University of Southern California Dissertations and Theses

Microheterogeneity of fetuin

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Microheterogeneity of fetuin

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Content MICROHETEROGENEITY OP FETUIN by Yuki O shiro A D i s s e r t a t i o n P r e se n te d to th e FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In P a r t i a l F u l f i l l m e n t o f the R equirem ents f o r the Degree DOCTOR OF PHILOSOPHY (B io c h e m istr y ) A p r il 1967 UNIVERSITY O F S O U T H E R N C A L IF O R N IA T H E G R A D U A T E S C H O O L U N IV E R S IT Y P A R K L O S A N G E L E S , C A L IF O R N IA 9 0 0 0 7 This dissertation, written by ......................Y ^ i„.O sh irp_....................................... under the direction of h%&...Dissertation Com mittee, and approved by all its members, has been presented to and accepted by the Graduate School, in partial fulfillment of requirements for the degree of D O C T O R O F P H I L O S O P H Y e . A z & z g & g ................. Dean Date............................................................................. DISSERTATION COMMITTEE o c t 1 q ; i where T ] = th e v i s c o s i t y of th e s o l u t i o n \ i ri0 = th e v i s c o s i t y of th e s o l v e n t j ! i c = th e c o n c e n t r a t i o n in g/m l : | : j I t can be e s t im a t e d from a s e r i e s of m easurem ents at’ d i f f e r e n t c o n c e n t r a t i o n s w ith a w e ll-k n o w n e m p i r i c a l eq u a - i I ; 47 t i o n c a l l e d H u g g in Ts e q u a t io n : j H z H o i . = [ -T|] + [ Tl] 2 C ! ! n0 c | ! 1 i jwhere K i s a c o n s t a n t . Assuming the p r o t e i n m o le c u le i s a Ir ig id e l l i p s o i d of r e v o l u t i o n , th e a x i a l r a t i o can be e s t i - i |mated from th e i n t r i n s i c v i s c o s i t y a c c o r d in g to the 2 1 f o llo w in g e q u a tio n :* ^ ! [r|] = v Cl + (6 ) vp 100 ! where p = the d e n s i t y o f the s o l v e n t i vh = the v i s c o s i t y increm ent j ! i A fter c a l c u l a t i n g the v i s c o s i t y increm ent vh w ith j i | 'th is e q u a tio n the a x i a l r a t i o of the e l l i p s o i d o f revo lu tion ! 46 49 i s ob tain ed from t a b l e s . ’ The a x ia l r a t i o would g iv e an japproximate shape o f the m o le c u le . I f a change in shape o c - i i icurs accompanying th e removal o f s i a l i c acid r e s i d u e s , i t j j ' i jmight be r e f l e c t e d in a change in th e a x ia l r a t i o . Further-! . . . . . * more, an in c r e a s e in the i n t r i n s i c v i s c o s i t y might be in te r-! p r e te d as e it h e r th e s w e llin g o f the m olecule due to a con- i fo rm a tio n a l change from a r i g i d g lo b u la r s tr u c t u r e to a ran-i dom c o i l or the fo rm a tio n o f an e lo n g a te d r ig i d rod . This kind o f change in th e i n t r i n s i c v i s c o s i t y was dem onstrated w ith r ib o n u c le a s e in urea and in 2 - c h l o r o e t h a n o l . ^ ^ It was hoped th a t changes in the m olecu la r conform ation o f f e t u i n upon removal of s i a l i c acid r e s id u e s might be a s s e s s e d ! in a s im ila r manner. i ! 3. O p tic a l R otatory D i s p e r s i o n . The change in the j se c o n d a r y -s tr u c tu r e can be s t u d i e s w ith o p t i c a l r o t a t i o n at j i 1 jlow u l t r a v i o l e t wave l e n g t h . The h e l i c a l c o n ten t o f protein; * 52 ! can be rou gh ly e stim a te d by the method o f Simmons e t a l . j i jWith the use of the reduced mean r e sid u e r o t a t i o n at 233 mp,j ; ! '[mT] 2 33 v a lu e s fo r p o ly -L -g lu ta m ic a cid o f -1 5 ,0 0 0 ° fo r I jthe 1 0 0 % h e l i c a l form and - 2 ,0 0 0 ° f o r random -coil were j 22 53 r e p o r t e d . The reduced mean r o t a t i o n i s e x p r e ss e d as f o l low s : .54 [m*] = ( _ ! _ ) (MRW) a ( 7 ) n +2 dc A where n = the r e f r a c t i v e in d ex o f th e s o l v e n t at the wave le n g th (MRW) = The mean r e s id u e w e ig h t (a p p r o x im a te ly 114) | d = the path le n g t h in d e c im e ter j c = the c o n c e n tr a tio n in g/lOO ml = the observed r o t a t i o n a t X . I i T h is method o f e s t i m a t i o n of h e l i c a l c o n te n t in p r o t e i n mol-j t i e c u l e s i s n ot n e c e s s a r i l y an a c c u r a te method. But o th e r j I 55 | imethods which use Drude e q u a tio n or the M o f f i t t and Yang | l e q u a t i o n ^ are a ls o c o n sid e r e d somewhat empir i c a l. ^ Thej r e s u l t i n g v a lu e o f h e l i c a l c o n t e n t , t h e r e f o r e , must be con- ! s id e r e d as a rough e s t i m a t e . MATERIALS AND METHODS Mater i a l s ! i I 3 The f e t a l c a l f serum from which f e t u i n was i s o l a t e d I jwas pu rchased from Hyland L a b oratory , Los A n g e le s , C a l i f o r - \ jnia. I t was a m ixtu re o f f r e s h f e t a l c a l f s e r a c o l l e