n r e p e a te d ch an g es o f t a p w a te r t o rem ove unbound s t a i n . The g e l s w ere p r e s e r v e d i n 7 p e r c e n t a c e t i c a c id . U re a d i s c g e l e l e c t r o p h o r e s i s (1 0 2 ) I . S o l u t i o n s A. 24 ml IN KOH 8 . 6 ml g l a c i a l a c e t i c a c id 2 ml N ,N ,N r , N’ - t e t r a m e t h y l e t h y l e n e d i a m i n e b r i n g t o f i n a l volum e 50 ml w ith HgO B. 24 ml IN KOH 1 .9 3 ml g l a c i a l HAc 0 .2 3 ml N,N,N* , N '- t e t r a m e t h y l e t h y l - e n e d ia m in e b r i n g t o f i n a l volum e 50 ml w ith H^O C. 15 gm a c r y la m id e 0 . 4 gm N jN '-m e th y le n e b is a c r y la m id e b r i n g t o f i n a l volum e 50 ml w ith H^O D. 5 gm a c r y la m id e 1 .2 5 gm N ,N '- m e th y le n e b is a c ry la m id e b r i n g t o f i n a l volum e 50 ml w ith H,,0 E. 2 mg r i b o f l a v i n b r i n g t o f i n a l volum e 50 ml w ith H2O P . 140 mg ammonium p e r s u l f a t e b r i n g t o f i n a l volum e 50 ml w ith H,,0 49 G. b u f f e r - 3 1 .2 gm - a l a n i n e 9 ml g l a c i a l a c e t i c a c id b r i n g t o f i n a l volum e 1 l i t e r w ith HgO I I . P r e p a r a t i o n o f g e l A. u p p e r g e l B iD i E i H ^ O ::1 :2 :1 :4 B. lo w e r g e l A=2ml, C=4 m l, F=4 m l, w a te r* 4 .5 m l, p l u s 1 .9 gm u r e a P o ly m e riz e lo w e r g e l on e h o u r w ith l a y e r o f w a te r on to p i n th e u s u a l f a s h i o n , t h e n remove t h e w a te r and add u p p e r l a y e r . P u t a n o th e r w a te r l a y e r on t o p , and p o ly m e riz e tw e n ty m in u te s w ith f l u o r e s c e n t l i g h t . Run sam p le f o u r h o u rs a t f o u r m illia m p e r e s p e r t u b e . P r e p a r e sa m p le by s a t u r a t i o n w ith u r e a , o r h e a t i n g to 40° C, o r b o t h . NaBH), ( % ) R e d u c tio n ( l 0 3 ) C o lla g e n p r e p a r a t i o n s w ere d i s s o l v e d i n 0 .4 5 M N acl ♦ .02 M Na-PO^ b u f f e r , pH J.O. A 1 0 0 -2 0 0 f o l d m o la r e x c e s s o f NaBH^ r e l a t i v e t o a ld e h y d e s was added and d i s s o l v e d on a m e c h a n ic a l a g i t a t o r . pH was m a in ta in e d b e tw e e n 7*0 and 8 . 0 by a d d i t i o n o f 0 .1 M NCI. The pH was m o n ito re d w ith a Beckman E x pand om atic pH m e te r e q u ip p ed w ith a c o m b in a tio n e l e c t r o d e . About 95 p e r c e n t o f t h e r e a c t i o n , a s i n d i c a t e d by r i s e i n pH, was c o m p le te i n t h e f i r s t 1 0 -1 5 m in u te s . R e a c tio n was a llo w e d t o c o n tin u e f o r an a d d i t i o n a l 90 m i n u t e s . The r e a c t i o n was te r m in a te d 50 by a d d i t i o n o f 1 M NCI, b r i n g i n g th e pH t o 4 . 0 . The r e a c t i o n m ix tu r e was th e n d i a l y z e d a g a i n s t ru n n in g c o ld t a p w a te r o v e r n i g h t . The d i a l y s i s was c o n tin u e d a g a i n s t two ch an g e s o f d e io n iz e d w a t e r . I f t h e p r o d u c t was to be s t o r e d , i t was t h e n l y o p h i l i z e d . T r i t i a t e d sodium b o r o h y d r id e was p u rc h a s e d from N u c le a r C hicago and th e s p e c i f i c a c t i v i t y was a d ju s te d t o 9 .3 mc/mmol by d i l u t i o n w ith u n l a b e l l e d sodium b o r o h y d r id e . The m a t e r i a l was t h o r o u g h ly m ixed u s in g a m o r ta r and p e s t l e . The p r o d u c t was t h e n used f o r r e d u c t i o n w ith th e same p ro c e d u re t h a t was used f o r n o n r a d i o a c t i v e r e d u c t i o n s . MBTH A ldehyde E s t i m a t i o n s (1 0 4 ,1 0 5 ) C o lla g e n sa m p le s w ere th o r o u g h ly d ia ly z e d a g a i n s t 0 .1 M g ly c in e -H C l b u f f e r , pH 4 . 0 . They w ere t h e n d e n a tu r e d f o r one h o u r a t 60° C t o i n s u r e t h a t a l l c o l l a g e n p r e s e n t was i n a s o l u b l e fo rm . Then 0 .8 ml o f sa m p le , 1 .0 ml o f 0 .1 M g l y c i n e , pH 4 .0 , and 0 . 2 ml o f N -m eth y lb en z o - t h i a z o l o n e h y d ra z o n e h y d r o c h l o r i d e i n 0 .1 M g l y c i n e b u f f e r , pH 4 . 0 , w ere mixed and in c u b a te d a t 40° C f o r t h i r t y m i n u t e s . A f t e r i n c u b a t i o n , v i s i b l e a b s o r p t i o n s p e c t r a o f t h e r e a c t i o n m ix tu r e s w ere sc an n e d i n a Beckman DBG s p e c tr o p h o to m e te r a tt a c h e d t o a S a r g e n t l i n e a r - l o g r e c o r d e r . S p e c t r a w ere scan n ed b e tw e e n 4 0 0 -3 0 0 mu u s in g t h e a u to m a tic s c a n n in g mode o f t h e i n s t r u m e n t . A ldehyde c o n te n t was e s t im a t e d u s in g a c e ta ld e h y d e s t a n d a r d s , and 51 a l l v a lu e s g iv e n w ere c a l c u l a t e d a s a c e ta ld e h y d e e q u iv a l e n t s from th e p o i n t o f maximum a b s o rb a n c e o f each s p e c tru m . F o r ty p e s u b u n i t s o f c o l l a g e n , 100 mg = 1 umol o f ^ c h a i n . H y d r o ly s is f o r Amino Acid A n a ly s is Acid h y d r o l y s i s (1 0 6 ) Sam ples w ere h y d ro ly z e d i n 6 N HC1 i n 15 x 100 m m i g n i t i o n tu b e s a f t e r b e in g th o ro u g h ly p u rg e d w ith n i t r o g e n g a s and s e a le d w ith an o x y g e n - n a tu r a l g a s t o r c h . Sam ples w ere in c u b a te d o v e r n i g h t a t 105° C. At th e end o f th e r e a c t i o n , tu b e s w ere rem oved from th e oven and a llo w e d t o c o o l . The s e a l was b ro k e n and th e sam p le was d r i e d i n a h e a t i n g b lo c k w ith a b lo w e r c i r c u l a t i n g a i r o v e r th e t u b e s . B ase h y d r o l y s i s (1 0 7 ) S am ples w ere h y d ro ly z e d i n 2 N KOH i n 15 * 100 m m i g n i t i o n tu b e s a f t e r p u r g in g w ith n i t r o g e n g a s and s e a l i n g o f tu b e s w ith an o x y g e n - n a t u r a l g a s t o r c h . A f t e r o v e r n ig h t i n c u b a t i o n a t 105° C, t h e tu b e s w ere a llo w e d t o c o o l. When c o o l, t h e tu b e s w ere o p en ed , and t h e c o n t e n t s t i t r a t e d t o pH 4 .0 w ith p e r c h l o r i c a c id w hich had b e en d i l u t e d 1 :1 w ith d i s t i l l e d w a te r . The sa m p le s w ere th e n c o o le d i n i c e f o r t e n m in u te s and c e n t r i f u g e d i n a c l i n i c a l c e n t r i f u g e a t h ig h speed f o r 52 f i v e m in u te s . The s u p e r n a t a n t was c o l l e c t e d and t h e p r e c i p i t a t e was re s u s p e n d e d i n d i s t i l l e d w a t e r . B oth s u p e r n a t a n t and su sp en d e d p r e c i p i t a t e w ere c o o le d an a d d i t i o n a l t e n m in u te s i n i c e and c e n t r i f u g e d f o r f i v e m in u t e s . The s u p e r n a t a n t s and w a sh e s w ere com bined and d r i e d i n a h e a t i n g b lo c k u n d e r a s tr e a m o f a i r from a b lo w e r . R e a c tio n w ith DNPH(4 2 ) S am ples w ere d ia ly z e d t o rem ove a l l s a l t and t h e n 6 N HC1 was added to g iv e a f i n a l c o n c e n t r a t i o n o f 2 N. A s o l u t i o n o f DNFH, 2 mg/ml i n 2 N HC1, was added and t h e r e a c t i o n was a llo w e d to p r o c e e d a t room t e m p e r a tu r e f o r 1 5 -3 0 m in u te s . E x c e ss DNPH was rem oved by d i a l y s i s a g a i n s t r u n n in g t a p w a te r o v e r n i g h t , f o llo w e d by two c h a n g e s o f d e io n iz e d w a t e r . I f m a t e r i a l was n o t t o be u se d im m e d ia te ly , i t was l y o p h i l i z e d . Amino a c i d a n a l y s i s o f r a d i o a c t i v e sa m p le s S am ples w ere h y d ro ly z e d w ith b a s e a c c o r d i n g to t h e m ethod d e s c r i b e d a b o v e . A n a ly s e s w ere c a r r i e d o u t w ith a J e o l JLC-5AH amino a c id a n a l y z e r u s i n g a s p l i t s tr e a m sy s te m t o c o l l e c t colum n e f f l u e n t f o r a n a l y s i s o f r a d i o a c t i v i t y . S am ples w ere a p p li e d d i r e c t l y t o t h e colum n by l a y e r i n g o n to t h e p r o t e c t i v e f i l t e r im m e d ia te ly a b o v e t h e r e s i n . The r a t e o f flo w i n t o t h e r e s i n w ith o u t pum ping was v i r t u a l l y n i l , so t h e flo w a d a p t e r was 53 hooked up t o th e pump, a tt a c h e d t o t h e f r a c t i o n c o l l e c t o r , and t h e r e c o r d e r was s t a r t e d . T im ing was begun im m edi a t e l y . The colum n ( 0 .9 x 65 cm) had p r e v i o u s ly b e e n e q u i l i b r a t e d w ith pH 3*25 b u f f e r , and t h e colum n te m p e r a t u r e was .55° C. F iv e m in u te f r a c t i o n s w ere c o l l e c t e d . A f t e r 3 5 m in u te s , colum n te m p e r a tu r e was changed t o 60° C. A f t e r two h o u r s and tw e n ty m in u te s , b u f f e r was changed from pH 3 - 2 5 t o 4 .2 5 . S in c e t h e p o i n t o f change o f b u f f e r pH was o b s e r v a b le on t h e r e c o r d e r c h a r t , t h e s y n c h r o n i z a t i o n o f p o s i t i o n o f f r a c t i o n e l u t i o n i n th e f r a c t i o n c o l l e c t o r w ith t h a t o f th e amino a c id a p p e a rin g a t t h a t p o i n t was e s t a b l i s h e d by m e a su rin g t h e pH o f f r a c t i o n s e l u t e d and e x t r a p o l a t i n g d i r e c t l y from t h e p o i n t o f pH c h a n g e . S o u rc e s o f I m p o r ta n t R e a g e n ts M atheson Colem an & B e l l 2 , 4 d i n i t r o p h e n y l h y d r a z i n e p o ta s s iu m h y d r o x id e , c h l o r a - m ine T, p -d im e th y la m in o b e n z - a ld e h y d e , p e r c h l o r i c a c id M atheson S c i e n t i f i c a c e t i c a c id K & K L a b o r a t o r i e s , I n c . n - m e th y lb e n z o th ia z o lo n e h y d ra z o n e HC1 C albiochem M a llin c k r o d t C hem ical Works E astm an O rg a n ic C h e m ica ls E astm an Kodak C an alco N u c le a r C hicago J . T . B a k er C hem ical Co. P a c k ard I n s tr u m e n t C o. , I n c . 5 4 2 -m e rc a p to e th y la m in e ammonium p e r s u l f a t e , h y d ro c h l o r i c a c i d , sodium c h l o r i d e , f o rm ic a c i d , u r e a , a c e t - a ld e h y d e , t o l u e n e , n - p r o p y l a lc o h o l cy an o g en b ro m id e , a c r y la m id e , N ,N ,N ',N , - t e t r a m e t h y l - e th y le n e d ia m in e , N ,N ’ - m e th y le n e b is a c r y la m id e , g l y c i n e , c ro to n a ld e h y d e p -d io x am e r i b o f l a v i n , / 5 - a l a n l n e sodium b o ro h y d rid e -^ H l a b e l l e d sodium c i t r a t e , sodium p h o s p h a te d i b a s i c , sodium h y d r o x id e , n a p h th a le n e d i p h e n y l o x a z o l e , 1 ,4 b i s - ( 2 - ( 4 - m e th y l- 5 - p h e n y lo n a z o ly l j]- b e n ze n e P i e r c e C hem ical C o. m e t h y l c e l l o s o l v e Whatman c a rb o x y m e th y l c e l l u l o s e , p h o s p h o c e llu lo s e P h a rm a c ia Sephadex G-50 B io Rad L a b o r a t o r i e s B io -G e l P - 2 , B io -G e l P-200 S o u rc e o f T is s u e P e t a l c a l f s k i n s g e n e r o u s ly s u p p lie d by A t l a s Meat P a c k in g C o ., V ernon, C a l i f o r n i a . CHAPTER IV RESULTS E x t r a c t i o n o f C o lla g e n The s u s c e p t i b i l i t y o f f e t a l c a l f s k i n c o l l a g e n t o e x t r a c t i o n i n t h e v a r i o u s m edia used f o r t h e s e e x p e r i m ents i s i l l u s t r a t e d by T a b le I I I . T h ese v a lu e s w ere o b ta in e d by t h e s e p a r a t e s e r i a l e x t r a c t i o n o f s i x t e e n sa m p le s o f .5 gm (w et w e ig h t) e ac h o f c le a n e d f e t a l c a l f d e rm is t a k e n from t h e same s o u r c e . T hese d a t a a re co m p a rab le t o t h o s e o b ta in e d by o t h e r i n v e s t i g a t o r s f o r s k in s from v a r i o u s young a n im a ls ( 1 0 8 ,1 0 9 ) . As i s i l l u s t r a t e d by e a c h o f t h e r e p e a t e x t r a c t i o n s w ith any o f t h e m ed ia, a t l e a s t 90 p e r c e n t o f th e c o l l a g e n w hich i s e x t r a c t a b l e w ith a p a r t i c u l a r medium i s removed from th e t i s s u e d u r i n g t h e f i r s t e x t r a c t i o n p r o c e d u r e . T h is i n d i c a t e s t h a t r e p e a te d e x t r a c t i o n s w ith any p a r t i c u l a r medium would be o f v e ry l i m i t e d u s e f u l n e s s i n p r e p a r a t i v e s t u d i e s . So, s u b s e q u e n t s t u d i e s w ere g e n e r a l l y done o n ly on th e f i r s t e x t r a c t i o n w ith any p a r t i c u l a r medium. A lth o u g h t h e d a t a i n T a b le I I I w ere o b ta in e d from o n ly one f e t u 3 a s t h e t i s s u e s o u r c e , i t was foun d i n s u b s e q u e n t 5 6 57 TABLE III EXTRACTABILITY OP COLLAGEN PROM PETAL CALF SKIN s t a r t i n g w e ig h t-5 0 0 mg - 10 w et w e ig h t S o lv e n t C o lla g e n e x t r a c t e d 0 .1 5 M N aC l........................................................... 1 .1 6 ± .0 7 mg 0 .4 5 M NaCl + .0 2 M Na-PO^ pH 7 .0 1 s t e x t r a c t i o n . 2nd e x t r a c t i o n .................. 0 .5 M a c e t i c a c i d 1 s t e x t r a c t i o n ........................ 6 1 .7 - 2 .0 mg 2nd e x t r a c t i o n ........................ 6 .2 - .9 5 mg o r : 0 .2 M 2 -m e r c a p to e th y la m in e + 0 .4 5 M NaCl + .0 2 M N a-P 04 pH 7 .0 1 s t e x t r a c t i o n . 4 7 .3 - 1 .4 mg 2nd e x t r a c t i o n . . . . . . . . . 3 .81 - 3 .1 mg i n s o l u b l e r e s i d u e ............................................... 7 3 .7 - 5 .2 mg 5 .2 2 ± .11 mg 0 .6 1 - .3 7 mg s t u d i e s t h a t t h e e x t r a c t a b i l i t y o f c o ll a g e n s from s k i n s o f o t h e r f e t u s e s conform ed t o th e same g e n e r a l p a t t e r n a s i s i l l u s t r a t e d h e r e . The 90 p e r c e n t d e c l i n e i n e x t r a c t a b i l i t y o f c o l l a g e n from t h e s k i n w ith a r e p e a t a l i q u o t o f any o f t h e m edia i n d i c a t e s t h a t 0 .1 5 M NaCI, 0 .4 5 M NaCl, 0 .5 M a c e t i c a c i d , and 0 .2 M c y s te a m in e i n 0 .4 5 M N aCl, e a c h e x t r a c t s an o p e r a t i o n a l l y d i s t i n c t p o p u l a t i o n o f c o l l a g e n m o le c u le s . I t s h o u ld be n o te d , h ow ever, t h a t a change i n o r d e r o f e x t r a c t i o n w ith th e v a r i o u s m edia w i l l r e s u l t i n a p a r t i a l o v e r l a p o f t h e p o p u l a t i o n s . T h is s u g g e s ts t h a t t h e o r d e r o f e x t r a c t i o n s t e p s i s s i g n i f i c a n t . Such o r d e r can be u n d e rs to o d i n te rm s o f an i n c r e a s i n g s e v e r i t y o f e x t r a c t i o n c o n d i t i o n s . I n t h i s m anner, 0 .1 5 M NaCl i s a l e s s s e v e r e c o n d i t i o n th a n 0 .4 5 M N aC I. 0 .4 5 M NaCl i s i n t u r n a l e s s s e v e r e c o n d i t i o n th a n 0 . 5 M a c e t i c a c id o r 0 - 5 M c i t r a t e (sodium b u f f e r , pH J . 6 . T hese two m edia e x t r a c t s i m i l a r p o p u l a t i o n s o f m o le c u le s . S i m i l a r l y , 0 .2 M 2 -m e rc a p to e th y la m in e i n 0 .4 5 M NaCI ♦ 0 .0 2 M Na-PO^, pH 7 - 0 , i s a more p o w e rfu l e x t r a c t i o n c o n d i t i o n t h a n e i t h e r 0 . 5 M a c e t i c a c id o r 0 .5 M c i t r a t e (so d iu m ) b u f f e r , pH 3 . 6 . A ldehyde C o n te n t o f E x t r a c t s The amount o f a ld e h y d e r e a c t i v e m a t e r i a l p r e s e n t i n e ac h o f t h e p u r i f i e d e x t r a c t s i s i n d i c a t e d i n T a b le IV. TABLE IV ALDEHYDE CONTENT OP SELECTED EXTRACTS S o lv e n t 0.45M NaCI ♦ .02M Na-PO^ pH 7 .0 0 . 5 M a c e t i c a c id DNPH umol/lOOmg o f C o lla g e n I .69 1.831 MBTH umol/lOOmg o f C o lla g e n 2 .6 7 2.30 H - r e d u c t i o n cpm/ugm o f C o lla g e n 210 1 5 195 - 4 .8 0 .2 M 2 -m e rc a p to e th y la m in e - 0 .4 5 M NaCI ♦ .02M Na-P04 pH 7 -0 i n s o l u b l e r e s i d u e ( g e l a t i n ) 1 .9 9 3 .3 0 2 .3 0 230 i 6 .5 60 The am ounts o f a ld e h y d e s d e t e c t e d by r e a c t i o n w ith DNPH w ere c o n s i s t e n t l y lo w e r t h a n by r e a c t i o n w ith MBTH. The r a t i o o f a ld e h y d e s d e t e c t e d by MBTH t o a ld e h y d e s d e t e c t e d by DNPH ran g e d from a b o u t 1 .3 t o 1 .8 . The g e n e r a l p a t t e r n o f d e t e c t a b l e a ld e h y d e s i n t h e v a r i o u s f r a c t i o n s d i d p e r s i s t from one ty p e o f a s s a y t o th e n e x t . One p o s s i b l e e x p l a n a t i o n f o r t h i s a r i s e s from th e n a t u r e o f t h e s t a n d a r d used f o r DNPH d e t e r m i n a t i o n s . S ta n d a rd c u rv e s w ere made u s i n g u n r e a c te d 2 ,4 d in itr o p h e r \y lh y d r a z in e . I t was assum ed t h a t th e m o la r e x t i n c t i o n c o e f f i c i e n t o f t h e u n re a c te d m a t e r i a l was e q u a l t o t h a t o f r e a c te d m a t e r i a l . Any v a r i a t i o n from t h i s a s s u m p tio n would a l t e r t h e f i n a l v a lu e s o b t a i n e d . The g e n e r a l r e l a t i o n s h i p among th e v a lu e s o b ta in e d s h o u ld be in d e p e n d e n t o f t h e c o n s t a n t used f o r c o n v e r t i n g raw d a t a t o num ber o f a ld e h y d e s p e r m o le c u le . The a b s o r p t i o n s p e c t r a o f t h e p r o d u c t s o f r e a c t i o n o f e a c h o f th e e x t r a c t s w ith MBTH o r DNPH a r e p r e s e n t e d i n F i g u r e s 3 and 6 . I n t h e r e a c t i o n w ith MBTH, a t a b o u t 350 mji, more o f t h e s h o u ld e r i s fo u n d i n p r e p a r a t i o n s w hich w ere e x t r a c t e d w ith more s e v e r e c o n d i t i o n s . T h is i n d i c a t e s t h a t t h e r e was a p r o g r e s s i v e l y g r e a t e r amount o f ^ S - s u b u n its i n t h e e x t r a c t s w ith p r o g r e s s i v e l y more s e v e r e c o n d i t i o n s . T h is p a t t e r n i s s i m i l a r t o t h a t o b se rv e d by o t h e r i n v e s t i g a t o r s (2). S t i l l , t h e a ld e h y d e c o n te n t o f e ac h f r a c t i o n w as g r e a t e r t h a n t h a t o b s e rv e d by o t h e r i n v e s t i g a t o r s . FIGURE 5 6 1 l i m i t e d c le a v a g e l i m i t e d c le a v a g e 0 .1 5 NSS l i m i t e d c le a v a g e l i m i t e d c le a v a g e on- CysSC ASC Absorption Spectra from Reaction of Selected Extracts with KBTH(l mg c o l l a g e n e a c h ) FIGURE 6 DNPH Spectra of Extracts o f F e t a l C a l f S k in (n o rm a liz e d ) 980 375 11m cleav 11m c le a v lim c le a v 11m cleav 500 900 40o 300 0. 15 HSS 0.45 HS5 ASC CysSC ^ P u b lis h e d s t u d i e s o f a ld e h y d e c o n t e n t by o t h e r i n v e s t i g a t o r s w ere p e rfo rm e d w ith t i s s u e s fro m p o s t n a t a l a n im a ls ( 110). A lth o u g h -^H u p ta k e c o u ld n o t be c o n v e r te d t o m oles o f a ld e h y d e p e r m ole o f c h a i n i n t h e e x t r a c t s , i t was fo u n d t h a t t h e p a t t e r n o f u p ta k e was c o n s i s t e n t w ith th e a ld e h y d e c o n t e n t from t h e o t h e r m e th o d s. F i g u r e s 5 and 6 a l s o i n c l u d e t h e s p e c t r a o f t h e MBTH and DNPH d e r i v a t i v e s o f e x t r a c t s w hich had b een s u b j e c t e d t o l i m i t e d c le a v a g e w ith CNBr. L im ite d c le a v a g e i s a c c o m p lis h e d by t r e a t m e n t o f c o l l a g e n w ith t h e r e a g e n t u n d e r c o n d i t i o n s w hich do n o t d e n a t u r e t h e p r o t e i n . When t h i s i s d o n e , c le a v a g e o c c u r s a t o n ly t h o s e m e th io n y l g r o u p s w hich a p p e a r i n t h e n o n - h e l i c a l r e g i o n o f t h e m o le c u le . E q u a l am ounts o f p r o t e i n w ere used f o r e a c h MBTH s p e c tr u m . The s p e c t r a o f D N P H -treated e x t r a c t s w ere n o r m a liz e d f o r e a s e o f c o m p a ris o n . The s p e c tru m o f e a c h o f t h e CNBr l i m i t e d c le a v e d e x t r a c t s was n o rm a liz e d r e l a t i v e t o i t s p a r e n t , u n c le a v e d e x t r a c t . The s p e c tru m o f e a c h e x t r a c t e x h i b i t s a c o m p a ra b le ch an g e i n d e m o n s tra b l e a ld e h y d e s b e tw e e n t h e u n c le a v e d and l i m i t e d c le a v e d s t a t e . F o r MBTH s p e c t r a , t h e e x t r a c t s e x h i b i t v a r y i n g am ounts o f d e m o n s tr a b le JL, ^ - u n s a t u r a t e d a ld e h y d e , a s i n d i c a t e d by t h e s h o u l d e r a p p e a r i n g a t 3 50 mji. A r e l a t i v e l y g r e a t e r am ount o f t h i s s h o u l d e r i s p r e s e n t i n ASC and CysSC t h a n i n 0 .1 5 NSS o r 0 .4 5 NSS. A f t e r l i m i t e d 64 c le a v a g e w ith CNBr, th e s h o u l d e r a t 350 mji was c o n s i d e r a b ly d im in is h e d f o r e ach e x t r a c t . T h is i s c o n s i s t e n t w ith t h e c o n c e p t t h a t i n t r a m o l e c u l a r c r o s s l i n k i n g i s e f f e c t e d th r o u g h t h e f o r m a tio n o f a l d o l c o n d e n s a tio n p r o d u c ts from a l y s i n e - d e r i v e d a ld e h y d e a p p e a r in g i n t h e non h e l i c a l amino t e r m i n a l end o f each o f tw o a lp h a c h a i n s . T h is a l d o l c o n d e n s a tio n p r o d u c t , w hich fo rm s i n t r a m o le c u la r jS s u b u n i t s , i s removed from t h e m o le c u le d u r i n g c le a v a g e w ith CNBr. F u r t h e r i l l u s t r a t i o n o f t h i s p o i n t comes from i n s p e c t i o n o f F ig u r e 6 . The p e a k s a t a b o u t 380 mji f o r ASC and CysSC a r e c h a r a c t e r i s t i c o f th e p r e s e n c e o f - u n s a t u r a t e d a ld e h y d e s ( s e e s p e c tru m o f c r o t o n a l d e - hyde-DNPH i n F ig u r e 7 ) . B ut t r e a tm e n t w ith CNBr c a u s e s a s h i f t i n th e p e a k o f t h e a b s o r p t i o n s p e c tru m to a b o u t 3 5 0 -3 6 5 mji ( s e e g lu ta ra ld e h y d e -D N P H sp e c tru m i n F ig u r e 8). F ig u r e 9 i l l u s t r a t e s t y p i c a l a c r y la m id e g e l e l e c t r o p h o r e s i s p a t t e r n s f o r e a c h o f t h e p a r a t i o n s . I t c a n be s e e n t h a t a b o u t 85 t o 90 p e r c e n t o f t h e ^ and s u b u n i t s a r e d e g ra d e d i n t h e l i m i t e d CNBr d i g e s t , w ith t h e com ponents i n c r e a s i n g t o a c o r r e s p o n d in g e x t e n t . T h is i s c o n s i s t e n t w ith t h e s e l e c t i v e c le a v a g e o f t h e n o n - h e l i c a l p e p t i d e s , l e a v i n g t h e h e l i c a l p o r t i o n s u b u n i t s i n t a c t . T h ese s u b u n i t s h av e a m o le c u la r w e ig h t a p p r o x i m a te ly 1 p e r c e n t l e s s t h a n cA c h a i n s , m aking them 390 3fO HfO 65 FIGURE 8 f G lu ta r a ld e h y d e 2 ,4 DNPH 210 - 220“- * ' ' * 0 ^ 2 4 0 400 300 225 600 500 400 300 67 FIGURE 9 A c ry la m id e g e l e l e c t r o p h o r e s i s o f ( a ) NSS, and (b ) CysSC e x t r a c t s and t h e i r r e s p e c t i v e l i m i t e d CNBr d i g e s t s : ( c ) and (d). A c ry la m id e g e l e l e c t r o p h o r e s i s o f a c id s o l u b l e c o l l a g e n ( e ) , and i t s l i m i t e d CNBr d i g e s t (f). 68 i n d i s t i n g u i s h a b l e from cA c h a in s on a c ry la m id e g e l e l e c t r o p h o r e s i s . T a b le V p r e s e n t s th e amino a c id c o m p o s itio n s o f t h e v a r i o u s e x t r a c t s and o f t h e ^ s u b u n i t s d e r iv e d from one o f them ( a c i d s o l u b l e c o l l a g e n ) . Only m inim al v a r i a t i o n o c c u r s from on e e x t r a c t to th e n e x t, i n d i c a t i n g t h a t i t i s l i k e l y t h a t t h e s u b u n i t s o f one e x t r a c t a r e l i k e l y t o be s i m i l a r t o t h e s u b u n i t s o f any o f t h e o t h e r e x t r a c t s . The amino a c id c o m p o s itio n s o f t h e ©4 s u b u n i t s o f a c id s o l u b l e c o l l a g e n a r e s i m i l a r t o th e v a lu e s found by o t h e r i n v e s t i g a t o r s u s in g b o v in e c o l l a g e n from p o s t n a t a l s o u r c e s ( 1 1 1 ,1 1 2 ) . R e s o l u ti o n o f S u b u n its o f Acid S o lu b le C o lla g e n by d a rb o x y m e th y l C e l l u l o s e C hrom atography F i g u r e 10 i s an exam ple o f t h e ty p e o f c h ro m a to gram o b t a i n e d . The p e a k s r e p r e s e n t i n g ^ , and n w ere w e ll s e p a r a t e d from t h e p e a k s r e p r e s e n t i n g and The r e s o l u t i o n o f from was i n c o m p l e t e . The r e s o l u t i o n o f from <^v was b e t t e r t h a n t h a t f o r from , b u t i t was a l s o in c o m p le te . The y i e l d o f p e p t i d e s from t h e colum n was t y p i c a l l y a b o u t 60 p e r c e n t , a s com pared t o th e amount o f m a t e r i a l a p p l i e d . T h is y i e l d i s c o m p a ra b le t o t h a t o b ta in e d i n s i m i l a r s t u d i e s o f c o l l a g e n s from o t h e r s o u r c e s ( 1 1 1 ,1 1 2 ) . The s m a ll p e ak t h a t e l u t e s b e tw e e n , and a p p e a re d c o n s i s t e n t l y i n t h e s e ch ro m ato g ram s, b u t was n o t s t u d i e d any f u r t h e r . TABLE V AMINO ACID COMPOSITION OP EXTRACTS AND SELECTED SUBUNITS Amino Acid 0 .1 5 NSS 0 .4 5 NSS CysSC ASC ABC ASC cX V h y d ro x y ly s in e 7 .1 6 .0 6 .4 6 .6 4 .1 9 -5 l y s i n e 2 6 .2 2 9 .4 2 3 .8 2 4 .2 3 0 .9 2 0 .3 h i s t i d i n e 3 .1 1 .8 4 .2 5 .1 2 .9 8 .5 a r g i n i n e 4 6 .3 4 7 .9 4 5 .1 4 6 .6 5 0 .5 55-0 m e th io n in e ♦ s u l f o x i d e s 4 .6 3 .9 5 -7 5 .1 6 .5 5 .5 a s p a r t i c a c id 4 8 .9 4 1 .0 4 6 .0 4 4 .2 4 7 .5 4 6 .8 th r e o n in e 16 .2 1 8 .0 1 7 -3 18.1 1 7 .1 2 0 .0 s e r i n e 3 1 -7 . 3 6 .5 3 1 .8 3 5 .3 3 0 .7 3 4 .0 g lu ta m ic a c id 6 7 .0 7 6 .3 70.1 6 8 .0 7 9 .1 7 8 .7 p r o l i n e 109.0 1 1 7 .2 126.0 105.0 129.0 1 2 5 .0 TABLE V Amino Acid 0 .1 5 NSS 0 .4 5 NSS h y d ro x y p ro lin e 1 1 1 .0 1 0 6 .4 g l y c i n e 3 2 4 .3 3 3 7 .0 a la n in e 1 1 2 .1 107 -9 v a l i n e 2 0 .7 2 1 .9 i s o l e u c i n e 1 0 .9 1 2 .3 l e u c in e 2 5 -3 2 6 .8 t y r o s i n e 1 .8 3*2 p h e n y la la n in e 1 0 .8 1 2 .1 (continued) CysSC ASC ASC ASC 9 6 .3 1 1 8 .0 1 0 8 .0 1 1 5 .0 3 3 4 .2 3 4 1 .0 3 5 1 -0 3 3 2 .0 1 1 6 .3 1 2 0 .0 1 2 1 .2 1 0 7 .0 1 9 .1 2 2 .1 2 0 .1 3 3 -0 1 0 .5 1 1 .0 6 .5 1 7 .4 2 4 .1 2 5 -5 2 2 .3 3 1 .2 2 .4 2 .5 1 .6 3 .4 1 2 .3 1 2 .7 1 2 .6 1 6 .1 o 71 FIGURE 10 0 .5 0 0 . 0 .4 0 0 . < 0 . 200- 200 4 0 0 i Volum e o f e f f lu < 100 E l u t i o n p r o f i l e o f c a r b o x y m e th y l c e l l u l o s e cl c o l l a g e n a s d e s c r i b e d i n t e x t ( s e e M e th o d s : o f C h a i n s ) . S h ad ed a r e a s i n d i c a t e f r a c t i o n s 71 FIGURE 10 400 Volum e o f e f f l u e n t ( m l) 3 0 0 600 800 500 700 e f f l u e n t p r o f i l e o f c a r b o x y m e th y l c e l l u l o s e c h ro m a to g ra p h y o f a c i d s o l u b l e i a s d e s c r i b e d i n t e x t ( s e e M e th o d s: CM C e l l u l o s e C h ro m a to g ra p h y i s ) . S h ad ed a r e a s i n d i c a t e f r a c t i o n s p o o l e d f o r f u r t h e r s t u d y . m m 72 I n no c a s e d id t h i s f r a c t i o n amount t o more t h a n 2 p e r c e n t o f t h e e l u t e d m a t e r i a l . T h ese c h ro m a to g ra p h ie s w ere t y p i c a l l y a llo w e d t o p ro c e e d o v e r n i g h t w ith t h e b u f f e r pump c o n n e c te d t o an a u to m a tic t i m e r . The t i m e r was s e t t o t u r n th e pump o f f when t h e r e was s t i l l 1 00-200 ml o f b u f f e r l e f t i n th e g r a d i e n t . The c o n ta n t te m p e r a tu r e b a th was n o t tu r n e d o f f , th o u g h . I n t h e m o rn in g , t h e pump was r e s t a r t e d and t h e re m a in d e r o f t h e g r a d i e n t was u s e d . The g r a d i e n t was fo llo w e d by w a sh in g t h e colum n w ith 0 . 5 M NaCl + .01 N NaOH. I t was g e n e r a l l y found t h a t a l a r g e p eak e lu t e d when pum ping was s t a r t e d t o c o m p le te t h e re m a in d e r o f t h e g r a d i e n t . An a d d i t i o n a l , s u b s t a n t i a l p eak was o b ta in e d w ith t h e sodium h y d r o x i d e - s a l t w ash. The p eak t h a t e l u t e d w ith t h e re m a in d e r o f t h e g r a d i e n t a t th e end o f th e o v e r n i g h t r u n s was n o t s e e n when c h ro m ato g rap h y was done from s t a r t t o f i n i s h i n one c o n tin u o u s r u n . I n such c a s e s , th e p e a k t h a t e l u t e d w ith .5 M NaCl ♦ .01 N NaOH was i n c r e a s e d i n s i z e . B ut th e i n c r e a s e was n o t s u f f i c i e n t t o a c c o u n t f o r t h e d e c l i n e i n t h e a p p e a ra n c e o f t h e p e a k a t t h e end o f t h e r u n . When t h e amount o f m a t e r i a l t h a t e l u t e d i n t h e s e two p e a k s was added t o t h e amount o f m a t e r i a l i n t h e more t y p i c a l p a r t o f th e chrom atogram , t h e t o t a l y i e l d o f 230 mji a b s o r b in g m a t e r i a l a p p ro a c h e d 90 p e r c e n t o f t h e amount o r i g i n a l l y 73 a p p lie d to t h e colum n. T a b le VI sum m arizes t h e d i s t r i b u t i o n o f o4 c h a in s i n th e ch ro m ato g ram . T h is c a l c u l a t i o n was done by c o n s i d e r i n g t h e m a t e r i a l e l u t i n g u n d e r th e p e a k as t o t a l l y . S i m i l a r l y , m a t e r i a l e l u t i n g u n d e r t h e f i t , p e ak was c o n s id e r e d a s one h a l f and one h a l f S in c e t h e volum e o f t h e f r a c t i o n s was c o n s t a n t , i t was p o s s i b l e t o i n t e g r a t e u n d e r e ac h p e ak by t o t a l l i n g th e 230 m)i a b s o rb a n c e v a lu e s o f a l l tu b e s c o rr e s p o n d in g t o a p a r t i c u l a r p e a k . The i n t e g r a t e d v a lu e s w ere t h e b a s i s f o r c a l c u l a t i n g t h e r e l a t i v e d i s t r i b u t i o n o f ^ 4 , v e r s u s T hese v a lu e s c o n t r a s t s h a r p ly w ith th e s ta n d a r d c o n c e p t o f c o l l a g e n a s h a v in g two c h a in s and one c h a i n . They a r e a l s o i n c o n s i s t e n t w ith t h e r e c e n t r e p o r t s o f a l t e r n a t i v e fo rm s o f c o ll a g e n c o n s i s t i n g p u r e ly o f a l t e r n a t i v e t y p e s o f ^4, c h a in s ( 1 4 , 1 7 , 1 8 ) . I f th e c o l l a g e n w ere p u r e l y o f th e norm al ty p e , th e r a t i o o f t o w ould be 2 .0 0 . P r e s e n c e o f any o f t h e r e p o r te d a l t e r n a t i v e fo rm s o f c o l l a g e n would i n c r e a s e t h e <Ai t o *4>. r a t i o n above 2 .0 0 . T hese d a t a i n d i c a t e t h e p r e s e n c e o f an e l e v a t e d amount o f ^ v . The c o m p o s itio n o f t h e p e a k s e l u t i n g a t t h e end o f t h e chrom atogram was n o t d e te r m in e d . I n g e n e r a l , t h e r e was enough m a t e r i a l i n th e s e p e a k s t o i n c r e a s e th e r a t i o t o n e a r l y 2 i f a l l o f i t w ere o r i g i n a l l y c h a i n s . TABLE VI ALPHA SUBUNIT COMPOSITION OP ACID SOLUBLE COLLAGENS OP PETAL CALF SKIN E x t r a c t i o n medium T o ta l (£ ) T o ta l o <2 (# ) R a tio 0 .5 M N a C itr a te b u f f e r pH 3 .6 5 4 .5 - 1 .1 ( 4 6 .9 - 6 3 .3 ) 4 5 .5 * 1 .1 ( 3 6 .7 - 5 3 .1 ) 1 .2 2 * .0 6 ( .8 8 - 1 .7 2 ) 0 .5 M a c e t i c a c i d 5 3 .7 * 1 .2 ( 4 8 .9 - 6 0 .4 ) 4 6 .3 * 1 .2 ( 3 9 .6 - 5 1 .1 ) 1 .1 8- . 0 6 ( . 9 6 - 1 . 5 2 ) N ote - P a r e n th e s e s i n d i c a t e ra n g e o f v a lu e s o b ta in e d . Method o f c a l c u l a t i o n d e s c r ib e d i n t e x t . ■ f i - 75 O th e r i n v e s t i g a t o r s have foun d an r a t i o s i g n i f i c a n t l y l e s s th a n 2 ( 9 5 , 1 1 2 ) . T hese r e p o r t s i n d i c a t e d t h a t t h e d i f f e r e n c e was w i t h i n e x p e r im e n ta l e r r o r o f a r a t i o o f 2 . The v a lu e s r e p o r t e d h e re w ere c a l c u l a t e d on t h e b a s i s o f 26 c h ro m a to g ra p h ic r u n s : 15 o f c o l l a g e n e x t r a c t e d w ith c i t r a t e and 11 o f c o ll a g e n e x t r a c t e d w ith a c e t i c a c i d . Of t h e s e , t h e r e w ere o n ly t h r e e ru n s w hich g a v e a n r a t i o g r e a t e r t h a n 1 . 5 . N e a rly two t h i r d s o f t h e r u n s g a v e an r a t i o o f l e s s th a n 1 .5 * F ig u r e 11 i l l u s t r a t e s t y p i c a l MBTH a ld e h y d e s p e c t r a o b ta in e d from t h e v a r i o u s s u b u n it f r a c t i o n s i s o l a t e d by c a rb o x y m e th y l c e l l u l o s e c h ro m a to g ra p h y . The and s u b u n i t s e a c h e x h i b i t s an s o r p t i o n maximum a t 3 0 7 -3 1 0 mji, t y p i c a l o f s im p le a l i p h a t i c a ld e h y d e s . The and s u b u n i t s e a c h had an a b s o r p t i o n maximum a t 3 0 8 -3 1 3 mji, s u g g e s t i n g th e p r e s e n c e o f a n h y d ro x y l g ro u p a d j a c e n t t o t h e a ld e h y d e f u n c t i o n . I n a d d i t i o n , a s u b s t a n t i a l s h o u l d e r a t a p p ro x im a te ly 350 m)i was foun d w ith t h e jS s u b u n i t s . T h is s h o u l d e r i s t y p i c a l o f , ^ - u n s a t u r a t e d a ld e h y d e s . S in c e and w ere n o t w e ll r e s o l v e d on t h e ch ro m ato g ram , i t i s n o t s u r p r i s i n g t h a t e x h i b i t s a s m a ll amount o f t h e 350 mp s h o u l d e r . The s p e c tru m o f e x h i b i t e d a s u b s t a n t i a l l y g r e a t e r amount o f t h e 350 mji s h o u l d e r t h a n was found w ith . The tu b e s p o o le d fro m t h e p e a k w ere d e l i b e r a t e l y 76 FIGURE 11 0 . Wo 0 . 1- 0.1 - 400 400 300 300 A b s o r p tio n S p e c t r a from R e a c tio n o f S u b u n its o f A cid S o lu b le C o lla g e n w ith MBTH 77 s e l e c t e d t o m in im ize c o n ta m in a tio n by f i ^ s u b u n i t s . No e f f o r t was made to m in im iz e t h e c o n ta m in a tio n o f th e p o o le d sa m p le s o f by f i )f . The h e i g h t o f th e f i n p e a k r e l a t i v e to th e p e a k was g e n e r a l l y g r e a t e r th a n t h e h e i g h t o f th e p e a k r e l a t i v e t o th e p e a k . T h ese f a c t s , p l u s t h e r e l a t i v e l y g r e a t e r r e s o l u t i o n o f o4v from com pared t o t h e r e s o l u t i o n o f from f i n , s u g g e s t t h a t t h e 550 mji s h o u l d e r o f t h e ^ ^ p e a k c a n n o t be e x p la in e d s o l e l y on t h e b a s i s o f c o n ta m in a tio n by The p e a k was c o n s i s t e n t l y much b r o a d e r th a n any o f t h e o t h e r p e a k s i n th e c h ro m a to g ra m . I t may be t h a t t h e r e i s some com ponent i n t h e t r a i l i n g s h o u ld e r o f t h e p e a k . T a b le V II shows t h e am ounts o f a d le h y d e s i n e ac h o f t h e i s o l a t e d s u b u n i t s . The v a lu e s o b ta in e d w ith MBTH w ere c o n s i s t e n t i n r e p e a te d d e t e r m i n a t i o n s t o w i t h i n t .2 m ol/m ol s u b u n i t . W ith DNPH, t h e r e p r o d u c i b i l i t y was good w i t h i n t . 