c t e d j j jwith h e p a rin as an a n t ic o a g u la n t from many f e t a l c a l v e s at d | ; jlo c a l s la u g h t e r house and s e n t to us in f r o z e n s t a t e . I t iwas a y e l l o w i s h s o l u t i o n th a t c o n ta in e d a very sm a ll amount I o f f l o a t i n g p a r t i c l e s . A fter the f r o z e n mass o f serum thawed, i t was f i l t e r e d through Whatman No. 1 f i l t e r p a p er. j The u n f i l t e r a b l e m a t e r ia l c o n ta in e d some g e l - l i k e m a te r ia l which cou ld n o t be se p a r a te d by c e n t r i f u g a t i o n . The f i l t r a t e was s u b s e q u e n tly used f o r th e p r e p a r a tio n o f f e t u i n . For th e stu d y o f m ic r o h e t e r o g e n e it y o f f e t u i n in inn d i v i d u a l f e t u s e s , we c o l l e c t e d i n d iv i d u a l s e r a from f i v e fe-j jtal c a l v e s a t a l o c a l s la u g h t e r h o u se . The f e t a l b loo d was ' ( c o l l e c t e d by h e a r t p u n ctu re w it h in 10 m in u tes a f t e r the j (mother cow was k i l l e d * and im m ed ia tely mixed w ith h e p a r in ! j : J so lu tio n . The b lo o d was c e n t r if u g e d at lOOOg f o r 10 m in u te s ! j i ! I i - - - ■ ■ - i i | | *The generou s h e lp in c o l l e c t i n g th e s e s e r a by Mr. ! IF. Yurohko of Dr. J . S a l k ’ s la b o r a t o r y i s d e e p ly a p p r e c i a te d . 24 and the r e s u l t i n g serum was kep t in a c o ld room u n t i l i t was u sed fo r s t a r c h g e l e l e c t r o p h o r e s i s on the same day. j A f e t u i n sample which was i s o l a t e d by ammonium s u l f a t e p r e c i p i t a t i o n was a g i f t from Dr. T. T. P u c k ,* D enver, jColorado. T h is sample was s e n t to us as a s t e r i l e s o l u t i o n jwithout f r e e z i n g . I t was im m e d ia te ly l y o p h i l i z e d on a r r i v a l land the dry sam ple was k ep t a t - 2 0 ° u n t i l u s e . i H ig h ly a c t i v e n eu ra m in id a se was o b ta in e d from Beh- irin g w erk e, A. G. Marburg, Germany. I t was n e g a t i v e when jtested f o r p r o t e o l y t i c (w ith A z a c o l) and g l y c o s i d i c a c t i v i - I I I t i e s . D ehydrated s t a r c h fo r p r e p a r a t io n of s t a r c h g e l was o b ta in e d from Connaught M edical R esearch L a b ., T oron to, Can ada, through F is h e r S c i e n t i f i c Co. Sodium t e t r a b o r a t e fo r pH sta n d a rd was o b ta in e d from N a tio n a l Bureau o f S tan d a rd , D ep t, of Commerce, W ashington, D. C. A l l o th e r c h e m ic a ls , o th e r than s p e c i f i c a l l y d e s i g n a t e d , were r e a g e n t grade and o b ta in e d from com m ercial s o u r c e s . i i Methods I ( 1 ) I s o l a t i o n and P u r i f i c a t i o n o f F e t u in S in c e th e low tem p era tu re e th a n o l p r e c i p i t a t i o n {method o f S p ir o1'5 seemed to produce th e most homogeneous f e - i t u i n as judged by u l t r a c e n t r i f u g a t i o n , f r e e boundary j *The a u th or i s in d e b te d to Dr. T. T. Puck f o r h i s jgenerous g i f t o f a sample o f f e t u i n . ; 25 i j e l e c t r o p h o r e s i s at s e v e r a l pHs and im m u n o lo g ica l exam in a- i : j t io n , t h i s method was c h o se n to p r e p a r e f e t u i n i n our l a b o r a t o r y . I t i s b a sed on Method 10 o f plasm a f r a c t i o n a t i o n o f j j jCohn e t a l . ^ j j F e t u in was i s o l a t e d from a p o o le d f e t a l c a l f serum , j The f r a c t i o n p r o c e d u r e i s shown i n the f o l l o w i n g diagram ; jCDiagram 1 ) . The f r a c t i o n a t i o n was c a r r i e d o u t in low tem - jp era tu re w a ter b a th w h ich was l o c a t e d in a c o ld room a t 4° 'and c o o le d by adding NaCl and i c e (or a c e to n e and i c e ) . The 1 i jtem p erature was m a in ta in e d w i t h i n *1 ° o f th e s p e c i f i e d tern- I jp era tu re f o r th e r e q u ir e d p e r i o d s of t i m e . The b a th was made o f a g l a s s ja r o f 12 in c h d ia m e te r and 8 in c h h e i g h t , j A 3 1 E rlen m eyer f l a s k was i n s e r t e d in t h e ja r and th e solu -; t i o n w as s t i r r e d by a m e c h a n ic a l s t i r r e r i n s e r t e d from th e t o p . R e a g e n ts were made a t room te m p e r a tu r e and m o s t ly p r e c o o le d b e f o r e a d d i t i o n . In order to r e d u c e th e r i s e o f tem p e r a t u r e , when 95% e t h a n o l was added, i t was p r e c o o le d to ; a p p r o x im a t e ly -4 0 ° and added d r o p w ise w i t h c o n s t a n t m ix in g , j !a therm om eter was i n s e r t e d i n t o th e f l a s k t o ch eck th e tern- I i i 1 i jp eratu re and th e r a t e o f e t h a n o l a d d i t i o n was a d j u s t e d t o 1 i ; jm aintain th e r e q u i r e d t e m p e r a t u r e . pH d e t e r m i n a t i o n s were j j ■ made w i t h a g l a s s - e l e c t r o d e a t room te m p e r a tu r e on th e sam- | i p i e d i l u t e d w i t h fo u r volum es o f d i s t i l l e d w a te r ( t o red u c e j ithe a l c o h o l c o n c e n t r a t i o n ) . C e n t r i f u g a t i o n was perform ed in j p l a s t i c b o t t l e s in th e S o v a l l c e n t r i f u g e . The f i r s t s t e p i jremoved m ost o f th e p lasm a p r o t e i n s i n c l u d i n g c o lo r e d 26 DIAGRAM 1 I s o l a t i o n o f f e t u i n from f e t a l c a l f serum F e t a l Serum Add 2 volum es 0 .0 3 M ZnC A c^, 28.57o e th a n o l a t -5 ° A d ju st to pH 6 .4 by 1 M NH4 0H-NH4 C1 b u f f e r pH 1 0 . 4 , in 19% e th a n o l L et sta n d 12 to 16 hours C e n t r ifu g e j P r e c i p i t a t e A i (D is c a r d ) S u p ern a ta n t A ( 0 . 0 2 M Zn (Ac) 19% e t h a n o l) Add 1 .0 M Ba(Ac)o and 95% e th a n o l to g i v e 0 . 0 2 M Ba , 25% e th a n o l a t - 5 ° , pH 6 .7 Let stan d 2 hours C e n tr ifu g e P r e c i p i t a t e B (D is c a r d ) S u p ern a ta n t B Add 95% e th a n o l to g iv e 40% e th a n o l a t -10 o L et sta n d 12 to 16 hours C e n tr ifu g e P r e c i p i t a t e C ( F e t u in ) S u p ern a ta n t C (D is c a r d ) 2 1 hem oglob in w h ich a p p a r e n tly had come from broken red c e l l s . i The p r e c i p i t a t e B was s l i g h t in amount and th e p r e c i p i t a t e C, f e t u i n , was a c o p io u s w h ite r e c i p i t a t e . I t was d i s s o l v e d in 1 M t r is o d iu m c i t r a t e and d i a l y z e d a g a i n s t d i s t i l l e d wa t e r a t 4° f o r a few d a y s ch angin g w ater once a day t o remove f i n e and barium i o n s . To a s s u r e the c o m p le te rem oval o f j s a l t s , th e d i a l y z e d s o l u t i o n was p a s s e d through a mixed bed ; b f io n -e x c h a n g e r e s i n s o f A m b e r lite MB-3, Dowex-1 x 8 (OH“ ' ; j , ; form ) and Dowex 50-W x 8 (H fo r m ) . The in n e r d ia m e ter of jthe column was 1 . 8 cm and th e h e ig h t o f e a ch r e s i n was 15 , 7; I • i and 10 cm, r e s p e c t i v e l y . The d i a l y z e d f e t u i n was p a s s e d ! | through t h i s column a t 4° a t a r a t e o f 15 drops per m in u te , j I i The d e i o n i z e d e l u e n t s o l u t i o n was f i n a l l y l y o p h i l i z e d and j S t o r e d a t - 2 0 ° u n t i l u s e . I ( 2 ) S t a r c h Gel V e r t i c a l E l e c t r o p h o r e s i s The r e s o l v i n g power o f s t a r c h g e l e l e c t r o p h o r e s i s was amply d e m o n stra te d by S m i t h i e s ^ in a n a l y s i s o f human serum p r o t e i n s sh ow ing most of th e s u b d i v i s i o n s o f th e f r a c - | ; j It io n s s e p a r a t e d by paper e l e c t r o p h o r e s i s . (For i n s t a n c e , 12| h a p t o g lo b in z o n e s were d i s t i n g u i s h e d . ) E l e c t r o p h o r e s i s on | | such m edia as paper or s t a r c h b lo c k are p r i m a r i l y dep en d en t 1 Ion th e ch a rg e d e n s i t y o f p a r t i c l e s b e in g s e p a r a t e d . The I 1 I R e p a r a tio n on s t a r c h g e l i s f u r t h e r e f f e c t e d by th e s i z e o f s i i ; [ | j ithe p a r t i c l e s due to the s i e v i n g e f f e c t c r e a te d by th e m o l- I i 39 I jecular netw ork o f th e g e l . S m a lle r p a r t i c l e s p a s s through! j j jthe m o le c u la r s i e v e more r e a d i l y than l a r g e r p a r t i c l e s , j 28 hence, the r e s o l u t i o n on the g e l i s in c r e a s e d . B e s id e s the above m entioned two f a c t o r s , the charge and s i z e , t h a t con t r i b u t e to the d i s s o l v i n g power, th e r e seems to be another f a c t o r which depends upon a b s o r p tio n o f p a r t i c l e s to the g e l . The natu re o f t h i s e f f e c t i s s im ila r t o th a t of ab- j I 5 9 . ■ isorp tion chromatography. P r o t e in m o le c u le s seem to form a! com plex w ith sta r c h g e l . The str o n g e r the complex r e a c t io n : jbetween p r o t e in m o le c u le s and the g e l , the slow er the m igra t i o n . In f a c t , t h i s complex r e a c t i o n works as a d isa d v a n ta g e when e l u t i o n o f p r o t e in from s ta r c h g e l must be p e r - i i 1 ! i form ed. ! ; | The apparatus fo r s t a r c h g e l v e r t i c a l e le c t r o p h o r e - j Isis was ob ta in ed from Buchler In stru m en t, I n c . , N. J . I t ! was d esig n ed in such a way th a t the g e l s o l i d i f i e d in b e tween a g l a s s p l a t e and a L u c ite mold and the g e l formed had) a uniform t h ic k n e s s of 0 .5 cm and a s i z e of 12 cm by 30 cm. ; A com b -lik e mold was removed from one end of the g e l to form! s l o t s fo r i n s e r t i o n of p r o t e in s o l u t i o n . For the prep ara- 6 0 : t i o n of sta r c h g el th e i n s t r u c t i o n g iven in the manual was; J . \ I | adapted to s u i t our p u rp o se. The b u ffe r s o l u t i o n f o r e l e c - | i trode chambers was made by d i s s o l v i n g s o l i d sodium a c e t a t e to 0.2M and adding g l a c i a l a c e t i c acid t o d e s ir e d pH so that) I the i o n i c s t r e n g th would remain c o n sta n t a t 0 . 