5 m ol/m ol s u b u n i t . N ote t h a t b e f o r e c r o s s l i n k f o r m a tio n , a s u b u n i t w ould be e x p e c te d t o have an a ld e h y d e c o n te n t e q u a l t o t h e sum ( n o t th e a v e ra g e ) o f i t s c o n s t i t u e n t </, s u b u n i t s . C o v a le n t i n t r a m o le c u la r c r o s s l i n k f o r m a t io n w ould be e x p e c te d t o d e c r e a s e t h e a ld e h y d e c o n t e n t p e r f i s u b u n i t by 1 . T hus, fin w ould be e x p e c te d t o e x h i b i t a n a ld e h y d e c o n te n t b e f o r e c r o s s l i n k f o r m a tio n o f 2 x ^ , (2 x 1 .7 * i n th e MBTH a s s a y ) . C r o s s l i n k f o r m a t io n w ould d e c r e a s e TABLE VII S u b u n it <*1 / * „ A 2 °^ 2 ALDEHYDE CONTENT OP SUBUNITS PROM CITRATE SOLUBLE COLLAGEN MBTH DNPH H ^ -r e d u c tio n m ol/m ol s u b u n it m ol/m ol s u b u n it cpm/ugm 1 .7 1 .2 80 2 .3 1.8 57 3 .0 2 .7 82 2 .7 1.8 137 oo 79 t h i s c o n te n t by 1 t o 2 .4 m ol/m ol s u b u n i t . The e x p e r i m e n ta l v a lu e o b ta in e d f o r was 2 .2 , i n good ag reem en t w ith th e e x p e c te d v a l u e . S i m i l a r l y , would be e x p e c te d t o hav e a v a lu e e q u a l t o ( 1 .7 ) p l u s ( 2 . 7 ) m inus 1, o r 2 . 4 . The e x p e r im e n ta l v a lu e f o r was 2 . 0 , i n a p p ro x im a te a g re em e n t o f th e e x p e c te d v a l u e . T hese v a lu e s a r e s i g n i f i c a n t l y h i g h e r t h a n th o s e o b ta in e d by o t h e r i n v e s t i g a t o r s w ith o t h e r t i s s u e s ( 5 5 , 1 1 0 ) . A c o n t r o l sam p le o f n e u t r a l s a l t s o l u b l e c o l l a g e n from t h e s k i n o f l a t h y r i t i c r a t s was f r e q u e n t l y a n a ly z e d as a c o n t r o l t o i n s u r e t h a t t h e a s s a y was done c o r r e c t l y . T h is p r e p a r a t i o n was c o n s i s t e n t l y found t o have l e s s th a n 0 .2 mol o f a c e ta ld e h y d e e q u i v a l e n t s p e r mole o f c h a in s , i n good a g re em e n t w ith t h e v a lu e s o b ta in e d by o t h e r i n v e s t i g a t o r s ( 1 1 2 ,4 4 ,4 5 ) . T h is c o n t r o l i n d i c a t e s t h a t t h e a ld e h y d e l e v e l s m easured w ith f e t a l c a l f s k i n e x t r a c t s a r e v a l i d m e a su re m e n ts. The above r e s u l t s a r e g iv e n f o r t h e s u b u n i t s f o r a c id s o l u b l e c o l l a g e n . R e p ea te d a tte m p ts t o r e s o l v e th e s u b u n i t s o f s a l t - and c y s te a m in e s o l u b l e c o ll a g e n s w ere u n ifo rm ly u n s u c c e s s f u l . I t was c h a r a c t e r i s t i c a l l y o b s e rv e d t h a t a s i n g l e , v e ry l a r g e p e a k e l u t e d a t th e p o i n t o f a p p e a ra n c e o f u n a b so rb e d m a t e r i a l . No o t h e r m a t e r i a l was e l u t e d th r o u g h o u t t h e c o u rs e o f p a s s a g e o f th e u s u a l g r a d i e n t . More m a t e r i a l was e l u t e d from th e colum n o n ly when t h e colum n r e g e n e r a t i o n was begun w ith 80 .01 N NaOH f 0 .5 M NaCl. Changes i n pH o r i o n i c s t r e n g t h o f t h e e l u t i o n b u f f e r d id n o t p r o v id e any im provem ent i n t h e s e r e s u l t s . The i n a b i l i t y t o r e s o l v e th e s u b u n i t s o f n e u t r a l s a l t - o r 2-m e rc a p to e th y la m ln e s o l u b l e c o lla g e n o n a p r e p a r a t i v e s c a l e made i t im p o s s ib le t o a n a ly z e t h e a ld e h y d e c o n te n t o f su ch s u b u n i t s s e p a r a t e l y . The p a t t e r n o f u p ta k e o f ^H by th e i n d i v i d u a l s u b u n i t s o f a c id s o l u b l e c o l l a g e n was s i m i l a r t o th e p a t t e r n o f a ld e h y d e c o n te n t as d e te rm in e d by r e a c t i o n w ith MBTH o r DNPH. No e f f o r t was made to c o n v e r t th e d a t a f o r u p ta k e o f ^H to m oles o f a ld e h y d e i n t r i n s i c to e a c h s u b u n i t . Such c a l c u l a t i o n would r e q u i r e a p r i o r know ledge o f t h e r e l a t i v e c o m p o s itio n o f e a c h s u b u n it w ith r e s p e c t t o l y s y l - and h y d r o x y l y s y l - d e r i v e d a ld e h y d e s and t o a ld o l c o n d e n s a tio n p r o d u c t . T h is in f o r m a ti o n w ould t h e n need t o be com bined w ith d a t a o b ta in e d from t h e r e d u c t i o n o f model compounds u n d e r s i m i l a r c o n d i t i o n s . The p r e s e n c e o f any S c h i f f b a se c r o s s l i n k s would n u l l i f y t h e s i g n i f i c a n c e o f any v a lu e s o b ta in e d by su c h s t u d i e s . C hrom atography o f Cyanogen B rom ide D i g e s t s I . D ig e s t o f p lu s /Sti f r a c t i o n The p a t t e r n o b ta in e d by c h ro m a to g ra p h y on c a rb o x y m e th y l c e l l u l o s e e q u i l i b r a t e d w ith c i t r a t e b u f f e r i s p r e s e n te d i n F ig u r e 1 2 . The p e p t i d e s w hich e l u t e i n t h e f i r s t 250 ml have b e e n shown t o be r e l a t i v e l y Absorbance at 250 FIGURE 12 o.H |HOo 200 HOO Soo iooo 1 2 0 0 Volume of effluent (cm3) Carboxymethyl cellulose chromatography of CNBr digest of acid soluble Carboxymethyl ________ collagen c C f f a subunits cellulose 82 s m a l l . In c lu d e d i n t h i s r e g i o n a r e t h e p e p t i d e s from th e amino t e r m i n a l end o f t h e m o le c u l e . T h e re i s a l a r g e f r a c t i o n w hich a p p e a rs b e tw ee n 300 t o 1 ,5 0 0 ml o f e f f l u e n t . By a n a lo g y w ith ch rom atog ram s o f c o l l a g e n c y an o g e n bro m id e d i g e s t s from o t h e r s o u r c e s , c n is r e g i o n s h o u ld be c o m p rise d s o l e l y o f p e p t i d e s from t h e h e l i c a l r e g i o n o f t h e m o le c u le ( 1 0 0 ,2 ) . T h is f r a c t i o n was l y o p h i l i z e d and d e s a l t e d on B io -G e l P - 2 . A f t e r l o y p h i l i - z a t i o n o f t h e d e s a l t e d m a t e r i a l , p a r t o f t h e f r a c t i o n was re c h ro m a to g ra p h e d on a c a rb o x y m e th y l c e l l u l o s e colum n e q u i l i b r a t e d w itn a c e t a t e b u f f e r . The r e s u l t s a re shown i n F ig u r e 1 3 . At l e a s t t h r e e m a jo r com ponents o f t h e f r a c t i o n w ere v i s u a l i z e d by t h i s p r o c e d u r e . A c ry la m id e g e l e l e c t r o p h o r e s i s o f th e o r i g i n a l f r a c t i o n s u g g e s t s t h a t t h e r e may be a d d i t i o n a l com ponents ( F ig u r e 1 4 ) . The r e s o l u t i o n o f com ponents shown i n F i g u r e 12 i s n o t as good a s t h a t o f o t h e r i n v e s t i g a t o r s w o rk in g w ith c o l l a g e n from o t h e r t i s s u e s o u r c e s ( 2 ) . B ut th e p r i n c i p a l g o a l o f r e s o l v i n g t h e i n t r a m o l e c u l a r c r o s s l i n k - r e g i o n p e p t i d e s from t h e p e p t i d e s o f t h e h e l i c a l r e g i o n was a c c o m p lis h e d . . F r a c t i o n V from F ig u r e 12 was f u r t h e r c h ro m a to g ra p h e d on c a rb o x y m e th y l c e l l u l o s e u n d e r t h e c o n d i t i o n s used f o r r e s o l v i n g s u b u n i t s o f t h e o r i g i n a l a c id s o l u b l e c o l l a g e n p r e p a r a t i o n ( F ig u r e 1 3 )• At l e a s t t h r e e com ponents a r e a p p a r e n t i n t h e c h ro m a to g ra m . MBTH a n a l y s i s 0.0- 0.4- e f f l u e n t volum e « P FIGURE 12 C arboxym ethyl c e l l u l o s e c h ro m ato g rap h y ( a c e t a t e p ro c e d u r e ) o f f r a c t i o n 1 from c arb o x y m eth y l c e l l u l o s e c h ro m ato g rap h y ( c i t r a t e ) o f CNBr d i g e s t o f * ^ . A I 7 5 o 155 00 FIGURE 14 A c ry la m id e G el E l e c t r o p h o r e s i s o f F r a c t i o n V from F ig u r e 7 85 o f F r a c t i o n Vc from F ig u r e 15 d e m o n s tra te d th e p r e s e n c e o f a p p ro x im a te ly 1 . 7 m oles o f a ld e h y d e p e r 1 0 0 ,0 0 0 m o le c u l a r w e ig h t ( F ig u r e 1 5 ) . Amino a c id a n a l y s i s o f t h i s f r a c t i o n i s p r e s e n t e d iri T a b le V I I I . T h is c o m p o s itio n i s c o n s i s t e n t w ith e i t h e r o f two p r e v i o u s ly d e s c r ib e d s u b u n i t s o f ©(,CB7 o r (7^CB8 . I I . F r a c t i o n F ig u r e 16 i l l u s t r a t e s th e r e s o l u t i o n o b ta in e d by ch ro m a to g ra p h y o f a c o m p le te CNBr d i g e s t o f com ponents on c a rb o x y m e th y l c e l l u l o s e e q u i l i b r a t e d w ith c i t r a t e b u f f e r . As w ith t h e chrom atogram o f th e d i g e s t o f^ < ,/ , t h e r e s o l u t i o n o f t h e i n d i v i d u a l f r a c t i o n s from th e h e l i c a l r e g i o n was p o o r . The p e p t i d e s from t h e r e g i o n w here i n t r a m o l e c u l a r c r o s s l i n k s a r e form ed w ere w e ll s e p a r a t e d from t h e h e l i c a l r e g i o n p e p t i d e s . I I I . P h o s p h o c e llu lo s e C hrom atography o f CNBr D ig e s t o f 2 -M e rc a p to e th y la m in e S o lu b le C o l la g e n . The p a t t e r n o b ta in e d i s i l l u s t r a t e d i n F ig u r e 1 7 . The p a t t e r n o f a b s o r p t i o n a t 250 mji i s g e n e r a l l y s i m i l a r i n m ost r e s p e c t s t o t h a t f o r d i g e s t s o f o t h e r c o l l a g e n p r e p a r a t i o n s e x c e p t f o r t h e u n u s u a lly h ig h p e a k o b ta in e d b e tw ee n 50 and 110 ml o f e f f l u e n t . However, t h i s f e a t u r e i s n o t n e a r ly so s u r p r i s i n g a s t h e p a t t e r n o b ta in e d by w a sh in g t h e colum n w ith 0 . 5 M NaCl ♦ .01 N NaOH, a t th e end o f t h e g r a d i e n t . D ig e s ts o f u n m o d ifie d c o l l a g e n FIGURE 15 o ( , F r a c t i o n Vc o .io H 0 .0 5 H J500 MBTH A ldehyde S p e c tru m o f F r a c t i o n Vc TABLE VIII AMINO ACID COMPOSITION OP FRACTION Vc FROM <*, OF ACID SOLUBLE COLLAGEN <*,Vc d,CB7* <A,CB8 * h y d r o x y ly s in e 1 .8 1 .1 1 .2 l y s i n e 7 -5 9 -4 9 -3 h i s t i d i n e 0 .9 - - a r g i n i n e 1 2 .5 1 3 .0 1 5 .0 h y d r o x y p r o lin e 2 9 .7 2 3 .0 2 7 .0 a s p a r t i c a c id 1 1 .8 1 2 .0 1 0 .0 th r e o n i n e 5 .0 5 -0 5 .2 s e r i n e 8 . 3 7 .4 8 .2 g lu ta m ic a c id 1 9 .8 1 7 .0 1 9 .0 p r o l i n e 3 2 .8 3 8 .0 3 3 .0 g l y c i n e 8 9 .0 8 9 .0 8 9 .O a l a n i n e 3 5 .0 3 6 .0 3 4 .0 v a l i n e 3*8 5 -3 4 .6 i s o l e u c i n e 2 .4 2 .8 1 .9 l e u c i n e 4 .5 4 .3 4 .4 t y r o s i n e - - - p h e n y la la n i n e 2 .8 3 .1 3 .1 h o m o se rin e - l a c to n e 0 .7 7 1 .0 1 .0 * d a t a o b ta in e d by B io c h e m is try 10: : V o lp in , D ., 1751 ( 1 9 7 1 ). and V e is , A. : Absorbance at 230 FIGURE 16 d l rr *— 0.8 0.6 0.4 0.2 400 100 1600 700 1300 1000 1900 2200 Carboxymethyl cellulose chromatography of CNBr digest of subunits of fetal calf skin, acid soluble collagen. oo 89 FIGURE 17 3 s 8 r o , p 3 B R C M •P c d 0) o § .n o c o S JE HL 1.6 1.2 o .a 0 A 400 600 200 P h o s p h o c e l l u l o s e c h ro m a to g r a p h y o f 2 ,4 - r e a c t e d 2 - m e r c a p t o e t h y l a m l n e - s o l u b l e f< P r o c e d u r e d e s c r i b e d i n M ethod s s e c t i o n 89 FIGURE 17 3 1 h l e n d o f g r a d i e n t - s t a r t w ash w i t h 0.5M N aCl ♦ 0.01M NaOH 800 600 4 00 1200 200 1000 o s p h o c e l l u l o s e c h r o m a to g r a p h y o f 2 , 4 - d i n i t r o p h e n y l h y d r a z i n e - a c t e d 2 - m e r c a p t o e t h y l a m i n e - s o l u b l e f e t a l c a l f s k i n c o l l a g e n , o c e d u r e d e s c r i b e d i n M e th o d s s e c t i o n o f t e x t . E f f l u e n t v o lu m e (cm ^) c h a r a c t e r i s t i c a l l y g i v e a v e ry l a r g e p e a k o f a b s o r p t i o n i n t h i s r e g i o n o f t h e ch ro m ato g ram . T h is p e a k r e p r e s e n t s m a t e r i a l from t h e h e l i c a l r e g i o n o f t h e c o l l a g e n m o le c u le and i s n o rm a lly r e s o lv e d by ch ro m a to g ra p h y on c a rb o x y m e th y l c e l l u l o s e . The a b se n c e o f a l a r g e p e a k i n t h i s r e g i o n s u g g e s ts t h a t t h e m a t e r i a l w hich e l u t e d a t t h e b e g in n in g o f th e g r a d i e n t in c lu d e d m a t e r i a l from th e h e l i c a l r e g i o n o f th e m o le c u le . T h is l a r g e e a r l y p e a k was th e o n ly p l a c e i n t h e chrom atogram t h a t a s i g n i f i c a n t 360 mp a b s o r b in g p e a k was f o u n d . The c o in c id e n c e o f th e l a r g e 360 mp a b s o r b in g p e a k w ith t h e l a r g e 230 mp a b s o r b i n g p e a k i m p l i e s t h a t th e d is p la c e m e n t o f l a r g e h e l i c a l r e g i o n p e p t i d e s from t h e end t o t h e b e g in n in g o f t h e chrom atogram was due t o a m o d i f i c a t i o n i n n e t c h a rg e a r i s i n g from r e a c t i o n w ith DNPH. S p e c tr a o f p e p t i d e s The s p e c tru m f o r f r a c t i o n I i s shown i n F i g u r e 18. At pH 1, a p e a k i s fo und a t a b o u t 235 mp, w ith s h o u l d e r s a t 210 t o 220 mp, 245 t o 265 mp, and 250 t o 255 mp* At pH 13, a b ro a d p e a k a p p e a rs from 230 t o n e a r l y 3 0 0 mp, w ith s h o u l d e r s a t 210 t o 225 mp and a t 380 t o 395 mp* T h is i s c o n s i s t e n t w ith t h e p r e s e n c e o f th e DNPH d e r i v a t i v e o f a n a l i p h a t i c a d e lh y d e ( F ig u r e 1 9 ) . The s p e c tru m f o r f r a c t i o n I I i s s e e n i n F i g u r e 2 0 . At pH 1 , p e a k s a r e o b s e rv e d a t 210 mp, 260 t o 265 mp, and 335 mp. At pH 1 3 , p e a k s a r e found a t 225 mp, 2 4 3 nyu, FIGURE 18 210 225 CysSC DNPH CNBr D ig e s t P h o s p h o c e llu lo s e F r a c t i o n I B io -G e l P -2 F r a c t i o n I pH = 500 200 210-220 FIGURE 19 F orm aldehyde 2 ,4 DNPH 355 245 *26 3 60 -36 325-330 230 600 200 500 300 400 2 1 0 ^ , 1 * '22260-265 FIGURE 20 CysSC DNPH CNBr D ig e s t B io -G e l P -2 F r a c t i o n I P h o s p h o c e llu lo s e F r a c t i o n I I '3 0 0 pH = pH = 4oo 2qo 94 and 403 mp w ith s h o u l d e r s a t 210 t o 220 mp, 340 t o 350 mp, and a b o u t 500 mp. T h is sp e c tru m i s d i s t i n c t l y d i f f e r e n t from t h e s p e c tru m o f any o f th e DNPH a ld e h y d e s t a n d a r d s . I t i s s i m i l a r i n some r e s p e c t s t o th e s p e c tru m o f a c e to n e DNPH ( F ig u r e 2 1 ) . I n t h i s s t a n d a r d , pH 1 p e a k s w ere o b s e rv e d a t 325 mp, 265 mp, and 205 mp. At pH 13, p e a k s a p p e a r a t 530 mp, 425 mp, 238 mp, and a t 212 mp, w ith a s h o u l d e r a t a b o u t 260 to 265 mp. A lth o u g h t h e r e a r e c l e a r s i m i l a r i t i e s b etw een t h e s p e c t r a o f a c e to n e DNPH and f r a c t i o n I I , th e y a re d e f i n i t e l y n o t i d e n t i c a l . The pH 1 p e a k o f f r a c t i o n I I a t 3 35 mp i s 10 mp d i f f e r e n t fro m t h e 325 mp p e a k o f a c e to n e DNPH. The pH 13 p eak o f f r a c t i o n I I a t 403 mp i s 22 mp d i f f e r e n t fro m th e p e a k a t 425 mp o f a c e to n e DNPH. A ceto n e DNPH shows a c l e a r p e ak a t 530 mp a t pH 13> b u t f r a c t i o n I I o n ly r e v e a l s an i n d i s t i n c t s h o u ld e r a t a b o u t 500 mp. At pH 1, f r a c t i o n s I I I and IV ( F i g u r e s 22 and 23) show s p e a k s a t 235 mp and 275 mp, w ith a c l e a r s h o u ld e r a t 215 t o 220 mp and a n i n d i s t i n c t s h o u l d e r a t a b o u t 350 mp. At pH 1 3 , a p e a k i s s e e n a t 230 t o 235 mp, w ith s h o u l d e r s a t 215 t o 220 mp, 270 t o 275 mp, a b o u t 300 mp, and a b o u t 400 mp. The sp e c tru m i s c o n s i s t e n t w ith th e p r e s e n c e o f a s m a ll amount o f m a t e r i a l w ith DNPH bound i n c o m b in a tio n w ith a l a r g e amount o f m a t e r i a l w hich h a s no DNPH b o u n d . FIGURE 21 A c eto n e 2 , 4 DNPH 425 2.65 325 530 \ t - 290 230 4 9 0 300 290 4 - 275 FIGURE 22 CysSC DNPH CNBr D ig e s t P h o s p h o c e llu lo s e F r a c t i o n I I I B io -G e l P -2 F r a c t i o n I pH = 200 3 0 0 400 500 600 f i i i » 275 210 FIGURE 23 CysSC DNPH CNBr D ig e s t P h o s p h o c e llu lo s e F r a c t i o n IV B io -G e l P -2 F r a c t i o n I pH = 400 500 300 200 98 The sp e c tru m o f f r a c t i o n V ( F ig u r e 24) a t pH 1 r e v e a l s a p e a k a t 2 2 0 mp w i t h an i n d i s t i n c t s h o u ld e r a t a b o u t 260 mp and a n o t h e r s h o u l d e r a t 215 t o 220 mp. A v e ry s i m i l a r sp e c tru m was o b t a i n e d a t pH 12, e x c e p t t h a t t h e r e was a r e l a t i v e l y g r e a t e r a b s o r p t i o n a t pH 12, e x c e p t t h a t t h e r e was a r e l a t i v e l y g r e a t e r a b s o r p t io n a t pH 12 a t w a v e le n g th s g r e a t e r t h a n a b o u t 285 mp. T h is sp e c tru m i s m ost s i m i l a r t o t h a t o f th e CNBr d i g e s t o f u n m o d ifie d c o l l a g e n . The a p p a r e n t l y g r e a t e r co n c e n t r a t i o n o f p e p ti d e i n t h e sp e c tru m o f f r a c t i o n V c o u ld a c c o u n t f o r t h e a b se n c e o f a v a l l e y b etw een th e p e a k s a t 210 mp and 220 mp f o r pH 1 , and b etw een th e p e a k s a t 215 and 240 mp f o r pH 12- The sp e c tru m o f f r a c t i o n VI ( F ig u