2 . For the j b u ffe r fo r s ta r c h g e l the above b u ffe r was d i l u t e d to l / 2 0 i by adding d i s t i l l e d w a te r . The io n ic s tr e n g th changed to p . 01 w h ile the pH remained c o n s t a n t . For some g e l th e j [buffer was made by d i s s o l v i n g NaH2PC>4 and H3 P0 4 at 0 . 2 i o n i c j jstrength fo r e l e c t r o d e chambers and l / 2 0 d i l u t i o n of i t fo r s ta r c h g e l b u f f e r . 6 2 .5 gm o f h y d ro ly zed s t a r c h was s u s pended in 500 ml o f the b u ffe r in a 1000 ml s u c t i o n f l a s k |and h e a ted to b o i l i n g s lo w ly w h ile s t i r r i n g c o n tin u o u s ly by la b ig m agn etic s t i r r e r fo r about 30 m in u te s. When th e so lu -| ! I it ion became c l e a r , th e f l a s k was con n ected to water l i n e and; f h e p r e ssu r e i n s i d e the f l a s k was reduced t o b u r s t th e bub b l e s . The hot s ta r c h s o l u t i o n was poured in the L u c ite mold: which was p la c e d h o r i z o n t a l l y on bench and i t was covered j i | ■with a p reh ea ted g l a s s p l a t e c a r e f u l l y a v o id in g any bubble I [form ation. A fte r th e g e l s o l i d i f i e d , sample s o l u t i o n s were | I ' I lin s e r te d in t o the s l o t s and covered w ith warm p etro leu m g e l . The sta r c h g e l was u s u a l l y used on th e same day i t was made.; O v ern ig h t s to r a g e a t 4° r e s u l t e d in a f r i a b l e g e l which was ' g e n e r a l ly u n s a t i s f a c t o r y fo r e l e c t r o p h o r e s i s . The sample s o l u t i o n s were made o f ap p ro x im a tely 1 to 2 % p r o t e i n s o l u - j i t i o n in the g e l b u f f e r or in d i s t i l l e d w a te r . In some cases; ■the w hole serum was used w ith o u t any d i l u t i o n . Each s l o t ! i ! Iwas f i l l e d w ith about 50 (j.1 of sample s o l u t i o n . A c o n s ta n t j i | [voltage of 200 to 210 v o l t s was a p p lie d by a B uchler power I ! ; 1 [supply f o r 15 h o u rs. I t corresponded to ap p ro x im a tely 7 j i 1 j : Iv o lts per cm o f g e l . The p o s i t i v e e le c t r o d e was th en con- 1 s n e c te d t o th e lower chamber and th e n e g a t i v e to the upper sd Ithat most serum p r o te in f r a c t i o n s m igrated upward and f e t u i n [downward at pH 4 .2 to 4 . 5 . The e l e c t r o d e chamber b u f f e r was ~..... 30 p sed s e v e r a l tim e s b e fo r e d i s c a r d i n g . The b u f f e r in two chambers were mixed b e fo r e use e v e r y tim e . A ll e l e c t r o p h o r e s i s was run in a c o ld room at 4° to p r e v e n t o v e r h e a t in g . The g e l tem p eratu re in c r e a s e d s l i g h t l y d u rin g e l e c t r o p h o r e - j s is , bu t o n ly to th e e x t e n t th a t i t f e l t a l i t t l e warm by Touch. A fte r c o m p le tio n of e l e c t r o p h o r e s i s , th e g e l was I i ■ dyed in 4% N ap hth alene B lack i n a c e t i c a c id , m eth a n o l, and j I Water ( 1 : 5 : 5 by volum e) fo r 30 t o 40 m in u te s and th e e x c e s s ! : dye was d e s t a in e d by th e same s o l v e n t . The s o l v e n t was r e - ! I | jused a f t e r rem oving the dye on c h a r c o a l. j I j S ta r c h g e l in 6 M urea was made a cco rd in g to th e f\ 1 .method d e s c r ib e d by P o u l ik w it h a l i t t l e a l t e r a t i o n . |62.5g o f h y d r o ly z e d s t a r c h was added s l o w l y to a s o l u t i o n of u rea (2 4 0 g /5 0 0 m l w a t e r ) . A fte r the b u b b le s were b u r s t under; reduced p r e s s u r e , th e g e l was poured i n t o the mold and a l lowed t o stan d o v e r n ig h t fo r s o l i d i f i c a t i o n . The f i n a l co n c e n t r a t i o n o f s t a r c h was a p p ro x im a te ly 8 g / l0 0 ml and the urea; c o n c e n t r a t io n a p p r o x im a te ly 6 M. The s o l i d g e l was transpar-; lent and to o weak to be h e ld in a v e r t i c a l p o s i t i o n f o r the i ! I p e r io d o f e l e c t r o p h o r e s i s . C o n s e q u e n tly , h o r i z o n t a l e l e c t r o p h o r e s i s was run f o r 15 hou rs at 200 v o l t s a t 4 ° . S in c e | th e s l o t s were n o t formed w e l l , sm all p i e c e s o f t h i c k f i l t e r i paper soaked w ith sam ple s o l u t i o n s were i n s e r t e d i n t o th e ! j . \ i s l o t s in th e g e l . One sample o f f e t u i n fo r s t a r c h g e l - u r e a j ! ,e l e c t r o p h o r e s i s was prepared by d e n a tu r in g in 8 M u r e a b e - i f o r e a p p l i c a t i o n to th e g e l . Another sample was b o th 31i d e n a tu re d and r ed u ced w ith 2 -m e r c a p to e th a n o l in 8 M u r e a a c c o r d in g to th e p r o c e d u r e d e s c r i b e d by S p i r o . ^ F e t u in was d i s s o l v e d i n 0 .1 M T r is b u f f e r a t pH 8 . 5 t h a t c o n ta in e d 8 M u rea and 200 f o l d e x c e s s o f 2 - m e r c a p t o e t h a n o l. D i s u l f i d e i bond r e d u c t i o n p r o c e e d e d in a co v er ed t e s t tu b e d e a e r a t e d by n i t r o g e n at room te m p e r a tu r e f o r 4 . 5 h o u r s . The red u ced fe -j ’ t u i n was d i a l y z e d a g a i n s t th e g e l b u f f e r w it h 2 - jm ereap toeth an ol f o r e l e c t r o p h o r e s i s in a c o ld room fo r 24 h o u r s b e f o r e a p p l i c a t i o n to u r e a - s t a r c h g e l . S t a i n i n g and j d e s t a in in g p r o c e s s e s were same as n o n -u r e a s t a r c h g e l . ! • ' i ( 3 ) Im m u n o lo g ica l S t u d i e s I A. P r e p a r a t io n o f a n t i - f e t u i n s e r a * j For th e p r e p a r a t i o n o f a n t i - f e t u i n s e r a , the method ; /L d e s c r i b e d by Campbell e t a l . was a d o p te d . 1 .0 ml o f 0.2% ■ f e t u i n i n s a l i n e s o l u t i o n was i n j e c t e d i n t r a v e n o u s l y i n t o t h r e e r a b b i t s w e ig h in g 3 t o 4 k g . The i n j e c t i o n c o n tin u e d f o r a w eek . The f i r s t s e r a w ere c o l l e c t e d t h r e e w eeks la te r , by h e a r t p u n c tu r e and th e f o r m a t io n o f a n tib o d y a g a i n s t f e - j I j Ituin was q u a l i t a t i v e l y ch eck ed in a c a p i l l a r y t u b e . A 1% ' j | I f e t u in s o l u t i o n in 0 . 1 5 M NaCl was drawn i n t o a c a p i l l a r y , j i ; j • ; jfo llo w e d by the a n t is e r u m . A fte r one or two h ou rs o f stand-: in g a t room te m p e r a tu r e a w h it e p r e c i p i t a t e was s e e n . Since! f !the p r e c i p i t a t e was n o t c o p i o u s , a n o th er i n j e c t i o n o f f e t u i n *The author i s in d e b t e d t o Mr. R uppert P e r r e n f o r jh is g e n e r o u s h e lp w i t h t h e i n j e c t i o n and b l e e d i n g . 32 was g iv e n to r a b b i t s . The se co n d , t h i r d , and fo u r th s e r a were c o l l e c t e d one, two and th r e e weeks a f t e r the b o o s te r i i s h o t . At the tim e o f th e l a s t c o l l e c t i o n of serum the rab - { i b i t was s a c r i f i c e d . The c o l l e c t e d blood was allow ed to sta n d fo r a few hours f o r c o a g u la t io n , and th e serum was I • : l c o l l e c t e d a f t e r c e n t r i f u g a t i o n and s to r e d at - 2 0 ° . j i : I B. Im m unodiffusion j : | Im m unodiffusion was perform ed a ccord in g to the meth- jod d e s c r ib e d by O u c h te rlo n y . 1% Agar (B a ltim o r e B i o l o g i - ! j I |cal L a b ., B a ltim o r e , Md. ) g e l was prepared in phosphate j I | b u f f e r a t pH 7 .2 w ith the i o n i c s t r e n g t h o f 0 .1 5 o f which j 2 /3 was due to NaCl. A t r a c e o f m e r t h io la t e was added as an' a n t i b a c t e r i a l a g e n t. Some agar g e l was made w ith a f i v e h o le mold and o t h e r s by p u n c tu r in g h o l e s w ith a s i x h o le F ein b erg agar g e l c u t t e r (C o n s o lid a te d L ab ., I n c . , 1 1 1 .) on ■ sem i-solid g e l . The g e l th u s formed was s e a le d w ith S c o tc h | tap e and s to r e d at 4° u n t i l fu r th e r u s e . F e tu in was d is ~ ; s o lv e d in warm 1 % agar at about 0 .2 % and c a r e f u l l y p la c ed in! j lone h o l e . The r a b b it an tiseru m was p i p e t t e d l i k e w i s e i n t o I J 1 Ian a p p ro p ria te h o l e . D i f f u s i o n and p r e c i p i t a t i o n r e a c t i o n j [took p la c e in a s e a le d P e t r i - d i s h a t room tem p era tu re. The j p r e c i p i t i n developm ent was observed e v e r y day fo r a week and; jphotographs were taken at a p p r o p r ia te t im e s . I 1 C. Im m u n oelectro p h o resis j ; T h is tec h n iq u e i s claim ed t o g iv e b e t t e r d i f f e r e n t i - } ; |a tio n of a h e te r o g e n e o u s p r o t e i n m ixtu re than the sim p le j r " . . . . . . . . . . . . . . . . . ■ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ■ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ' ■ ■ - ■ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ■ ■ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ' . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ■ 33 h o u b le d i f f u s i o n b e c a u s e th e a n t i g e n s are e l e c t r o p h o r e t i c a l - l y s e p a r a t e d b e f o r e r e a c t i n g w ith a n t ib o d y . The p r o c e d u r e | 3 8 I Was s i m i l a r t o t h a t r e p o r t e d by Schmid e t a l . S ta r c h g e l j ! v e r t i c a l e l e c t r o p h o r e s i s of f e t u i n a t pH 4 . 