r e 25) r e v e a l s a p e a k a t 29 0 mp and a s h o u l d e r a t 2 2 0 t o 2^-0 mp a t pH 1 2 . At pH 1, th e o n ly f e a t u r e o f n o te i s a p e ak a t 240 mp. T h is sp e c tru m i s s i m i l a r t o t h a t o f a sim p le a l i p h a t i c a ld e h y d e , a lth o u g h t h e pH 1 p e a k i s s h i f t e d 15 to 20 n p to w ard th e u l t r a v i o l e t , and th e 2 9 0 p e a k i s r e l a t i v e l y more p ro m in e n t t h a n t h e 250 p e a k a t pH 12 when com pared t o a s im p le a l i p h a t i c a ld e h y d e s ta n d a r d DNPH d e r i v a t i v e . FIGURE 24 225 CysSC DNPH CNBr D ig e s t P h o s p h o c e llu lo s e F r a c t i o n V B io -G e l P-2 F r a c t i o n I 400 500 2<J0 100 I__ 200 FIGURE 25 3{*0 CysSC DNPH CNBr D ig e s t P h o s p h o c e llu lo s e F r a c t i o n IV B io -G e l P -2 F r a c t i o n I 390 300 5 oo 5^*5 6db 101 IV . B io -G e l P -2 C h ro m ato g rap h y o f CNBr D i g e s t o f DNPH- R e a c te d 2 - m e r c a p to e th y la m ln e S o lu b le C o l l a g e n . The e l u t i o n p r o f i l e i s i l l u s t r a t e d i n F i g u r e 2 6 . Two m a jo r p e a k s o f 360 mp a b s o r b in g m a t e r i a l a r e s e e n . One p e a k c o r r e s p o n d s t o m a t e r i a l e l u t i n g w i t h t h e v o id v o lu m e . The o t h e r m a jo r p e a k e l u t e s b e tw e e n 118 t o 157 ml o f e f f l u e n t . Two o t h e r 560 mp a b s o r b in g p e a k s a p p e a r i n t h e c h ro m a to g ra m . One p e a k e l u t e s b e tw e e n 75 t o 100 ml o f e f f l u e n t . The o t h e r p e a k a p p e a rs b e tw e e n 155 to 170 ml o f e f f l u e n t . About on e q u a r t e r (2 6 p e r c e n t ) o f t h e 3 6 0 mp a b s o r b i n g m a t e r i a l a p p e a re d i n t h e f i r s t p e a k , a b o u t one s e v e n th (1 4 p e r c e n t ) i n t h e se c o n d p e a k , o v e r h a l f (5 7 p e r c e n t ) i n t h e t h i r d p e a k , and t h e b a la n c e ( 5 - 4 p e r c e n t ) a p p e a re d i n th e f o u r t h p e a k . The f i r s t p e a k had a much l a r g e r a b s o r p t i o n a t 250 mp t h a n th e o t h e r p e a k s . The se c o n d p e a k a l s o had much 250 mp a b s o r b i n g m a t e r i a l . The t h i r d and f o u r t h p e a k s had r e l a t i v e l y much m ore 5 6 0 mp a b s o r p t i o n t h a n a t 2 5 0 mp. The s p e c t r a o f t h e I n d i v i d u a l f r a c t i o n s a r e p r e s e n t e d i n F i g u r e s 27 t o 5 0 . F r a c t i o n I ( F ig u r e 27) shows a b ro ad a b s o r p t i o n maximum b e tw e e n 250 to 515 up* T h e re a p p e a re d t o be some s p e c i f i c a b s o r p t i o n a t 5 5 0 t o 5 6 0 mp a t pH 1 . T h is a b s o r p t i o n was d i f f i c u l t t o d i s t i n g u i s h fro m th e s h o u l d e r o f t h e 250 t o 515 mp p e a k . At pH 15, h o w e v er, a d i s t i n c t s h o u l d e r was o b s e rv e d a t 400 t o 410 mp. 102 FIGURE 26 0 . 5 0 . 4 §• & 0 . 3 K\ 4- 3 a a > I o .a V i O 1 0 < 0.1 2 .0 t - f t C V J rt 1.2 0.8 0 . 4 E f f l u e n t volum e B io - G e l P - 2 g e l f i l t r a t i o n c h ro m a to g r c y 8 t e a m i n e s o l u b l e c o l l a g e n . "¥ 102 FIGURE 2 6 .0 A + «- 4- 7T T TO — ---------- * 4------± = ---------- 4 THO V TBo B o E f f l u e n t v o lu m e (cm ^ ) »-G el P - 2 g e l f i l t r a t i o n c h r o m a to g r a p h y o f CNBr d i g e s t o f ► teamine s o l u b l e c o l l a g e n . I 200 P l G tn tB 2 7 0jr8S0 SXPB C JrB ■ ® l o -C e i p r 4 l « e s t P S . , ' a c t io « 1 p b . ; 3 4 00 500 # 6 0 0 r \ 104 FIGURE 28 CysSC DNPH CNBr d i g e s t Bio-Gel P-2 fraction II 440 300 400 500 ' 600 1 1 1 200 I 105 5 60 FIGURE 29 CysSC DNPH CNBr D ig e s t B io -G e l P -2 F r a c t i o n I I I 600 400 500 5 0 0 r\ i—228 106 FIGURE 30 210—^j/j CysSC DNPH CNBr D ig e s t B io -G e l P -2 F r a c t i o n IV 200 3 0 0 i 400 i 500 i 600 I 107 S i m i l a r s h o u ld e r s w ere o b s e rv e d a t pH 13 I n th e s p e c t r a o f s t a n d a r d DNPH d e r i v a t i v e s o f a l i p h a t i c a ld e h y d e s . F r a c t i o n I I ( F ig u r e 28) g i v e s a p e a k a t 308 m p and a d i s t i n c t s h o u ld e r a t 395 mp a t pH 1 . At pH 13, a p e a k was s e e n a t 3 0 8 mp, a d i s t i n c t s h o u l d e r a t 3 5 0 mp, and a n o th e r s h o u l d e r a t a b o u t 440 mp. The pH 1 s h o u l d e r a t 3 9 5 mp i s c o m p a rab le t o th e s h o u l d e r a t 3 8 0 -3 8 5 mp o b t a i n e d w ith s t a n d a r d croto naldehyde-D N P H d e r i v a t i v e ( F i g u r e 7 ) • C rotonaldehyde-D N PH g a v e a pH 1 p e ak a t 275 t o 2 8 0 mp w ith an a p p a r e n t s h o u l d e r a t a b o u t 300 t o 305 mp. At pH 1 3 , crotonaldehyd e-D N P H e x h i b i t e d a d i s t i n c t s h o u l d e r a t a b o u t 440 mp and a p e a k a t 280 to 285 mp. F r a c t i o n I I I ( F ig u r e 2 9 ) e x h i b i t e d a pH 1 p eak a t a b o u t 3 40 mp, w ith no o t h e r d i s c e r n i b l e s p e c t r a l f e a t u r e s b e tw e e n 3 0 0 to 600 mp. At pH 13, t h e r e was a p e a k a t 3 6 0 mp and a s i g n i f i c a n t s h o u l d e r a t 395 mp. T h ese f e a t u r e s a r e s i m i l a r to t h o s e o b t a i n e d w ith s t a n d a r d a l i p h a t i c a ld e h y d e DNPH d e r i v a t i v e s . F o rm a ld e hyde DNPH f i v e s a pH 1 p e a k a t 35 5 mp ( F i g u r e 19)* At pH 1 3 , a p e a k i s s e e n a t 360 t o 3 6 5 mp and a s h o u ld e r a p p e a r s a t 3 9 5 t o 400 mp. G lu ta r a ld e h y d e i s an a l i p h a t i c d i a l d e h y d e . I t s DNPH d e r i v a t i v e g i v e s a pH 1 p e a k a t 3 55 mp ( F i g u r e 13) • At pH 13, a p e a k a p p e a rs a t 340 t o 345 mp, and a s h o u l d e r i s found a t 3 9 5 t o 400 npi. 108 F r a c t i o n IV ( F ig u r e 3 0 ) e x h i b i t e d p e a k s a t 228 and 355 mp a t pH 1 . S h o u ld e rs w ere fo u n d a t 2 1 5 mp and 2 4 0 mp. At pH 1 3 , p e a k s w ere fou n d a t 2 1 5 and 2 4 0 mp, w ith a s h o u l d e r a t 3 2 5 t o 3 3 0 mp. The pH 1 p e a k , 2 2 8 mp and s h o u l d e r a t 2 10 mp, a s w e ll a s t h e pH 13 p e a k s a t 2 1 0 m p-24o n p , a re c o m p a rab le t o t h e p e a k s o b ta in e d w ith a CNBr d i g e s t o f u n m o d ifie d a c id s o l u b l e c o l l a g e n . But t h e d i g e s t o f u n m o d ifie d c o l l a g e n d o e s n o t p r e s e n t any p e a k s o r s h o u l d e r s i n t h e r e g i o n 3 2 0 t o 3 6 0 mp. pH 1 p e a k a t 3 5 5 mp o f f r a c t i o n IV i s s i m i l a r t o t h a t o f s t a n d a r d DNPH d e r i v a t i v e s o f a l i p h a t i c a ld e h y d e s . But t h e pH 13 s h o u l d e r a t 3 2 8 mp i n f r a c t i o n IV i s n o t c o m p a ra b le t o any s p e c tru m fou n d w ith o u r s t a n d a r d a ld e h y d e DNPH d e r i v a t i v e s . F r a c t i o n I fro m B io -G e l P -2 c h ro m a to g ra p h y o f CNBr d i g e s t o f CysSC-DNPH was c h ro m a to g ra p h e d on c a r b o - x y m e th y l c e l l u l o s e u s i n g th e c i t r a t e b u f f e r sy s te m . The e l u t i o n p r o f i l e i s shown i n F i g u r e 3 1 . Two p r i n c i p l e f e a t u r e s a r e a p p a r e n t i n t h i s c h ro m a to g ra m . F i r s t , a l l o f t h e d e t e c t a b l e 3 6 0 mp a b s o r b in g m a t e r i a l i s e l u t e d i n t h e f i r s t 1 5 0 m l. T h is i s t h e a r e a w here m a t e r i a l e l u t e s w h ich i s n o t a b s o rb e d t o t h e colum n. S econd, e s s e n t i a l l y no m a t e r i a l i s e l u t e d b e tw e e n 4 0 0 t o 1 ,7 0 0 ml o f e f f l u e n t . T h is i s t h e r e g i o n w here m ost o f t h e p e p t i d e s from t h e h e l i c a l p o r t i o n o f th e c o l l a g e n m o le c u le a r e o r d i n a r i l y e l u t e d . T h e re i s a l a r g e 2 3 0 mp a b s o r b in g 109 FIGURE 31 I * Ic 0.8 1.2 i i i i i ' o . 6 8 Id o 0 . 4 § J Q b O ® 0.2 4 do ldoo 7 0 0 100 C a rb o x y m e th y l c e l l u l o s e c h ro m a to g r a p h y o f < fro m B lo - G e l P -2 c h ro m a to g r a p h y o f CNBr d ij r e a c t e d 2 - m e r c a p t o e t h y l a m l n e s o l u b l e c o l l a j 109 FIGURE 31 u 7 0 0 ldoo 4 d o c y m e th y l c e l l u l o s e c h r o m a to g r a p h y o f v o i d v o lu m e f r a c t i o n ( f r a c t i o n I ) i i o - G e l P -2 c h r o m a to g r a p h y o f CNBr d i g e s t o f 2 , 4 - d i n i t r o p h e n y l h y d r a z i n e id 2 - m e r c a p t o e t h y l a m i n e s o l u b l e c o l l a g e n . 110 p e a k a t t h e b e g in n i n g o f t h e c h ro m a to g ra m . T h is p e a k i n c l u d e s t h e r e g i o n w here t h e 3 6 0 mji a b s o r b i n g m a t e r i a l e l u t e s . I n ch ro m a to g ra m s o f CNBr d i g e s t s o f u n m o d ifie d c o l l a g e n , t h i s i s t h e r e g i o n w h ere th e s m a l l e r p e p t i d e s e l u t e , i n c l u d i n g t h e c r o s s l i n k r e g i o n p e p t i d e s . B ut c r o s s l i n k r e g i o n p e p t i d e s u s u a l l y a p p e a r i n t h e in c lu d e d volum e when s u b j e c t e d t o c h ro m a to g ra p h y o n B io -G e l P - 2 . The s p e c t r a o f f r a c t i o n s l a , l b and I c a r e p r e s e n t e d i n F i g u r e s 32 t o 3 4 . T he s p e c tru m f o r f r a c t i o n l a ( F i g u r e 3 2 ) e x h i b i t s a b ro ad a b s o r p t i o n maximum b e tw ee n 230 t o 300 my a t pH 1 . A s h o u l d e r a t a b o u t 3 5 0 mji a l s o a p p e a r s a t t h i s pH. At pH 13* a b ro a d p e a k a p p e a rs b e tw e e n 23? t o 315 m^i, and a s h o u l d e r i s s e e n a t 400 t o 410 mji. The s p e c tr u m i s c o n s i s t e n t w ith t h e p r e s e n c e o f a l i p h a t i c a ld e h y d e i n a l a r g e amount o f p e p t i d e m a t e r i a l . The s p e c tru m f o r f r a c t i o n l b ( F i g u r e 3 ? ) g i v e s a p e a k a t 2 3 0 nyi and s h o u l d e r s a t 210 m y, 2 70 t o 275 nya, and 3 ? 5 t o 3 4 0 mji, a t pH 1 . At pH 13, a p e a k i s a g a i n found a t 23 0 mp, w i t h d i s t i n c t s h o u l d e r s a p p e a r in g a t 210 mji and 3 0 0 m)i. L ess c l e a r s h o u l d e r s a l s o o c c u r r e d a t a b o u t 3 5 0 mji and 400 m)i. The s p e c tru m f o r f r a c t i o n I c ( F i g u r e 3 4 ) g i v e s a p e a k a t 2 3 0 to 2 5 0 mp and s h o u l d e r s a t 215 mja, 2 8 5 mji, and a t a b o u t 3 5 0 mji a t pH 1 . At pH 13, a p e a k a p p e a r s a t 230 my, w ith s h o u l d e r s a t 2 1 5 njn, 295 mji, and l e s s d i s t i n c t l y , a t a b o u t 3 4 0 toy. F r a c t i o n s l b and I c a p p e a r t o be p r i n c i p a l l y D N PH -free, b u t e a c h I l l FIGURE 32 CysSC DNPH CNBr D ig e s t N o n d ia ly z a b le C arboxy- l0 m e th y l C e l l u l o s e F r a c t i o n I , B io -G e l P -2 F r a c t i o n l a 600 400 500 300 200 230 - > 112 FIGURE 33 210 CysSC DNPH CNBr D ig e s t B io -G e l P -2 F r a c t i o n I C arbox ym ethyl C e ll u l o s e F r a c t i o n l b 223 s . V 400 500 300 200 f i t « \ pH pH FIGURE 34 CysSC DNPH CNBr D ig e s t B lo -G e l P -2 F r a c t i o n I C arboxym ethyl C e l l u l o s e F r a c t i o n I c i-— I 113 3 0 0 I 400 t 500 I 600 i 114 seem s to have some s m a ll amount o f m a t e r i a l w ith DNPH b o u n d . V. C arboxym ethyl C e l l u l o s e C hrom atography o f C N B r-D igest o f NaB-^Hjj- Reduced 2 -m e r c a p to e th y la m in e S o lu b le C o lla g e n . The e l u t i o n p r o f i l e i s shown t h e F ig u r e 55* The b u lk o r t h e r a d i o a c t i v i t y i s e l u t e d b etw een 280 t o 2 ,0 0 0 m l. I n a d d i t i o n , a s u b s t a n t i a l amount o f r a d i o a c t i v i t y i s e l u t e d b e tw e e n 2,50 t o 270 m l. Two a d d i t i o n a l p e a k s w ere e l u t e d a t t h e end o f t h e ch ro m ato g ram . One p e a k was e l u t e d w ith 1 .0 M NaCl i n c i t r a t e b u f f e r a t th e end o f th e g r a d i e n t ( 2 ,0 5 0 to 2 ,1 0 0 m l) . An a d d i t i o n a l f r a c t i o n was e l u t e d w ith 0 .5 M NaCl + .01 N NaOH ( 2 ,1 7 5 t o 2 ,2 2 5 m l ) . The p a t t e r n o f a b s o rb a n c e a t 250 mp was g e n e r a l l y s i m i l a r t o t h e p a t t e r n o f r a d i o a c t i v i t y . The r e g i o n a b o u t 900 ml o f e f f l u e n t a p p e a rs t o be a n e x c e p ti o n . I n t h i s r e g i o n t h e a b s o r p t i o n a t 250 mp i s i n c r e a s i n g w h ile th e r a d i o a c t i v i t y i s d e c r e a s i n g . T h is i m p l i e s t h a t a p e p t i d e a p p e a rs i n t h i s r e g i o n w hich d o e s n o t c o n t a i n N a B ^ Ify -re d u c ib le m o i e t i e s . P o o lin g o f t u b e s from t h e chrom atogram was done a c c o r d in g t o t h e r a d i o a c t i v i t y p r o f i l e . P o o le d p e a k s w ere l y o p h i l i z e d and d e s a l t e d o n B io -G e l P - 2 . The d e s a l t i n g f r a c t i o n s w ere l y o p h i l i z e d and s t o r e d i n t h e d ry fo rm u n t i l f u r t h e r u s e . 115 FIGURE 35 S h E h 0} ■ U c •H e S h < 1 ) f t a 4 - 3 O o b O O i — l G r a d i e n t b e g u n 0 .8 . 3.0 - ftO.6- 5- r x 0 - 0 . 2 - 3bo 900 E f f l u e n t volum e 12 C a r b o x y m e th y l c e l l u l o s e c h ro m a to g r a p h y c y s t e a m l n e s o l u b l e c o l l a g e n . P r o c e d u r e o f t e x t . 115 FIGURE 35 IE v ^UE> t UC -> «- ZL -► -ft jnr J r a d i e n t b e g u n 1 660 960 i2bo 1560 E f f l u e n t volum e (cm-5) ►xymethyl c e l l u l o s e c h ro m a to g ra p h y o f CNBr d i g e s t o f N a B T ^ -re d u ce d sam ine s o l u b l e c o l l a g e n . P r o c e d u r e d e s c r i b e d I n M e th o d s s e c t i o n > x t. * 116 Some m a t e r i a l from f r a c t i o n V was s u b j e c t e d t o b a s e h y d r o l y s i s . Com ponents o f t h e h y d r o l y s a t e were r e s o lv e d o n an amino a c id a n a l y z e r . F r a c t i o n s o f th e e f f l u e n t w ere c o l l e c t e d from th e a n a l y z e r u s in g a s p l i t - s tr e a m d e v i c e . A liq u o ts o f th e f r a c t i o n s w ere mixed w ith a s t a n d a r d s c i n t i l l a t o r and c o u n te d u s in g a Beckman LS 233 l i q u i d s c i n t i l l a t i o n c o u n te r . P o i n t s o f e l u t i o n o f known a ld e h y d e r e d u c t i o n p r o d u c t s i n o t h e r c o l l a g e n s o u r c e s have p r e v i o u s l y b e e n i d e n t i f i e d w ith t h i s s y s te m (5 5 * 1 0 3 ). F ig u r e 3 6 shows t h e r a d i o a c t i v i t y p a t t e r n o b ta in e d from a h y d r o l y s a te o f f r a c t i o n V fro m F ig u r e 3 5 . I t can be s e e n t h a t a s u b s t a n t i a l p e a k o f r a d i o a c t i v i t y e l u t e s i n th e r e g io n p r e v i o u s l y e s t a b l i s h e d f o r t h e a p p e a ra n c e o f h y d r o xy no r l e u c i n e . T h is i s th e r e d u c t i o n p r o d u c t o f th e a ld e h y d e d e r i v a t i v e o f l y s i n e . T h e re i s one o t h e r p e ak o f r a d i o a c t i v i t y . T h is a p p e a rs a t t h e s i t e o f e l u t i o n o f d ih y d r o x y n o r le u c i n e , t h e r e d u c t i o n p r o d u c t o f th e a ld e h y d e d e r i v a t i v e o f h y d r o x y l y s i n e . T h is p e a k am ounts to a b o u t l / 3 as much r a d i o a c t i v i t y as i s found w ith h y d r o x y n o r l e u c in e . 280- 260- 240- 220- 200. 