2 was run u s i n g j 50 p i o f 2% f e t u i n per s l o t as d e s c r i b e d p r e v i o u s l y . A long] i i ! ! p i e c e o f g e l near one s l o t was c u t out and s t a i n e d t o d e t e r - j mine how f a r th e f a s t e s t f r a c t i o n m ig r a te d from th e s l o t . W ith t h i s p i e c e as a g u i d e , a p o r t i o n o f s t a r c h g e t w hich j ; was known to c o n t a i n a l l th e s e p a r a t e d f r a c t i o n s was c u t out! 1 | and b u r ie d i n 1% agar g e l in a P e t r i - d i s h . A tro u g h in agarj I ’ ! j g e l was c u t p a r a l l e l to th e b u r ie d s t a r c h g e l and th e a n t i - j j ' j serum in 1% agar was p l a c e d in th e tr o u g h . The d e v e lo p m en t I | i o f a p r e c i p i t i n l i n e to o k p l a c e a t room t e m p e r a tu r e . As a c o n t r o l , a p o r t i o n of t h e same s t a r c h g e l where no f e t u i n m ig r a te d was a l s o c u t o u t and b u r ie d in 1 % agar g e l a lo n g th e o th e r s i d e o f th e a n t is e r u m . Any d e v e lo p m e n t o f a n o n - s p e c i f i c p r e c i p i t i n l i n e c o u ld be o b s e r v e d i n t h i s way. • j 1(4) D e s i a l i c i z a t i o n o f F e t u in ! ; | I Removal o f s i a l i c a c id may be a c c o m p lish e d e i t h e r byj j 2 34 . j jacid tr e a tm e n t or by n e u r a m in id a s e d i g e s t i o n . In t h i s ! i ! e x p e r im e n t b o t h m ethods were em ployed f o r rem oval of s i a l i c ; 1 a c id from f e t u i n . j ! A cid T r e a tm e n t- - 0 .1% F e t u in in 0 . 0 5 N H2 SO4 s o l u t i o n was in c u b a t e d a t 80° f o r one h o u r. The s o l u t i o n was im m edi a t e l y n e u t r a l i z e d and d i a l y z e d a g a i n s t d i s t i l l e d w a te r f o r | |4 d a y s ch a n g in g th e w a ter f r e q u e n t l y . The d i a l y z e d s o l u t i o n 34 was p a s se d through a column of d e i o n i z i n g r e s i n s as d e s c r ib e d b e f o r e . The d e io n iz e d p r o t e i n was l y o p h i l i z e d and s to r e d at - 2 0 ° . The e x t e n t o f d e s i a l i c i z a t i o n was d e t e r mined by comparing the s i a l i c acid l e f t in d e s i a l i c i z e d f e t u in w ith th a t o f n a t i v e f e t u i n by th e method o f Svenner- jholm ^ or W a r r e n .^ The a c id treatm en t showed alm ost com- I jp lete removal of s i a l i c acid from f e t u i n . I t was r e p o r te d ^ ! jthat such a trea tm en t o f f e t u i n in 0 .0 2 5 N did n o t ; jcause th e r e l e a s e of any o th er su g a rs or amino a c id s , as j Ijudged from paper chromatography o f the c o n c e n tr a te d d i a l y - j i 1 ;sate. j ■ i N euram inidase Treatm ent- - ! g of n a t i v e f e t u i n was j d i s s o l v e d in 30 ml o f 0 .0 5 M sodium a c e t a t e b u f f e r at pH 6.1i and 1000 u n i t s o f n euram inidase (B e h r in g w e rk e , Germany) was added t o the m ix tu r e. One u n i t o f n eu ram in id ase r e l e a s e s 1 ng o f N -a c e ty ln e u r a m in ic a c id in 15 m in u tes at 37° and pH j 5 .5 from a g ly c o p e p t id e s u b s t r a t e in 0 .0 1 M sodium a c e t a t e j t b u f f e r c o n ta in in g 1 g CaCl2 per 1. The m ixtu re was in c u - j jbated a t 37° f o r 24 h o u rs. The s o l u t i o n was d ia ly z e d ; i 1 ja g a in st d i s t i l l e d water a t 4° w ith fr e q u e n t changes o f wa- ! j Iter. I t was d e i o n i z e d , l y o p h i l i z e d and s t o r e d at - 2 0 ° . The | i jcomparison w ith n a t i v e f e t u i n showed a p p ro x im a tely 80 t o 90% jr e le a s e of s i a l i c a cid r e s i d u e s from f e t u i n by neuraminidase; j , I jtrea tm en t. No experim en t to remove the enzyme p r o t e i n was j attem p ted b eca u se the amount of p r o t e in due t o the enzyme j i , i was l e s s than l/lOOO o f the f e t u i n . j }(5) P a r t i a l F r a c t i o n a t i o n o f N a tiv e F e tu in on DEAE-Sephadex | | P r e p a r a t io n o f th e column- - 2 5 g o f DEAE-Sephadex (P h a rm acia, Sweden) was washed w it h 1 l i t e r o f 0 .1 N NaOH and 1 l i t e r o f 0 .1 N HC1 s o l u t i o n s . A fte r e x h a u s t iv e wash in g w ith d i s t i l l e d w a ter th e DEAE-Sephadex was su sp en ded in i ;0.01 M, pH 4 .5 0 Na^PO^ b u f f e r . A ft e r th e e q u i l i b r a t e d DEAE 5-S ep h a d ex had s e t t l e d , 2 g o f washed c e l i t e was added w h ile ; i | jmixing g e n t l y so th a t no b u b b le s were form ed. The p u rp ose iof the c e l i t e was to i n c r e a s e the f l o w r a t e o f th e column. jThe m ix tu r e was packed i n a column o f 4 cm d ia m e te r and j i ; '50 cm h e i g h t . F i r s t a sm a ll amount o f th e b u f f e r was placed; :in th e column and th e t h i c k s l u r y o f DEAE-Sephadex and c e - ! l i t e m ix tu r e was added. ( I f a l a r g e amount o f th e b u f f e r ! was u s e d , c e l i t e s e t t l e d down a t th e b o t to m .) B e fo r e ap p l i c a t i o n o f f e t u i n , 4 1 of the same b u f f e r , 0.01M Na^PO^ a t pH 4 . 5 0 , was p a s se d th rou g h the column in a c o ld room at the f l o w r a t e o f 1 ml per m in u te . I Chrom atography o f f e t u i n - - 3 . 0 0 g o f f e t u i n was d i s - j s o l v e d in 150 ml o f th e b u f f e r . The f e t u i n s o l u t i o n was j jplaced in a d i a l y s i s bag and e q u i l i b r a t e d w ith th e same buf-l jfer a t 4° o v e r n ig h t . The e q u i l i b r a t e d f e t u i n s o l u t i o n was j p i p e t t e d o n to the column and f o llo w e d by th e same b u f f e r . I The column was c o n n e c te d to a g r a d ie n t b u f f e r o f 2 1 o f j 0.05M , pH 4 .5 0 NaH0 PO„ b u f f e r c o n n e c te d t o 2 1 o f 0.01M pH j i | 4 .5 0 NaHoP0„ b u f f e r by a b r id g e . Both b u f f e r s , in 4 1 I \ Z Q t ’ i j f l a s k s , were p la c e d on th e same l e v e l . The c o n c e n t r a t i o n ofj jNaH2 PC>4 i s p r o p o r t i o n a l t o th e volume o f e l u t a n t in t h i s ' 6 6 [method o f m ix in g . When t h e s e b u f f e r s w ere e x h a u s t e d , th e i ! 1 c o n c e n t r a t i o n o f NaH PO. was in c r e a s e d from 0.05M t o 0.09M j 2 4 i u s in g 2 1 e a ch o f th e b u f f e r s , th e n , th e i n c r e a s e from 0.09Mj t o 0 . 1 5 M NaH^PO. was f o l l o w e d . A ll f e t u i n f r a c t i o n s w ere | j ! [washed o u t from th e column by th e end o f th e 0 . 1 5 M Nal^PO^ s b u f f e r a t pH 4 . 5 0 . The f l o w r a t e was a p p r o x im a te ly 1 . 4 ml ; | : ) ! per m in u te . A c t u a l l y the f lo w r a t e g r a d u a l l y d e c r e a s e d [probably due to g r a d u a l p a c k in g o f the DEAE-Sephadex. At ; I ’ i i ; ithe end o f th e ch rom atograp h y , th e h e i g h t o f DEAE-Sephadex ; ! ' I d e c r e a s e d to about h a l f o f th e o r i g i n a l h e i g h t . A ll th e j p r o c e s s e s of chrom atography w ere perform ed a t 4 ° . P r o t e i n j was f o l l o w e d by m easurem ent o f ab so rb a n cy a t 280 mp. The e l u e n t was d i v i d e d i n t o s i x p o r t i o n s . Each p o r t i o n was l y - [ b p h i l i z e d to d e c r e a s e th e v o lu m e, e x h a u s t i v e l y d i a l y z e d [a g a in st d i s t i l l e d w a te r a t 4° w ith f r e q u e n t change o f w a t e r , p a s s e d th rou gh a colum n o f d e i o n i z i n g r e s i n A m b e r lite MB-3, ; l y o p h i l i z e d , and s t o r e d in a f r e e z e r . I t ; i ; I j(6 ) U l t r a c e n t r i f u g a t i o n [ ! j The s e d i m e n t a t i o n c o n s t a n t s o f 1% f e t u i n f r a c t i o n s i i were d e te r m in e d i n sodium p h o sp h a te b u f f e r pH 7 . 2 , 0 . 1 5 ! i i o n i c s t r e n g t h , w it h sodium c h l o r i d e making up 2 /3 o f t h e j i o n i c s t r e n g t h . The in s tr u m e n t em ployed was a S p in c o m odel j E a t a sp eed o f 5 9 ,7 8 0 rpm a t 2 0 . 0 - 2 0 . 6 ° . The se d im e n ta tio n j ; I [c o n sta n ts o b ta in e d i n t h i s b u f f e r was c o r r e c t e d to th e j i ' ' i j i [standard v a lu e o f s on by th e e q u a tio n g i v e n by Svedb erg i I .....................................................................e v , W.........................-...............................................-........- -• ' and P ed erson I I ! S 37 67 2 0 ,w = S„ 7 - ? P20,w> ^20 ,w Cl - V > where ru and ri^ were v i s c o s i t i e s of s o l v e n t and w ater T ,s '20 ,w r e s p e c t i v e l y , v the p a r t i a l s p e c i f i c volume o f p r o t e i n , and pip g and P20 ,w d e n s i t i e s of s o l v e n t and w a te r , r e s p e c t i v e l y .! The nu m erical v a lu e s o f v i s c o s i t i e s and d e n s i t i e s were taken; or e s tim a te d from the v a l u e s g iv e n in l i t e r a t u r e . 6 7 » 1 5 , 6 8 . | : j(7) I s o i o n i c p o in t j \ \ | Most o f the pH measurem ents were done by u s in g a j iBeckman Zerom atic pH m eter or Radiometer t i t r a t o r a t room j ' i item p era tu re. The pH stan dard s o l u t i o n s used were pH 4 .0 0 ofj iBeckman, pH 6 . 8 6 o f Beckman, pH 7 .0 0 o f Beckman, and pH 9 .1 3 o f U. S. N a tio n a l Bureau of S ta n d a r d s. For a c c u r a te m eas- ' urement o f pH, d i s t i l l e d and d e io n iz e d w ater was used in making s o l u t i o n s . ( 8 ) U l t r a v i o l e t A b so rp tio n Measurement j U l t r a v i o l e t a b s o r p tio n was measured by u sin g a G i l - : jford sp e c tr o p h o to m e te r . L y o p h iliz e d f e t u i n was d r ied in an I I . ■ ; jAbderhalden p i s t o l at about 90° by b o i l i n g w ater under r e - j jduced p r e s s u r e to g e th e r w ith ^ 2 ^ 5 ^ o r h o u r s. A d d itio n a l j 5 and 12 hours of d ryin g were f o l l o w e d . A fter 13 hours o f jdrying o n ly s l i g h t changes in w e ig h t were o b ser v ed . The av-j | i ^erage w e ig h t r e d u c tio n a f t e r d ry in g fo r 30 hours was 8 %. i ; . 