1 8 a 160- 140- h yd ro x y n o rleu cin e A d ih y d r o x y n o rle u c in e I g l y c i n e FIGURE 56 F r a c t i o n V from c arb o x y m eth y l c e l l u l o s e c h ro m ato g rap h y o f CNBr d i g e s t o f N aB ^H ^-reduced CysSC. T r itiu m r a d i o a c t i v i t y p a t t e r n from amino a c i d a n a l y s i s o f b a s e . h y d r o l y s a t e . H atch ed p e a k s a r e s i t e s o f e l u t i o n o f s e l e c t e d amino a c i d s . CHAPTER V DISCUSSION I t i s w e ll e s t a b l i s h e d t h a t a g r e a t e r p r o p o r t i o n o f s o l u b l e c o l l a g e n i s foun d i n t h e s k i n s o f v e ry young a n im a ls t h a n i n o l d e r a n im a ls ( 1 1 4 ) . N e u t r a l s a l t s o l u b l e c o l l a g e n c o n s t i t u t e s a r e l a t i v e l y g r e a t e r p r o p o r t i o n o f t h e s o l u b l e c o l l a g e n from t h e s k i n s o f young a n im a ls t h a n fro m o l d e r a n im a ls ( 9 6 ) . The c o l l a g e n w hich i s e x t r a c t a b l e by a p a r t i c u l a r s o l v e n t fro m e a c h t i s s u e i s u s u a l l y fo u n d t o be e s s e n t i a l l y i n v a r i a b l e t o any d e t e c t a b l e e x t e n t r e g a r d l e s s o f t h e age o f t h e a n im a l. T h a t i s , n e u t r a l s a l t s o l u b l e c o l l a g e n fro m s k i n s o f t h r e e week o ld r a t s i s i n d i s t i n g u i s h a b l e fro m t h e n e u t r a l s a l t s o l u b l e c o l l a g e n from s k i n s o f t h r e e m onth o ld r a t s o r fro m one y e a r o ld r a t s . Some s t u d i e s have show n v a r i a t i o n s among th e e x t r a c t a b l e c o l l a g e n s o f t h e s k i n s o f a p a r t i c u l a r ty p e o f a n im a l, w h ich c a n be r e l a t e d t o e v e n t s w hich o c c u r r e d a f t e r p e p t i d e bond f o r m a t i o n . Such v a r i a t i o n s in c lu d e d t h e e x t e n t o f h y d r o x y l a t i o n o f l y s y l and p r o l y l r e s i d u e s ( 1 1 5 ) , t h e am ount o f g l y c o s y l a t i o n o f h y d r o x y ly s y l 118 119 r e s i d u e s ( l l 6 ) , and th e r e l a t i v e am ounts o f JL v e r s u s s u b u n i t s ( 1 2 ) . I t was g e n e r a l l y found t h a t t h e i n i t i a l amino a c id c o m p o s itio n and se q u e n c e o f c o l l a g e n from s k i n s was c o n s t a n t . E x te n s iv e s t u d i e s w ith c o l l a g e n i s o l a t e d from bone and te n d o n i n d i c a t e d t h a t t h e i n i t i a l amino a c id c o m p o s itio n and se q u e n c e o f t h i s m a t e r i a l was th e same as t h a t from s k i n ( 2 5 ) . T hese f i n d i n g s cau se d m ost i n v e s t i g a t o r s t o a c c e p t a s a w o rk in g h y p o t h e s is t h a t a l l c o l l a g e n s from a p a r t i c u l a r ty p e o f a n im al w ere t h e same when new ly s y n t h e s i z e d . A c c o rd in g t o t h i s h y p o t h e s i s , t h e new ly s y n t h e s i z e d c o l l a g e n m o le c u le was t h e n m o d ifie d a c c o r d in g to t h e r e q u ir e m e n ts o f t h e t i s s u e i n w hich i t was s y n t h e s i z e d . R e c e n t f i n d i n g s o f v a r i a n t ty p e s o f c o l l a g e n s i n s e l e c t e d t i s s u e s h a s n e c e s s i t a t e d a m o d i f i c a t i o n o f t h i s h y p o t h e s i s . V a r ia n t c o l l a g e n s have b e e n fo u n d i n a r t i c u l a r and s t e r n a l c a r t i l a g e s and i n c a r t i l a g i n o u s em b ry o n ic bond ( 1 4 ) . A d i f f e r e n t v a r i a n t c o l l a g e n h a s b e e n shown to o c c u r i n b asem en t membrane ( 1 8 ,1 9 ) . More r e c e n t s t u d i e s o f s k i n from new born humans and from h a t c h l i n g c h ic k s hav e fo u n d t h a t t h e r e i s an a d d i t i o n a l v a r i a n t o f c o l l a g e n ( 1 7 ) . T h is v a r i a n t a p p e a rs to o c c u r o n ly i n e m b ry o n ic , f e t a l , o r new born s k i n s . I t i s n o t d e t e c t a b l e i n s k i n s o f a d u l t humans o r a n im a ls . I t h a s lo n g b e e n known t h a t em b ry o n ic and f e t a l s k i n s e x h i b i t e d a c o l l a g e n - l i k e p r o t e i n w hich a p p e a rs t o form 120 b r a n c h in g n e tw o rk s when view ed w ith t h e e l e c t r o n m ic ro sc o p e . T h is m a t e r i a l h a s b e e n named r e t i c u l i n and a p p e a rs t o be a c o l l a g e n (1 1 7 )- I t h a s n o t y e t b e en r e p o r t e d w h e th e r t h e newly found c o l l a g e n v a r i a n t i s i n any way r e l a t e d t o r e t i c u l i n s i n c e t h e c o m p o s itio n o f t h e l a t t e r i s n o t known. The v a r ia n t type o f c o lla g e n in embryonic and f e t a l s k in has an amino acid com position th a t i s so d i s t i n c t from c o lla g e n from s k in o f o ld e r anim als th a t i t must have a d i f f e r e n t i n i t i a l amino a c id sequence ( 17). E arly r e p o r ts in d ic a te th a t as much as one h a l f of the c o lla g e n i n f e t a l o r embryonic s k in may be o f th e v a ria n t ty p e , w hile th e rem ainder o f th e c o lla g e n appears to be s im ila r to th e c o lla g e n from sk in s o f o ld e r a n im a ls. The e x i s t e n c e o f a n e n t i r e l y new ty p e o f c o l l a g e n n e c e s s i t a t e s c a u t i o n i n i n t e r p r e t a t i o n o f any d a t a o b ta in e d fro m su c h t i s s u e s . I t c a n n o t be im m e d ia te ly assum ed t h a t d a t a o b ta in e d w ith f e t a l s k i n s c a n be i n t e r p r e t e d o n t h e b a s i s o f i n f o r m a t i o n p r e v i o u s l y o b ta in e d from s k i n s o f o l d e r a n i m a l s . T h is s tu d y was i n i t i a t e d to exam in e t h e g e n e r a l s i g n i f i c a n c e o f t h e f i n d i n g s i n t h i s l a b o r a t o r y w ith r a t s k i n s t h a t s o l u b l e c o l l a g e n e x t r a c t e d w ith 2 -m e r c a p to e th y la m in e (CysSC) e x h i b i t e d s u b s t a n t i a l am ounts o f a ld e h y d e m o i e t i e s ( 2 ,3 ^ ) * S t u d i e s i n o t h e r l a b o r a t o r i e s w ith n e u t r a l s a l t and a c id e x t r a c t a b l e c o l l a g e n p r e p a r a t i o n s had foun d t h a t t h e a p p e a ra n c e o f 121 a ld e h y d e s i n c o l l a g e n was r e s t r i c t e d t o t a l l y t o th e cyan ogen b ro m id e p e p t i d e s w hich come from t h e n o n - h e l i c a l , amino t e r m i n a l end o f t h e m o le c u le ( 4 4 ,4 5 ) . B ut CysSC from r a t s k i n s h a s now b e en shown t o hav e s p e c i f i c cy anogen brom id e p e p t i d e s from t h e h e l i c a l r e g i o n i n w hich l y s y l o r h y d r o x y ly s y l r e s i d u e s a r e o x i d a t i v e l y d e a m in a te d to form a ld e h y d e m o i e t i e s ( 2 ) . R ecen t s t u d i e s by o t h e r i n v e s t i g a t o r s h a v e q u e s tio n e d th e g e n e r a l s i g n i f i c a n c e o f t h e s e f i n d i n g s ( 5 0 ) . The o n ly c r o s s l i n k s w hich have b e en c o n c l u s i v e l y shown t o o c c u r i n c o l l a g e n hav e a l l r e q u i r e d l y s y l - o r h y d r o x y ly s y l- d e r iv e d a ld e h y d e s f o r t h e i r f o r m a t i o n . Thus t h e p o s s i b l e o c c u r r e n c e o f h e l i c a l r e g i o n - d e r i v e d a ld e h y d e s h as im p o r ta n t i m p l i c a t i o n s r e l a t i n g t o t h e t o t a l c r o s s l i n k i n g p o t e n t i a l o f a c o ll a g e n s y s te m . B o vin e s k i n s w ere s e l e c t e d f o r s tu d y b e c a u s e t h i s s y s te m h a s p r e v i o u s l y b e e n e x t e n s i v e l y c h a r a c t e r i z e d . P e t a l s k i n was i n i t i a l l y s e l e c t e d b e c a u s e i t was a n t i c i p a t e d t h a t c o n s i d e r a b l e am ounts o f s o l u b l e c o l l a g e n c o u ld be o b ta in e d from t h i s s o u r c e . The d a t a f o r e x t r a c t a b i l i t y o f f e t a l c a l f s k i n i n d i c a t e t h a t o v e r h a l f o f th e t o t a l c o l l a g e n was s o l u b l e i n e i t h e r 0 . 5 M a c e t i c a c id o r 0 .2 N c y s te a m in e pH 7 * 0 . T h is i s much m ore th a n t h e r e p o r t e d e x t r a c t a b i l i t y o f s k i n from a d u l t s t e e r s ( 2 l ) . V a lu es o f 1 p e r c e n t o r l e s s o f th e t o t a l c o l l a g e n a r e t y p i c a l l y fou n d f o r t h e e x t r a c t a b i l i t y o f su c h t i s s u e s . I n hum ans, 1 p e r c e n t 122 o r l e s s o f t h e t o t a l c o l l a g e n i s e x t r a c t a b l e e v e n i n new borns ( 1 7 )- A ldehyde d a t a f o r t h e v a r io u s e x t r a c t s d i f f e r from v a lu e s o b ta in e d w ith o l d e r a n im a ls . M ost i n v e s t i g a t o r s f i n d t h a t n e u t r a l s a l t e x t r a c t a b l e c o l l a g e n from p o s t n a t a l a n im a ls h a s an a ld e h y d e c o n te n t o f l e s s th a n 1 mole p e r m ole o f e/* c h a i n s ( 1 1 2 ) . Such s t u d i e s w ere a b le to a c c o u n t f o r a l l o f t h e a ld e h y d e s found o n th e b a s i s o f d e t a i l e d e x a m in a tio n o f t h e n o n - h e l i c a l r e g i o n CNBr- d e r iv e d p e p t i d e s . I n t h i s s t u d y , s u b s t a n t i a l l y g r e a t e r am ounts o f a ld e h y d e s w ere fo u n d i n n e u t r a l s a l t s o l u b l e e x t r a c t s th a n a r e fo u n d i n o t h e r p u b lis h e d r e p o r t s ( 1 , 1 1 2 ) . C o n tr o l s t u d i e s w ere d one w ith c o lla g e n e x t r a c t e d from l a t h y r i t i c young r a t s k i n s . T h ese c o n t r o l s w ere found to be s i m i l a r i n a l l r e s p e c t s t o v a lu e s o b t a i n e d by o t h e r i n v e s t i g a t o r s ( 1 , 1 1 2 , 4 0 ) . T h is i n d i c a t e s t h a t t h e m ethodology u se d was n o t p r o d u c in g a r t i f a c t u a l l y e l e v a t e d d a t a . I n s i m i l a r f a s h i o n , c o l l a g e n e x t r a c t e d w ith a c id o r w ith 2 - m e r c a p to e th y la m in e e x h i b i t s a h i g h e r c o n te n t o f a ld e h y d e t h a n i s u s u a l l y fo u n d w ith p o s t n a t a l a n im a ls ( l ) . Acid s o l u b l e c o l l a g e n g e n e r a l l y e x h i b i t s an a ld e h y d e c o n te n t o f a b o u t 1 m ole p e r m ole o f c h a i n s . T h is i s g e n e r a l l y i n t e r p r e t e d a s m eaning t h a t e s s e n t i a l l y a l l o f t h e l y s y l r e s i d u e s i n th e am ino te r m in a l r e g i o n o f t h e m o le c u le have b e e n c o n v e r te d t o ald eh y d e m o i e t i e s . T h u s, i f t h e r e i s m ore t h a n one a ld e h y d e p r e s e n t p e r c h a in , 123 some amount o f a ld e h y d e must a p p e a r i n th e h e l i c a l p o r t i o n o f t h e m o le c u le . The s t i l l g r e a t e r amount o f a ld e h y d e found w ith c o l l a g e n e x t r a c t e d w ith 2 - m e r c a p to e th y la m in e i s c o n s i s t e n t w ith th e r e s u l t s found f o r t h e f r a c t i o n s o b ta in e d w ith l e s s s e v e r e c o n d i t i o n s . We h av e le a r n e d t h a t o t h e r i n v e s t i g a t o r s a re f i n d i n g u n u s u a lly h ig h am ounts o f a ld e h y d e s i n e x t r a c t s o f f e t a l s k i n s , a lth o u g h t h i s i n f o r m a t i o n i s n o t y e t i n p r e s s ( 1 1 8 ,1 1 9 ) . The c o n c lu s io n t h a t f e t a l c a l f s k i n c o ll a g e n s do in d e e d hav e e l e v a t e d l e v e l s o f a ld e h y d e s i s a p p a r e n t l y s o u n d . The u n u s u a l s u b u n i t d i s t r i b u t i o n foun d w ith a c id s o l u b l e c o l l a g e n i s q u i t e s u r p r i s i n g . O b s e r v a tio n s o f th e v a r i a n t c o ll a g e n s p r e s e n t i n c a r t i l a g e and i n b asem en t membrane r e v e a l e d s u b u n i t s w hich c h ro m a to g ra p h ed i n a m anner s i m i l a r t o t h a t o f c / , ( I ) . In d e e d , t h e f i r s t i n d i c a t i o n t h a t t h e r e w as a v a r i a n t c o l l a g e n i n c a r t i l a g e came from t h e o b s e r v a t i o n t h a t t h e r e was an u n u s u a lly l a r g e r a t i o o f to <Av i n c a rb o x y m e th y l c e l l u l o s e c h ro m a to g ra m s. T h is r a t i o was f r e q u e n t l y fo u n d to be 4 : 1 : : ^ , : and i n some e x t r a c t s was found a s h ig h a s 12 o r 13 t o 1 . ( 1 4 ) . The c l a s s i c a l c o n c e p t o f e ac h t r o p o c o l l a g e n c o n s i s t i n g o f two 9(1 and one c o u ld n o t p o s s i b l y a c c o u n t f o r a l l o f th e p r e s e n t , so i t was n e c e s s a r y t o p ro p o s e th e e x i s t e n c e o f c o l l a g e n m o le c u le s c o n s i s t i n g t o t a l l y o f s u b u n i t s . The f i n d i n g o f a v a r i a n t ty p e o f s u b u n i t i n n e o n a t a l 124 human s k i n I s b a se d upon an e x a m in a tio n o f th e CNBr p e p t i d e s from i n s o l u b l e c o l l a g e n ( 1 7 ) . The d e s i g n a t i o n o f th e v a r i a n t cA s u b u n i t a s I I I ) was based upon th e hom ology o f s e l e c t e d CNBr p e p t i d e s w ith t h e p e p t i d e s from ^ ( 1 ) . T h u s, i t was n o t shown t h a t t h i s v a r i a n t c / c h a in a c t u a l l y c h ro m a to g ra p h s i n a m anner s i m i l a r t o . I t i s t h e r e f o r e p o s s i b l e t h a t v a r i a n t c h a in s o f f e t a l s k i n would more c l o s e l y re s e m b le i n t h e i r c h ro m a to g r a p h i c p r o p e r t i e s . A ll o f t h i s i s c o n j e c t u r e , h o w ev er. What i s n o t c o n j e c t u r e i s t h a t th e e l u t i o n p r o f i l e r e v e a l s r e l a t i v e l y more t o t a l eXj, c o n te n t th a n i s e x p la in e d by e i t h e r ( « A ;) 0 **i> o r ( ^j ) . I t h a s n o t been r u le d 2 3 o u t t h a t th e m a t e r i a l w hich d id n o t e l u t e w ith t h e s a l t g r a d i e n t was p r i m a r i l y o r t o t a l l y <JL, i n o r i g i n . The m a t e r i a l w hich e l u t e d a t th e c o m p le tio n o f th e norm al p a r t o f t h e chrom atogram (p e a k s X / * fin > was c o l l e c t e d f o r f u r t h e r s t u d y . I t was fo u n d t h a t e x te n s i v e d e g r a d a t i o n had ta k e n p l a c e , w ith a s u b s t a n t i a l amount o f t h e m a t e r i a l a p p e a r in g as p e p t i d e s w hich w ere s m a l l e r t h a n ^ c h a in s on a c ry la m id e g e l e l e c t r o p h o r e s i s . Such d e g r a d a t i o n o f i s o l a t e d c o l l a g e n s u b u n i t s h a s b e e n o b s e rv e d p r e v i o u s l y ( 1 6 ) . The breakdow n t a k e s p l a c e s p o n t a n e o u s ly , and o c c u r s a t an i n c r e a s i n g r a t e w ith i n c r e a s i n g t e m p e r a t u r e . The m a in te n a n c e o f th e colum n o v e r n i g h t a t 4 0 ° C c o u ld th u s h av e c a u se d i n i t i a l l y p o ly m e r ic m a t e r i a l t o become d e g ra d e d . 125 P o ly m e ric m a t e r i a l i s c h a r a c t e r i s t i c a l l y a b so rb e d i n a m anner w hich i s i r r e v e r s i b l e u n d e r th e c o n d it i o n s em ployed f o r p a s s a g e o f th e g r a d i e n t ( 2 1 ) . Any s m a ll m o le c u la r w e ig h t m a t e r i a l w hich i s c r e a t e d by th e s u g g e s te d d e g r a d a t i o n p r o c e s s would be e l u t e d im m e d ia te ly upon r e s u m p tio n o f p a s s a g e o f th e g r a d i e n t . As a r e s u l t , a p e ak would a p p e a r i n th e e l u t i o n p r o f i l e w hich would s u g g e s t th e p r e s e n c e o f a n o v e l com ponent i n t h e p r o t e i n sa m p le . T h ere a re o t h e r p o s s i b l e e x p l a n a t i o n s f o r t h e a p p e a ra n c e o f t h e d e la y e d p e a k and t h e a p p e a ra n c e o f d e g ra d e d m a t e r i a l . One a l t e r n a t i v e e x p l a n a t i o n s t a r t s w ith th e p r e v i o u s l y m en tio n ed p o ly m e r ic m a t e r i a l . I t i s known t h a t th e r a t e o f d e s o r p t i o n o f p r o t e i n s from i o n ex ch an g e m ed ia d e c r e a s e s w ith i n c r e a s i n g m o le c u la r w e i g h t . I n te rm s o f s e p a r a t i o n o f c o l l a g e n s u b u n i t s t h i s means t h a t a t t h e flo w r a t e used f o r p a s s i n g th e g r a d i e n t , < 5 ^ and s u b u n i t s would r a p d i l y a c h ie v e e q u i l i b r i u m b e tw ee n t h e s t a t i o n a r y p h a se and t h e c h a n g in g i o n i c s t r e n g t h o f th e m o b ile p h a s e . The s u c c e s s i v e l y h i g h e r m o le c u la r w e ig h t s u b u n i t s ')/' and ^ w ould have s u c c e s s i v e l y lo w e r r a t e c o n s t a n t s f o r a c h i e v i n g e q u i l i b r i u m . I f t h e s e r a t e s a r e s u f f i c i e n t l y s m a ll , t h e r e w ould be a s t e a d y i n c r e a s e i n t h e d i f f e r e n c e from e q u i l i b r i u m f o r t h e h i g h e r m o le c u la r w e ig h t co m p o n e n ts. I n t h i s way, e l u t i o n o f JL, and £ s u b u n i t s would t a k e p l a c e b e f o r e th e h i g h e r m o le c u la r w e ig h t s u b u n i t s c o u ld be d i s p l a c e d . I f passag e of th e g ra d ie n t were th e n ceased , m aintenance of tn e generax c o n d itio n s o f te m p e ra tu re , pH, and io n ic s tr e n g th o f th e column would allow th e h ig h e r m o lecu lar w eight components to g ra d u a lly achieve e q u ilib riu m w ith th e m obile p h a se . Then when passage o f th e g r a d ie n t i s resumed, th e desorbed high m o le cu lar w eight m a te ria l would e l u te as a s in g le peak. The breakdown th a t i s observed i n t h i s l a s t peak could ta k e p la c e d u rin g th e d e s a lti n g and p r e p a r a tio n f o r acrylam ide g e l e l e c t r o p h o r e s is . These s tu d ie s have n o t shown which of th e s e p ro c e s se s i s involved n or have they shown w hether any o th e r p ro c e ss i s in v o lv e d , but su g g e st some unusual u nder ly in g problem o r b e h a v io r. The breakdown o f m a te ria l caused v a r ia b le lo s s e s d u rin g d e s a lti n g p ro cedures w hether by d i a l y s i s o r by g e l f i l t r a t i o n . So, a t t h i s tim e i t has not been c o n c lu s iv e ly shown w hether th e unusual d i s t r i b u t i o n o f su b u n its a r i s e s from a r e a l v a r ia n t d i s t r i b u t i o n i n th e s t a r t i n g m a te r ia l o r w hether th e v a r ia n t p a t t e r n a r is e s from th e te c h n ic a l p ro cess of chrom atography. I t should be n o ted , however, th a t o th e r in v e s tig a to r s have re p o rte d f in d in g e radiots le s s th a n 2 (9 5 ,1 1 1 ,1 1 2 ). In g e n e ra l, th e s e r e s u l t s have been ex p lain e d as w ith in ex p e rim en ta l e r r o r o f 2 o r have not ex p lain e d th e s e v alu es a t a l l . In view o f th e c o n s id e r a b le in v e s t i g a t i o n done in th e p a s t which e s ta b lis h e d th e concept o f a tro p o c o lla g e n m olecule th a t was 127 i n v a r i a b l y composed o f two s u b u n i t s and o n e s u b u n i t , o u r f i n d i n g s m e r it f u r t h e r c o n s i d e r a t i o n . One l i n e o f r e a s o n in g 'w hich h a s b e e n u sed i n th e p a s t i s t h a t e v e ry P s u b u n i t sh o u ld have a c o m p lim e n ta ry ck s u b u n i t . I n o t h e r w o rd s, i f a s u b u n i t i s fo u n d , th e n i t s p a r e n t m o le c u le m ust h a v e a l s o c o n ta in e d one s u b u n i t . T h e r e f o r e , a c o n s t r a i n t e x i s t s i n any chrom atogram t h a t th e com ponent c a n n o t e x ce ed tw ic e th e a ^ } com ponent. S i m i l a r l y , each chro m atogram m ust h av e no more th a n tw ic e th e amount o f a s t h e r e i s d y y I n t h e s e e x p e rim e n ts t h e r e a p p e a re d t o be c o n s i d e r a b ly m ore ^ ^ t h a n com p lim e n ta r y o i,,. T h e re w as, h o w ev er, c o n s i s t e n t l y enough ^ v . t o a c c o u n t f o r a l l o f t h e • I t i s g e n e r a l l y f e l t t h a t p u r i f i e d s o l u b l e c o l l a g e n p r e p a r a t i o n s w hich h a v e b e e n c e n t r i f u g e d a t 1 0 0 ,0 0 0 x g f o r 90 m in u te s have m in im al ( l e s s t h a n 3 p e r c e n t ) i n t e r m o l e c u l a r c r o s s l i n k s ( 9 )• U n le s s g r e a t e r am ounts o f i n t e r m o l e c u l a r c r o s s l i n k s a p p e a r i n t h e s e p r e p a r a t i o n s , t h e s e r e s u l t s f u r t h e r s u g g e s t t h a t t h e ( ^ , J g C ^ t r o p o - c o l l a g e n i s n o t a b s o l u t e . The i s o l a t e d and e A v s u b u n i t s d id a p p e a r t o be s i m i l a r i n t h e i r am ino a c id c o m p o s itio n s t o t h e s u b u n i t s p r e v i o u s l y r e p o r t e d by o t h e r i n v e s t i g a t o r s ( 1 0 9 ) . The aldehyde c o n te n t o f th e e x t r a c t s were uniform ly h ig h e r th a n e x t r a c ts o f s k in from p o s tn a ta l o r a d u lt b o v in es. R e so lu tio n o f acid s o lu b le c o lla g e n allowed 128 m easurem ent o f a ld e h y d e s i n t h e s e s u b u n i t s . The p a t t e r n o f a ld e h y d e d i s t r i b u t i o n was c o n s i s t e n t w ith t h e a ld e h y d e c o n te n t o f t h e t o t a l e x t r a c t . I n t h e t o t a l e x t r a c t a ld e h y d e c o n te n t m easured by MBTH e q u a l l e d an a v e ra g e o f 2 .J > ^ j n o l / 1 0 0 mg. The i s o l a t e d s u b u n i t s ran g e d i n a ld e h y d e c o n te n t b etw een 1 .2 t o 2 . 7 ju^m ol/lO O rag. The w e ig h te d a v e ra g e o f a ld e h y d e c o n te n t c a l c u l a t e d from t h e i n d i v i d u a l s u b u n i t s e q u a lle d 1 .8 t o 2 .0 j i j n o l / 1 0 0 mg. T h is v a lu e i s s l i g h t l y l e s s t h a n found w ith t h e t o t a l e x t r a c t . The d i f f e r e n c e may be r e l a t e d t o t h e l a r g e f r a c t i o n o f m a t e r i a l w hich rem ained a d so rb e d t o t h e colum n when th e p r i n c i p a l s u b u n its w ere e l u t e d . A s i m i l a r d i s t r i b u t i o n o f a ld e h y d e c o n te n t was found i n t h e DNPH r e a c t i o n o f th e s u b u n i t s as com pared w ith t h e o r i g i n a l a c id e x t r a c t . As i n d i c a t e d i n t h e r e s u l t s we w ere u n a b le to r e s o l v e t h e s u b u n i t s o f NSS o r CysSC on a p r e p a r a t i v e s c a l e . Such t e c h n i c a l d i f f i c u l t i e s p r e v e n te d c o m p a riso n o f s u b u n i t a ld e h y d e c o n te n t o f t h e s e e x t r a c t s . I t was p o s s i b l e to r e s o l v e t h e s e s u b u n i t s o f t h e s e e x t r a c t s by a c r y la m id e g e l e l e c t r o p h o r e s i s . The p a t t e r n s o b ta in e d w ere s i m i l a r t o p a t t e r n s r e p o r t e d f o r e x t r a c t s from a d u lt t i s s u e s ( 1 0 2 ,1 2 0 ) . An e x c e p t i o n w as t h a t n e u t r a l s a l t s o l u b l e c o ll a g e n s c o n ta in e d a l a r g e r f r a c t i o n o f s u b u n i t s b i g g e r t h a n J<( , > / , and J ) . E x a m in a tio n o f e x t r a c t s f o r a ld e h y d e c o n te n t a f t e r t r e a t m e n t w ith CNBr ( l i m i t e d c le a v a g e ) o r w ith p e p s i n show s t h a t t h e r e i s a s u b s t a n t i a l 129 f r a c t i o n o f t h e t o t a l a ld e h y d e c o n t e n t w h ich i s l o c a te d i n t h e h e l i c a l r e g i o n o f th e m o l e c u l e . A c ry la m id e g e l e l e c t r o p h o r e s i s i n d i c a t e d t h a t a t l e a s t 90 p e r c e n t o f t h e c r o s s l i n k e d s u b u n i t s had b een c o n v e rte d to s u b u n i t s th e s i z e o f T h is i n d i c a t e s t h a t rem o v a l o f t h e n o n - h e l i c a l r e g i o n w here i n t r a m o le c u la r c r o s s l i n k s a re form ed i s a b o u t 9 0 p e r c e n t c o m p le te . I f a d i f f e r e n c e i n t h e r e a c t i v i t y o f t h e non h e l i c a l r e g i o n o f < ? -< s u b u n i t s e x i s t s when com pared w ith f t s u b u n i t s , i t w ould be e x p e c te d t h a t f t s u b u n i t s would be l e s s r e a c t i v e t h a n a*, s u b u n i t s . The o n ly o b v io u s c h e m ic a l d i f f e r e n c e r e l a t e s t o t h e g r e a t e r mass and h en ce s t e r i c b lo c k a g e c a p a b i l i t y o f t h e n o n - h e l i c a l r e g i o n o f f t s u b u n i t s . T h e r e f o r e , i t i s r e a s o n a b l e to assum e c le a v a g e o f a t l e a s t 90 p e r c e n t o f t h e h e l i c a l r e g i o n o f s u b u n i t s , s i n c e a t l e a s t 9 0 p e r c e n t o f th e f t s u b u n its w ere shown t o be c o n v e rte d t o <yt.-sized s u b u n i t s . A f te r d i a l y s i s o r g e l f i l t r a t i o n t o rem ove th e n o n - h e l i c a l p o l y p e p t i d e s w hich had been c le a v e d , s u b s t a n t i a l am ounts o f a ld e h y d e s rem a in e d i n th e h e l i c a l m a t e r i a l . The s p e c t r a o f t h e h e l i c a l r e g io n s e x h i b i t e d s u b s t a n t i a l d e c r e a s e i n a l d o l c r o s s l i n k a ld e h y d e s as i n d i c a t e d by a b s o r p t i o n a t 250 mji. T h is i s f u r t h e r i n d i c a t i o n t h a t s u b s t a n t i a l am ounts o f th e f t com ponent had been d e g ra d e d t o * 4 - s i z e d s u b u n i t s . The r e s u l t s o b ta in e d w ith th e s e m a t e r i a l s u s i n g DNPH w ere s i m i l a r t o t h e MBTH r e s u l t s . 130 T h at i s , d i a l y z e d sa m p le s o f D N PH -reacted e x t r a c t s a f t e r c le a v a g e w ith CNBr o r p e p s i n g a v e l e s s a b s o r p t i o n i n th e w a v e le n g th s c h a r a c t e r i s t i c o f f t c r o s s l i n k s . But s u b s t a n t i a l am ounts o f a b s o r p t i o n re m a in a t w a v e le n g th s c h a r a c t e r i s t i c s o f a l i p h a t i c a ld e h y d e DNPH d e r i v a t i v e s . T h e r e f o r e , t h e m a j o r i t y , and p e rh a p s a l l o f th e a ld e h y d e s i n t h e h e l i c a l r e g io n a r e o f t h e a l i p h a t i c t y p e . Such a ld e h y d e s c o u ld be e i t h e r h y d r o x y l y s i n e - o r l y s i n e - d e r iv e d . The s p e c t r a w ith MBTH o f < A -h y d ro x y a ld e h y d e s a r e v e ry s i m i l a r t o th e s p e c t r a w ith a l i p h a t i c a ld e h y d e s . Some l y s i n e s and p r o l i n e s w hich become h y d r o x y la te d a r e fo u n d to be in c o m p le te ly h y d r o x y l a t e d . T h at i s to s a y , a p a r t i c u l a r s i t e o f a p p e a ra n c e o f h y d ro x y p r o lin e o r h y d r o x y ly s in e i n th e amino a c id se q u e n c e i s l i k e l y t o be in c o m p le te ly h y d r o x y la te d when m easured i n a l a r g e p o p u l a t i o n o f su c h m o le c u le s . The am ine o x id a s e w hich h a s b e e n found i n c o n n e c tiv e t i s s u e s t o be c a p a b le o f c o n v e r t in g l y s y l r e s i d u e s i n t o a l l y s y l r e s i d u e s i s a ls o f e l t t o be c a p a b le o f c o n v e r t i n g h y d r o x y ly s y l r e s i d u e s t o h y d r o x y a l l y s y l r e s i d u e s . So, i f a ld e h y d e s a re p ro d u c e d a t a p a r t i c u l a r s i t e fro m h y d r o x y ly s y l r e s i d u e s i t w ould be e x p e c te d t h a t o t h e r m o le c u le s w hich have n o t b e e n h y d r o x y la te d a t t h i s s i t e w ould be c o n v e rte d t o a l l y s y l r e s i d u e s . The m ixed p o p u l a t i o n o f a ld e h y d e s p ro d u c e d i n t h i s m anner w ould g i v e r i s e upon r e a c t i o n w ith MBTH o r DNPH t o s p e c t r a w hich hav e t h e c h a r a c t e r i s t i c s 131 o f b oth ty p e s o f a ld e h y d e s. The p ro x im ity of th e ab s o r p tio n peaks o f th e two ty p e s o f aldehydes makes i t d i f f i c u l t to d is c e r n th e r e l a t i v e c o n tr ib u tio n o f each. C hrom atography o f CNBr d i g e s t s o f v a r i o u s p r e p a r a t i o n s was a tte m p te d i n o r d e r t o f u r t h e r l o c a l i z e d th e a p p e a ra n c e o f a ld e h y d e s i n th e h e l i c a l r e g i o n o f th e m o le c u l e . F r a c t i o n V from c a rb o x y m e th y l c e l l u l o s e c h ro m a to g ra p h y o f a c o m p le te CNBr d i g e s t o f e^/ and f9 „ s u b u n i t s o f a c id s o l u b l e c o l l a g e n r e p r e s e n t s t h e l a r g e m a j o r it y o f t h e p e p t i d e s i s o l a t e d . I t was found to hav e an a ld e h y d e c o n te n t o f 1 .7 u m ol/100 mg. F r a c t i o n V i s n o t a s i n g l e p e p t i d e , h o w e v er. R ech ro m ato g rap h y o f p a r t o f t h i s f r a c t i o n on c arb o x y m eth y l c e l l u l o s e a t pH 4 .8 ( a c e t a t e b u f f e r ) d e m o n s tra te d th e p r e s e n c e o f a t l e a s t two and p r o b a b ly t h r e e m a jo r co m p o n en ts. S i m i l a r r e s u l t s w ere o b t a i n e d u s in g a c ry la m id e g e l e l e c t r o p h o r e s i s . T his method dem onstrated th e p rese n ce of s m a lle r amounts o f a number o f o th e r p e p tid e s , some o f which were probably d e g ra d a tio n p ro d u c ts of th e p r in c ip l e p eak s. Amino a c id a n a ly s is o f t h i s f r a c t i o n in d ic a te s t h a t i t i s not a s in g le pure p e p tid e . I t i s , however, s im ila r to s e v e r a l o f th e la r g e r p e p tid e s from th e h e l i c a l re g io n of th e m o le c u le . P h o sp h o ce llu lo se chromatography o f CNBr d ig e s t o f DNPH-reacted CysSC gave an unusual p a t t e r n . The la c k o f s u b s t a n t i a l peak a t the b eg in n in g of r e g e n e ra tio n o f 132 th e colum n i s m atched by an u n u s u a lly l a r g e p e ak e l u t i n g w ith u n a d so rb e d m a t e r i a l . T h is u n a d so rb e d p e ak a l s o c o n ta in e d a l l o f th e d e t e c t a b l e bound DNPH. T h is i m p l i e s t h a t a s u b s t a n t i a l amount o f t h e p e p t i d e s from th e h e l i c a l r e g i o n o f c o l l a g e n had r e a c t e d w ith DNPH. B in d in g o f DNPH a p p e a rs t o c a u se a s u b s t a n t i a l m o d i f i c a t i o n i n th e n e t c h a rg e c h a r a c t e r i s t i c s o f e a c h o f th e r e a c t e d p e p t i d e s . I t s h o u ld be n o te d t h a t t h e r e was a s m a ll b u t s i g n i f i c a n t a b s o r p t i o n a t 3 6 0 mp s e e n th ro u g h o u t th e g r a d i e n t and w ash . T h is a p p e a rs to be DNPH w hich h a s b een n o n s p e c i f i c a l l y a d so rb e d t o th e p e p t i d e s . G rad u al d e s o r p t i o n w ould a llo w t h e o b s e r v a t i o n o f t h i s b a s e l i n e a b s o r p t i o n a t 3 6 0 mp. The p r i n c i p l e p o i n t t o be made i s t h a t t h e e l u t i o n o f DN PH-free p e p t i d e s from th e colum n f o llo w s a n orm al p a t t e r n . The a p p e a ra n c e o f t h i s n orm al p a t t e r n i m p l i e s t h a t t h e c o n d i t i o n s used i n g e n e r a t i n g th e colum n w ere c o r r e c t f o r t h i s p r o c e d u r e . The a b se n c e o f p e p t i d e s w ith t h e wash m ust be s u r p r i s i n g s i n c e p r e v io u s p u b l i c a t i o n s h a v e u n ifo rm ly i n d i c a t e d th e p r e s e n c e o f s u c h * p e a k ( 4 4 , 4 5 ) . At p r e s e n t t h e o n ly l o g i c a l c o n c l u s i o n p o s s i b l e i s t h a t t h o s e p e p t i d e s have b een d i s p l a c e d t o t h e b e g in n in g o f t h e c h ro m ato g ram . N ote t h a t e v e n t h e p e p t i d e s from th e n o n - h e l i c a l r e g i o n o f th e m o le c u le do n o t a p p e a r i n th e m ain body o f t h e chrom atogram a s DNPH d e r i v a t i v e s . T h is i s c o n s i s t e n t w ith th e i d e a t h a t r e a c t i o n w ith DNPH c a u s e s a p e p t i d e 133 to e l u t e u n a b so rb e d on p h o s p h o c e l l u l o s e . B io -G e l P -2 g e l f i l t r a t i o n c h ro m a to g ra p h y o f CNBr d i g e s t o f D N PH -reacted CysSC g a v e r i s e t o f o u r p e a k s o f 3 6 0 a b s o r b in g m a t e r i a l . F r a c t i o n I a p p e a re d a t th e v o id volum e and c o n ta in e d t h e l a r g e m a j o r it y o f 230 my a b s o r b in g m a t e r i a l . The sp e c tru m was p r i m a r i l y t h a t o f an a l i p h a t i c a ld e h y d e . F r a c t i o n I I had th e g e n e r a l c h a r a c t e r i s t i c s o f an ©<, ^ - u n s a t u r a t e d a ld e h y d e and i s c o n s id e r e d t o c o n t a i n much o r a l l o f t h e non h e l i c a l p e p t i d e s o f s u b u n i t s . F r a c t i o n I I I c o n ta in e d a l a r g e amount o f DNPH- r e a c t e d m a t e r i a l w ith a sp e c tru m o f an a l i p h a t i c a ld e h y d e . I t i s f e l t t o c o n t a i n t h e n o n - h e l i c a l p e p t i d e s o f c h a i n s . F r a c t i o n IV c o n s i s t e d o f o n ly a s m a ll amount o f m a t e r i a l w ith a n anom alous s p e c tru m . We have n o t y e t d i s c e r n e d a s p e c i f i c i n t e r p r e t a t i o n o f t h e a p p e a ra n c e o f t h i s f r a c t i o n . F r a c t i o n I from t h e B io -G e l P -2 colum n was c h ro m a to g ra p h e d on c a rb o x y m e th y l c e l l u l o s e i n t h e c i t r a t e b u f f e r s y s te m . Only one p e a k o f 3 60 mji a b s o r b in g m a t e r i a l was d i s c e r n e d . T h is p e a k a p p e a re d a t t h e b e g in n in g o f th e chro m ato g ram w here u n a d so rb e d m a t e r i a l g e n e r a l l y e l u t e s . Such n o n a d s o r p tio n i s c o n s i s t e n t w ith t h e r e s u l t s o f c h ro m a to g ra p h y on p h o s p h o c e l l u l o s e . H ere a g a in , s e v e r a l norm al p e a k s w hich had no d e t e c t a b l e DNPH bound a p p e a re d a t t h e end o f t h e chro m ato g ram . T h ese p e a k s a re c h a r a c t e r i s t i c o f th e e l u t i o n p a t t e r n o f a l p B j , cA/CBq, ^ C B y and c I n c o n t r a s t w ith th e c h ro m a to g r a p h ic p a t t e r n o f c o m p le te CNBr d i g e s t o f o r su c h d i g e s t o f no 2J50 mp a b s o r b in g m atei,' J. was o b s e rv e d b e tw e e n 4 5 0 -1 ,7 0 0 ml o f e f f l u e n t . One p o s s i b l e e x p l a n a t i o n f o r t h i s would be t h a t w ith a t, o r ^ ^ o x i d a t i o n o f a l y ^ y l r e s i d u e t o an a ld e h y d e would d e c r e a s e t h e p o s i t i v e c h a rg e c h a r a c t e r i s t i c o f a p e p t i d e . A d e c r e a s e i n p o s i t i v e c h a rg e c a u s e s a d is p la c e m e n t tow ard lo w e r e l u t i o n volum e o f t h a t p e p t i d e . W ith s e v e r a l £ -NH2 g ro u p s in v o lv e d i n a ld e h y d e p r o d u c t i o n th e n e t d is p la c e m e n t o f t h e o v e r a l l p a t t e r n o f e l u t i o n o f p e p t i d e s w ould be to w a rd s m a l l e r e l u t i o n volum e. The r e a c t i o n o f a l d e h y d e - c o n t a i n i n g p e p t i d e s w ith DNPB c a u s e s f u r t h e r d is p la c e m e n t to w ard s t i l l l e s s e r e l u t i o n vo lu m e. I f th e D N PH -reacted p e p t i d e s a r e s u f f i c i e n t l y d i s p l a c e d any u n o x id iz e d p e p t i d e s ( a l d e h y d e - f r e e ) c o u ld t h e n form a more n orm al e l u t i o n p a t t e r n . The chrom ato gram on c a rb o x y m e th y l c e l l u l o s e o f a CNBr d i s g e s t o f r a d i o a c t i v e N aBH ^-reduced CysSC r e v e a le d a num ber o f p e p t i d e s w hich had become r e d u c e d . Some o f t h i s r a d i o a c t i v e u p ta k e as s e e n i n t h e chrom atogram c o u ld be a r e s u l t o f n o n s p e c i f i c t r i t i u m e x c h a n g e . F r a c t i o n V was s u b j e c t e d t o b a s e h y d r o l y s i s and a p p l i e d t o t h e amino a c id a n a l y z e r . Only two p e a k s o f 135 r a d i o a c t i v i t y w ere f o u n d . The l a r g e s t c o rre s p o n d e d t o t h e e l u t i o n p o i n t o f h y d r o x y n o r l e u c in e . A s m a l l e r p e a k a p p e a re d a t t h e p o i n t o f e l u t i o n o f d i h y d r o x y n o r l e u c i n e . T hese a r e t h e p r o d u c t s o f r e d u c t i o n o f t h e a ld e h y d e d e r i v a t i v e s o f l y s i n e and h y d r o x y l y s i n e , r e s p e c t i v e l y . The a b se n c e o f o t h e r r a d i o a c t i v e p e a k s i n d i c a t e s t h a t r e d u c t i o n was r e s t r i c t e d t o a ld e h y d e s i n t h i s r e g i o n . The f i n d i n g o f e i t h e r o f t h e s e p e a k s when ta k e n i n t h e c o n te x t o f t h e c a rb o x y m e th y l c e l l u l o s e chrom atogram i n d i c a t e s t h a t a t l e a s t one r e s i d u e o f l y s i n e o r h y d ro x y - l y s i n e a p p e a rs i n t h e h e l i c a l p o r t i o n o f t h i s c o l l a g e n w hich i s s u s c e p t i b l e to f o r m a tio n o f a l l y s i n e o r h y d ro x y - a l l y s i n e . T h e r e f o r e , c o l l a g e n from f e t a l c a l f s k i n i s c a p a b le o f fo rm in g a t l e a s t one i n t e r m o l e c u l a r c r o s s l i n k w ith o u t p a r t i c i p a t i o n o f i t s amino t e r m i n u s . T hese f i n d i n g s a re c o n s i s t e n t w ith p r e v i o u s f i n d i n g s f o r s o l u b l e c o ll a g e n s from r a t s k i n s ( 2 ) . CHAPTER VI SUMMARY S o lu b le c o l l a g e n was p re p a re d fro m f e t a l c a l k s k i n by e x t r a c t i o n w ith n e u t r a l s a l t s o l u t i o n , a c i d i c b u f f e r s , o r 2 - m e rc a p to e th y la m in e i n n e u t r a l s a l t s o l u t i o n . The amino t e r m i n a l , n o n - h e l i c a l r e g i o n p e p t i d e s w ere s e l e c t i v e l y removed fro m some p r e p a r a t i o n s by l i m i t e d c le a v a g e w ith cy an o g en brom ide (CN Br). C om parison o f t h e p r o d u c t s o f l im i te d c le a v a g e w ith u n m o d ifie d e x t r a c t s u s in g two a ld h y d e r e a g e n t s showed t h a t t h e r e w ere a ld e h y d e s i n t h e h e l i c a l r e g i o n o f e a c h e x t r a c t . R e s o l u t i o n o f t h e i n d i v i d u a l s u b u n i t s o f a c id s o l u b l e c o l l a g e n was a c c o m p lis h e d on a p r e p a r a t i v e s c a l e u s i n g c a rb o x y m e th y l c e l l u l o s e (CMC) c h ro m a to g ra p h y . Amino a c id a n a l y s i s o f a l l e x t r a c t s and o f t h e ^ s u b u n i t s o f a c i d s o l u b l e c o l l a g e n i n d i c a t e d t h a t t h e s e p r e p a r a t i o n s w ere p ro b a b ly t h e same a s s i m i l a r p r e p a r a t i o n s o f a d u l t c o l l a g e n . C om plete c le a v a g e o f t h e i s o l a t e d s u b u n i t s w ith CNBr and r e s o l u t i o n o f th e p r o d u c t s by CM C c h ro m a to g ra p h y a llo w e d i d e n t i f i c a t i o n o f a s u b u n i t w hich c o n ta in e d a n a ld e h y d e m o ie t y . The amino a c i d c o m p o s itio n o f t h i s s u b u n i t was 1JS6 137 s i m i l a r to t h a t o f e i t h e r o r ^ ; CBg a s d e te rm in e d by o t h e r i n v e s t i g a t o r s . R e d u c tio n o f 2 -m e rc a p to th y la m in e s o l u b l e c o l l a g e n w ith t r i t i u m - l a b e l l e d sodium b o r o h y d r id e allo w e d r e s o l u t i o n o f l a b e l l e d f r a c t i o n s from t h e h e l i c a l r e g i o n o f th e m o le c u le on CM C a f t e r co m p lete c le a v a g e w ith CNBr. One su c h p e p t i d e r e v e a le d th e l a b e l l e d r e d u c t i o n p r o d u c t s o f a ld e h y d e s d e r iv e d from h y d ro x y - l y s i n e and l y s i n e a f t e r b a s e h y d ro ly s is c h ro m a to g ra p h on an amino a c id a n a l y z e r . I d e n t i f i c a t i o n o f su c h a ld e h y d e s d e m o n s tr a te s t h e c a p a c i ty o f c o l l a g e n t o form c r o s s l i n k s w hich do n o t in v o lv e th e n o n - h e l i c a l r e g i o n o f th e m o le c u le . R E F E R E N C E S 138 REFERENCES 1 . Deshmukh, K ., and N im ni, M .E .: J . B i o l . Chem. 2 4 4 :1 7 8 7 (1 9 6 9 ) . 2 . Deshmukh. K ., and N im ni, M .E .: B io c h e m is try 1 0 :1 6 4 0 ( 1 9 7 1 ) . ““ 5 - S t r a t e s , B .S ., and U r i s t , M .R .: C l i n . O rth o p . 66: 226 ( 1 9 6 9 ) . — 4 . G lim c h e r, M .J ., and K ra n e , S .M .: i n T r e a t i s e on C o lla g e n , G.N. R am achandran and B.3Y Gould ( e d s . ) , Mew Y ork: A cad. P r e s s , ( 1 9 6 8 ) "B io lo g y o f C o lla g e n ," v o l . 2 , p . 6 8 . 5 . S t u a r t , E .S ., and M oscona, A.A. : S c ie n c e 157-'947 (1 9 6 7 ). 6 . W o essn er, J . F . , J r . : i n C h e m istry and M o le c u la r B io lo g y o f t h e I n t e r c e l l u l a r M a t r ix , E .A . 6 a l a z s ( e d . J , New Y ork: A cad. P r e s s , (1 9 7 0 ) v o l . 3 , p . 1665* 7 . S e r a f i n i - F r a c a s i n i , A ., W e lls , P . J . , and S m ith , J . W . : i n C h e m istry and B io lo g y o f t h e I n t e r c e l l u l a r M a tr ix , E. A. S a l a z s ( e d . ) , New Y ork: A cad. P r e s s , (I97& J v o l . 2 , p . 1 2 0 1 . 8 . K atzm an, R . L . : B iochem . B io p h y s . A c ta 2 2 9 •516 ( 1 9 7 1 ) . 9 . Wood, G .C .: i n I n t e r n a t i o n a l Review o f C o n n e c tiv e T is s u e R e s e a r c h , b . A. H a ll ( e d . ) , New Y ork: A cad. f r e s s , (T 9 6 4 ), v o l . 2 , p . 1 . 1 0 . N o ld , J . G . : S c ie n c e 1 J 0 :1 0 1 6 ( 1 9 7 0 ). 1 1 . T ra u b , W ., and P i e z , K .A .: i n A dvances i n P r o t e i n C h e m is tr y , C .B . A n fin s e n , J r . , j . T . E d s a l l , and P.M . R ic h a rd s ( e d s . ) , New Y ork: A cad. P r e s s , (1 9 7 1 ) v o l . 2 5 , P- 2 4 6 . 12. B akerm an, S .. and H e rs h , R . T . : N a tu re London 201: 190 ( 1 9 6 4 ) . 1 2 9 1 3 . 1 4 . 1 5 . 1 6 . 1 7 . 1 8 . 1 9 . 2 0 . 2 1 . 2 2. 2 3 - 2 4 . 2 5 . 2 6 . 2 7 . l 4 o M i l l e r , E . J . : B io c h e m is try 1 0 :1 6 5 2 (1 9 7 1 ). T r e l s t a d , R .L ., Kang, A .H ., I g a r a s h i , S . , and G ro ss, J . : B io c h e m is try 2 :4 9 9 2 ( 1 9 7 0 ) . P i e z , K .A .: i n T r e a t i s e o n C o lla g e n , G.N. R am achandran ( e d . ) , foew Y ork: A c ad . P r e s s , ( 1 9 6 7 ) v o l . 1 , p . 2 0 7 . P i e z , K .A .: B io c h e m is try 4 :2 5 9 0 ( 1 9 6 5 ). M i l l e r , E . J . , and E p s t e i n . E .H .. J r . : Biochem . B io p h y s . R e s. Commun. 4 2 :1 0 2 4 ( 1 9 7 1 ) . K e f a l i d e s , N .A .: B iochem . B io p h y s . R es. Commun. 4 ^ :2 2 6 (1 9 7 1 )- K e f a l i d e s , N .A .: B iochem . B io p h y s . R e s. Commun. 4 £ :1 1 5 1 ( 1 9 7 2 ). B o r n s t e i n , P . , M a rtin , G .R ., and P i e z , K .A .: S c ie n c e 1 4 4 :1220 ( 1 9 6 4 ) . V e is , A ., and A nesey, J . : J . B i o l . Chem. 2 4 0 :2899 ( 1 9 6 5 ) . P i e z , K .A .: i n Annu. R ev. B io ch em ., P .D . B oyer ( e d . ) , P a lo A lto , C a l i f .: A nnual R eview s, I n c . , ( 1 9 6 8 ) v o l . 2 7 , P . 547. Kang, A. H ., N a g ai, Y ., P i e z , K .A ., and G ro ss, J . : B io c h e m is tr y £ :5 0 9 ( 1 9 6 6 ) . R am achandran, G .N .: i n T r e a t i s e on C o lla g e n , G.N. R am achandran ( e d 7 ) , New Y ork: A cad. P r e s s (1 9 6 7 ) v o l . 1 , p . 102- B o r n s t e i n , P . , and K ang, A .H .: i n C h e m i s t ^ and M o le c u la r B io lo g y o f t h e I n t e r c e l l u l a r M a tr ix , Ei.A. B a la z s (e d T ), New Y ork: A cad. P r e s s (19 70) v o l . 1 , p . 9 9 • K a tz , E . P . , and Shu-Tung L i : B iochem . B io p h y s. R e s. Commun. 4 6: 268 (1 9 7 2 ) . T ra u b , W ., and P i e z , K .A .: i n A dvances i n P r o t e i n C h e m is try , C.B . A n f in s e n , J r . , 77T . E d s a l l , and P.M . R ic h a r d s ( e d s . ) . New Y ork: A cad. P r e s s (1 9 7 1 ) v o l . 2 5 , p . 2 4 2 . l 4 l 2 8 . M ech an ic, G .L ., and L evy, M .: J . Amer. Chem. S o c. 8 1 :1 8 8 9 ( 1 9 5 9 ) . 29- B en au aan , H .G .: i n C h e m istry and M o le c u la r B io lo g y o f t h e I n t e r c e l l u l a r M a t r ix , E .A . B a la z s ( e d . ) . New Y ork: A cad. P r e a a , ( l$ 7 0 ) v o l . 1 , p . l 4 l . 3 0 . V e ia , A ., and S c h l u e t e r , R . J . : N a tu re London 1 9 3 : 204 ( 1 9 6 3 ) . 3 1 . S c h l u e t e r , R . J . , and V e ia , A .: B io c h e m ia try 3 :1 6 5 7 (1 9 6 4 ). 3 2 . M ilc h , R .A .: G e r o n to lo g ia 7^129 ( 1 9 6 3 ) . 33* L a B e lle c , F . : B iochem . B io p h y s. R e s. Commun. J50:333 ( 1 9 6 8 ) . 3 4 . B e ll o , J . , and B e l l o s , A .R .: B iochem . B io p h y s . A c ta 6 2 :5 6 2 ( 1 9 6 3 ) . 35* G a llo p , P .M ., B lu m e n fe ld , 0 . 0 . , H enson, E ., and S c h n e id e r , A .: B io c h e m is try £ :2 4 0 9 ( 1 9 6 8 ) . 3 6 . G a llo p , P .M ., S e l f t e r , S ., and M eilm an, E .: N a tu re London 1 8 2 :1 6 5 9 (1 9 5 9 ). 37- B u t l e r , W .T .: J . B i o l . Chem. 2 4 4 :3 4 1 5 ( 1 9 6 9 ) . 3 8 . B o r n s t e i n , P . : B io c h e m is try 2 ; 24o8 ( 1 9 7 0 ) . 3 9 . B o r n s t e i n , P . : B iochem . B io p h y s. R e s . Commun. 3 6 : 957 (1 9 6 9 ) . 4 0 . L ev e n e, C . I . : J . E x p t l . Med. 1 1 6 :1 1 9 ( 1 9 6 2 ) . 4 1 . B lu m e n fe ld , 0 . 0 . , and G a llo p , P .M .: B io c h e m is try 1 :9 4 7 ( 1 9 6 2 ) ' 4 2 . R o jk in d , M ., B lu m e n fe ld , 0 . 0 . , G a llo p , P .M .: B iochem . B io p h y s . R e s . Commun. 1 7 3 2 0 ( 1 9 6 4 ) . 4 3 . R o jk in d , M ., B lu m e n fe ld , 0 . 0 . , and G a llo p , P .M .: J . B i o l . Chem. 2 41:1 5 3 0 ( 1 9 6 6 ) . 4 4 . B o r n s t e i n , P . , Kang, A .H ., and P ie z , K .A .: P r o c . N a t l . A cad. S c i . U .S .A . £ £ : 4 l 7 (1 9 6 6 ) . 142 45- B o r n s t e i n , P . , and P i e z , K .A .: B io c h e m is try 5 0 4 6 0 ( 1966). 4 6 . B o r n s t e i n , P . , Kang, A .H ., and P i e z , K .A .: B iochem i s t r y 5 :3 8 0 3 ( 1 9 6 6 ) . 4 7 . Hodge, A . J . , H ig h b e rg e r, J . H . , D e f f n e r , G .G .J ., and S c h m itt, P .O .: P r o c . N a t l . A cad. S c i . U .S .A . 4 6 :1 9 7 ( I 9 6 0 ) . 4 8 . R u b in , A .L ., D ra k e, M .P ., D a v iso n , P . P . , P f a h l , D ., Speakman, P . T . , and S c h m itt, P .O .: B io c h e m is try 4 : l 8 l ( 1 9 6 5 ) . 4 9 . R u b in , A .L ., P f a h l , D ., Speakm an, P . T . , D a v iso n , P . P . , and S c h m itt, P .O .: S c ie n c e N. Y. 139 ' • 37 ( 1963) • 5 0 . Kang, A .H .: B io c h e m is try 1 1 :828 (1 9 7 2 ) . 5 1 . S i e g e l , R .C ., P i n n e l l , S .R ., and M a r tin , G .R .: J . B i o l . Chem. 2 4 £ :6 l5 3 ( 1 9 7 0 ) . 5 2 . Deshmukh, K ., and N im ni, M .E .: P e d n . P ro c . P e d n . Am. S oc. Exp. B i o l . 2 J :7 6 8 ( 1 9 6 8 ) . 53- B o r n s t e i n , P . , and P ie z , K .A .: J . C l i n . I n v e s t . 4 3 :1 8 1 3 ( 1 9 6 4 ) . 5 4 . Deshmukh, K ., and N im ni, M .E .: B iochem . B io p h y s. Act 1^4: 258 ( 1 9 6 8 ). 5 5 . Deshmukh, K ., and N im ni, M .E .: B iochem . B io p h y s. R e s. Commun. 4 jJ :4 l 6 ( 1 9 7 1 ). 5 6 . M i l l e r . E. J . : Biochem . B io p h y s . R e s. Commun. 4 ^ :4 4 4 ( 1 9 7 1 ) . 57* Deshmukh, K ., and N im ni, M .E .: i n p r e p a r a t i o n 5 8 . G a llo p , P .M .: B io p h y s. J . 4 :8 0 ( 1 9 6 4 ) . 59* M i l l e r , E . J . , P i n n e l l , S .R ., M a r tin , G .R ., and S c h iffm a n , E .: B iochem . B io p h y s. R e s. Commun. 2 6 :1 3 2 ( 1 9 6 7 ) . 6 0 . S i e g e l , R .C ., and M a rtin , G .R .: J . B i o l . Chem. 2 4 ^ :1 6 5 3 ( 1 9 7 0 ) . 143 61 . M echanic, G . L . : I s r . J . Med. S c i . J : 4 58 ( 1 9 7 1 ) . 6 2 . P az, M .A ., L e n t, R.W ., P a r i s , B ., F r a n z b la u , C ., B lu m e n fe ld , 0 . 0 . , and G a llo p , P .M .: B iochem . B io p h y s . R e s. Commun. £ k :2 2 1 ( 1 9 6 9) . 6 3 . T a n z e r, M .L ., and M echanic, G .: B iochem . B io p h y s. R e s. Commun. 3 9 :183 (1 9 7 0 ). 6 4 . Kang, A .H ., P a r i s , B ., and F r a n z b la u , C .: B iochem . B io p h y s . R e s. Commun. 3 9 •175 ( 1 9 7 0 ). 6 5 . M ech an ic, G .L ., and T a n z e r, M .: B iochem . B io p h y s. R e s. Commun. 4 1 :1 5 9 7 (1 9 7 0 ). 6 6 . T a n z e r, M .L .: B iochem . B io p h y s. A cta 1 3 3 :584 ^1 9 6 7 ) . 6 7 . B a li a n , G .A ., Bowes, J . H . , and C a te r , C.W .: B iochem . B io p h y s. A c ta l 8 l : 331 ( 1 9 6 9 ) . 6 8 . DeLuque, 0 . , and M ech an ic, G .L .: B io c h e m is try £ :4 4 8 7 (1 9 7 0 ) . 6 9 . B a il e y , A . J . , and P e a ch , C.M .: B iochem . B io p h y s. R e s. Commun. ^ : 8 l 2 ( 1 9 6 8 ) . 7 0 . Zimmerman, B .K ., e t a l : E u r. J . B iochem . 1 6 :2 1 7 ( 1 9 7 0 ) . — 7 1 . M ech an ic, G .L .: i n C h em istry and M o le c u la r B io lo g y o f t h e I n t e r c e l l u l a r M a tr ix j E .A . B a la zS ( e d . ) , New Y o r k : A cad. P r e s s , (1 9 7 0 ) v o l . 1 , p . 3 0 5 . 7 2 . T a n z e r, M .L ., and M ech an ic, G .: B iochem . B io p h y s. R e s . Commun. £ 2 :8 8 5 ( 1 9 6 8 ) . 73* B a ile y , A . J . : B iochem . B io p h y s. A cta 2 2 1 :6 5 2 ( 1 9 7 0) . 7 4 . B a ile y , A . J . , F o w le r, L . J . , and P e a ch , C .M .: B iochem . B io p h y s . R e s. Commun. 3 5 »663 (1969)* 75- M cC lain, P . E . , C re e , G . J . , W iley , E .R .. and G e r r i t s , R . J . : B iochem . B io p h y s . A cta 2 2 1 :3 ^ 9 (1 9 7 0 ). 7 6 . T a n z e r, M .L .: i n C h e m istry and M o le c u la r B io lo g y o f t h e I n t e r c e l l u l a r M a tr ix , E .A . B a la z s (e d T ), New Y o rk : A cad. P r e s s (1970) v o l . 1 , p . 2 8 7 . 77* Kang, A .H ., B o r n s t e i n , P . , and P i e z , K .A .: B io c h e m is tr y 6 :7 8 8 ( 1 9 6 7 ) . 144 7 8 . L even e, C . I . , and G ro s s , J . : J . E x p tl . Med. 1 1 0 :7 7 1 ( 1 9 5 9 ) . 79. M a r tin , G .R ., G ro s s , J . , P ie z , K .A ., and L ew is, M .S .: B iochem . B io p h y s . A c ta 53•599 ( l § 6l ) . 8 0 . S i e g e l , R .C ., P i n n e l l , S .R ., and M a rtin , G .R .: B io c h e m is tr y £.*4486 (1 9 7 0 ). 8 1. N im ni, M .E ., and B e v e t ta , L .A .: S c ie n c e 1 5 0 :9 0 5 (1 9 6 5 ) . 8 2 . Nim ni, M .E .: B iochem . B io p h y s. A cta 1 1 1 :5 7 6 ( 1 9 6 5 ) . 835. N im ni, M .E ., G e r th , N ., and B e v e tta , L .A .: N a tu re 2 1 ^ :9 2 1 ( 1 9 6 7 ) . 8 4 . Nim ni, M .E .: J . B i o l . Chem. 2 4 ^ :1 4 5 7 ( 1 9 6 8 ) . 8 5 . Wood, G .C .: B iochem . J . 8 4 :4 2 9 ( 1 9 6 2 ) . 8 6 . Deshmukh, K .. and N im ni, M .E .: Biochem . J . 1 1 2 : 397 ( 1 9 6 9) . 8 7 . B o r n s t e in , P . : B io c h e m is try 6 :3082 ( 1 9 6 7 ) . 8 8 . M i l l e r , E . J . , L ane, J .M ., and P ie z , K .A .: B io c h e m is tr y 8:j50 (19& 9). 8 9 . B u t l e r , W .T .: i n C h e m istry and M o le c u la r B io lo g y o f t h e I n t e r c e l l u l a r M a t r ix . E.A. B a la z s (edT . Mew Y ork: A cad. P r e s s (1 9 7 0 ) v o l . 1, p . 149. 9 0 . S p ir o , K .C .: J . B i o l . Chem. 2 4 4 :6 0 2 ( 1 9 6 9 ) . 9 1 . B u t l e r , W .T ., P i e z , K .A ., and B o r n s t e in , P . : B io c h e m is try 6 :^ 7 7 1 ( 1 9 6 7 ) . 9 2 . H a rk n e s s , R .D ., M arko, A.M ., M uir, H.M ., and N e u b e rg e r, A .: B iochem . J . 5 6 :5 5 8 ( 1 9 5 4 ) . 93. J a c k s o n , D .S .: B iochem . J . 6 5 :2 7 7 (1957)* 9 4 . S i e g e l . R .C ., and M a r tin , G .R .: B io c h e m is try £ :4 4 8 6 (1 9 7 0 ). 9 5 . Chou, W .S ., S a v a g e , J . E . , and O 'D e ll, B . E . : J . B i o l . Chem. 2 4 4 :5 7 8 5 ( 1 9 6 9 ) . 145 9 6 . Nimni, M.E., Deshmukh, K ., and B a v e tta , L .A .: Arch. Biochem. B iophys. 122:292 ( 1967). 9 7 . P ie z , K .A ., E ig n e r , E .A ., L ew is, M .S .: B io c h e m is try 2 :5 8 ( 1 9 6 3 ) . 9 8 . B o r n s te in , P . , and P i e z , K .A .: S c ie n c e 1 4 8 :1353 ( 1 9 6 5 ). 99- N ordw ig, A ., and D ic k , Y .P .: B iochem . B io p h y s. A c ta 2 7 :1 7 9 (1 9 6 5 )• 1 0 0 . M i l l e r , E . J . , E p s t e i n , E .H ., J r . , and P i e z , K.A. : B iochem . B io p h y s . R e s. Commun. 4 2 :1 0 2 4 ( 1 9 7 1 ) . 1 0 1 . F e s s l e r , J . H . , and B a i l e y , A . J . : B iochem . B io p h y s. A c ta 1 1 2 0 6 8 (1 9 6 6 ) . 1 0 2 . S t a r k , M., and Kuhn, K .: E u r. J . B iochem . 6 :5 3 4 ( 1968). 103* Deshmukh, A ., Deshmukh, K ., and N im ni, M .E .: B io c h e m is tr y 1 0 :2 3 3 7 ( 1 9 7 1 ) . 1 0 4 . P az, M .A ., B lu m e n fe ld , 0 . 0 . , G a llo p , P .M .: A rch . B iochem . B io p h y s . 1 0 9 »548 ( 1 965) . 105- S a w ic k i, E ., H a u se r, T .R ., S t a n le y , T .W ., E l b e r t , W.: A n a l. Chem. ^ 3 :9 2 ( I 9 6 l ) . 106. P a s h le y , D .H ., Claycom b, C .K ., and Summers, G .W .: A n a l. B iochem . 1 £ :1 5 4 ( 1 9 6 6 ) . 107. L e n t, R.W ., S m ith , B . , S a lc e d o , L .L ., P a r i s , B ., and F r a n z b l a u , C .: B io c h e m is tr y 8 :2 8 3 7 ( 1 9 6 9 ) . 108. Bakerm an, S . : B iochem . B io p h y s . A cta 2 2 ; 621 (1 9 6 4 ). 109. Bakerm an. S . , and H e rsh , R .T .: N a tu re London 2 0 1 :190 ( 1 9 6 4 ). 110. G a llo p , P .M ., B lu m e n fe ld , 0 . 0 . , and P a z , M .: i n C h e m istry and M o le c u la r B io lo g y o f t h e I n t e r c e l l u l a r M a t r i x , E .A . B a la z s ( e d . ) , Mew Y ork: A cad, t r e s s (1 9 7 0 ) v o l . 1 , p . 179* 1 1 1 . V o lp in , D ., and V e is , A .: B io c h e m is try 1 0 :1 7 5 1 ( 1 9 7 1 ). 146 112. C la r k , C .C ., and V e ls , A .: B io c h e m is try 1 1 :4 9 4 (1 9 7 2 ). — 1 1 3 . P i e z , K .A ., M a r tin , G .R ., Kang, A .H ., B o r n s t e i n , P . : B io c h e m is try £ : 3 8 l 3 ( 1 9 6 6) . 114. H a rk n e s s , R .D .: B i o l . Rev. ^ 6 :3 9 9 (1 9 6 1 ) . 115. P ro c k o p , D . J . : i n C h em istry and M o le c u la r B io lo g y o f t h e I n t e r c e l l u l a r M a trix , M.A. BaHazs (ed.T. N e w Y o r k : A cad. P r e s s 11970 ) v o l . 1 , p . 3 3 5 . 116 . S p i r o , R.G.: J . B i o l . Chem. 2 4 2 :1917 ( 1 9 6 7 ) . 11 7 . S n e llm a n , 0 . : i n S t r u c t u r e and F u n c tio n o f C o n n e c tiv e and S k e l e t a l T i s s u e , S . F i t t o n J a c k s o n , S.M. f a r t r i d g e , Jt.D. H a rk n e s s, and G.R. T r i s t r a m ( e d s . ) , London: B u tte r w o r th s ( 1 9 6 5 ) , p . 3519. 118. P a z , M.A.: p e r s o n a l co m m u n icatio n . 11 9 . M e ch a n ic, G .: p e r s o n a l c o m m u n ica tio n . 1 2 0 . S tr a w ic h . E ., and N im ni, M.E.: B io c h e m is try 1 0 :3 9 0 5 ( 1 9 7 1 ).

Linked assets

University of Southern California Dissertations and Theses

Conceptually similar

PDF

Microheterogeneity And Crosslinking Of Salt Soluble Collagen

PDF

Application Of Rubber Elasticity Theory To Crosslinked Denatured Collagen

PDF

The inability of collagen cross-linking to influence the insolubilization of proteodermatan sulfate and a sialo-glycopeptide

PDF

The ferric complexes of citrate, ETDA and NTA: their nature and the ligand exchange reaction with human transferrin

PDF

Studies On The Viscoelastic Behavior Of Connective Tissue

PDF

The Effect Of Polyols On Placental Estradiol Dehydrogenase

PDF

Steric effects in the acid, amine, and glyceraldehyde-3-phosphate dehydrogenase catalyzed hydrolysis of acyl phosphates

PDF

Studies On Antigens Associated With Wilm'S Tumor (Nephroblastoma)

PDF

Physicochemical Studies Of High-Density Lipoproteins Of Bovine Plasma

PDF

Studies On The Cell-Free Synthesis Of Collagen On Polysomes From Chick Embryo Connective Tissues

PDF

Nucleation And Solubility Of Monosodium-Urate In Relation To Gouty Arthritis

PDF

Studies of human ɑ₂ macroglobin: physical and enzyme binding properties

PDF

Studies on the pulmonary surface-active lipoprotein: isolation and characterization of its peptide moiety

PDF

A Nonlinear Control Systems Model For Cellular Metabolism As Applied To The Adipocyte

PDF

Studies On The Quantitative Precipitin Reaction

PDF

Studies On A Modified Fat

PDF

Kinetic Studies Of Ultracentrifugal Demulsification

PDF

The RNA polymerases of Gymodinium breve and soybean hypocotyls

PDF

Changes in the rate of net synthesis of oral tissue collagen with age and wound healing

PDF

Studies On The Biosynthesis Of Collagen In The Rabbit Embryo Skin In Vitro

Asset Metadata

Creator Norby, David Paul (author)

Core Title Crosslink Precursors In Fetal Calf Skin Soluble Collagens

Degree Doctor of Philosophy

Degree Program Biochemistry

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag chemistry, biochemistry,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor Nimni, Marcel E. (committee chair), Allerton, Samuel E. (committee member), Friou, George J. (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c18-788936

Unique identifier UC11363430

Identifier 7309318.pdf (filename),usctheses-c18-788936 (legacy record id)

Legacy Identifier 7309318

Dmrecord 788936

Document Type Dissertation

Rights Norby, David Paul

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus, Los Angeles, California 90089, USA