1 U n f r a c tio n a te d f e t u i n was d r ie d f o r 15 hours under the same j 38 c o n d i t i o n s . Absorbance o f the p r o t e i n in Ho0 was measured | {using q u a rtz c e l l s of 1 cm p ath l e n g t h at room tem p eratu re |at 278 m(i. i i ^ |(9) Chem ical A n a ly s is o f C arbohydrates A n a ly s is f o r s i a l i c a c i d - - S i a l i c a c id was e s tim a te d I 5 4 by the r e s o r c i n o l a ss a y o f Svennerholm and by th e th io b a r -i ) i 65 b i t u r i c a c id a s s a y of Warren. The r e s o r c i n o l a s s a y was plaim ed t o be in d ep en d en t of a ld o h e x o s e and 6 - d e o x y h e x o s e , |and havin g a low stan d ard e r r o r o f * 1 . 0 %, but u n s u i t a b l e | ! i f o r m a te r ia l c o n t a in in g k e t o h e x o s e s , p e n t o s e s , or d eo x y h ex - ; o s e s . S in c e f e t u i n did not c o n t a in t h e s e i n t e r f e r i n g sugar j i com ponents, t h i s method of a s s a y was c h o se n . The p roced u re I was v ery s im p le , i n v o l v i n g o n ly one s t e p and the o p tim a l range o f a s s a y was 10 to 40 pg o f s i a l i c a c id . The c o lo r (developed from f e t u i n was i n t e r p o l a t e d from the graph f o r stan dard s o l u t i o n s of N -a c e ty ln e u r a m in ic a c id (S ig m a ). Ga l a c t o s e , m annose, and g lu c o s e a l s o r e a c t s l i g h t l y w ith t h i s | r e a g e n t and g iv e a maximum a b s o r p tio n a t 580 mp, s i m i l a r to j 69 I i s i a l i c a c i d . No c o r r e c t i o n fo r the c o n t r i b u t i o n by t h e s e j ■ i | i [neutral h e x o s e s was made b e c a u se th e e r r o r was l e s s than l%.j \ \ I The r e s u l t s from the r e s o r c i n o l a ssa y was compared w ith th e | 6 5 t h i o b a r b i t u r i c a s s a y o f Warren. Three sam p les o f each i \ f r a c t i o n were used in t h i s ex p erim en t and the average values! I I are p r e s e n t e d . > The t h i o b a r b i t u r i c a s s a y i n v o l v e s th e p r io r r e l e a s e | j | iof s i a l i c a c id from the g l y c o p r o t e i n , b e c a u se c o lo r j 3 9 jdevelopment occu rs o n ly w ith f r e e s i a l i c a c id . F etu in was jincubated in 0 .1 N at 80° fo r one hour and the r e s u l t ing s o l u t i o n was used d i r e c t l y fo r the a ssa y o f s i a l i c acid.I The t h i o b a r b it u r i c a c id a ssa y i s one o f the most s e n s i t i v e jmethods for the d e te r m in a tio n of s i a l i c a c id . 2 pg o f N- j I i lacetyln eu r aminic acid can be e stim a te d w ith an accuracy o f j 70 7 0 1 %. L-Fucose and 2 -d e o x y r ib o se i n t e r f e r e in t h i s a s s a y . iSince n e it h e r of t h e s e i n t e r f e r i n g su g a rs e x i s t in f e t u i n , jthis method was ch o se n . I t has been rep orted th a t the molar; je x tin c tio n c o e f f i c i e n t of N -g ly c o ly ln e u r a m in ic acid in t h i s ; r e a c t i o n i s 19% low er than th a t o f N -a c e ty ln e u r a m in ic acid.^~j Although f e t u i n was r e p o r te d to c o n ta in about 7% of i t s s i - j | 1 jalic a c id in th e form o f N -g ly c o ly ln e u r a m in ic a c id , no c o r - \ t r e c t i o n was made. The s i a l i c acid was e x p r e sse d in terms ofj N -a c e ty ln e u r a m in ic a c id . S in ce the r e a c t i o n i s claim ed to be q u it e s p e c i f i c , th e r e s u l t i n g v a lu e s o f s i a l i c acid con t e n t in f e t u i n f r a c t i o n s can be taken as a b s o lu te amounts. Three d e te r m in a tio n s were made. Three sam ples o f each fra c-i t io n were used in each d e te r m in a tio n . The r e s u l t s o f b oth i jthe r e s o r c i n o l a ssa y and the t h i o b a r b it u r i c a ss a y seem to j I ! jagree r e a so n a b ly w e l l . I i . i | A n a ly s is fo r h e x o s e — I d e a l l y sp e a k in g , q u a n t i t a t i v e i 1 jhexose d e te r m in a tio n should be done on in d iv id u a l h e x o se s se p a r a ted by column or paper chromatography because most m ethods a v a ila b le at p r e s e n t fo r n e u t r a l hex ose d e ter m in a t i o n are n o n - s p e c i f i c and in t e r f e r e n c e by o th e r su gars such I 40 | : |as s i a l i c a c id or even by p e p t id e seem to o c cu r. S p iro I r e p o r te d t h a t 8.3% of t o t a l h e x o se s was found by anthrone a s s a y on the w hole f e t u i n , w hereas 7.6% of g a l a c t o s e and mannose added to g e th e r when in d iv id u a l h e x o s e s were d e t e r mined a f t e r h y d r o ly s is and chrom atographic s e p a r a tio n on pa- ■ i p e r . S im ila r r e s u l t s were r e p o r te d w ith a ^ -a c id g ly c o p r o - * 37 ; t e in o f human serum; 14.7% hex ose by o r c i n o l r e a c t i o n on |the whole g ly c o p r o t e in and 13.1% by anthrone r e a c t i o n a f t e r ^chromatographic s e p a r a t io n . S in c e the purpose of t h i s e x p e r im e n t was to compare the c o n te n t o f carb oh yd rate in f e - j t 'tu in f r a c t i o n s , the sim p le method o f anthrone r e a c t i o n on the! • i j 'whole f e t u i n s u f f i c e d . N eu tral h e x o se s were e s tim a te d by j 72 ' th e anthrone reagen t of Roe on the w hole g l y c o p r o t e i n , ! ' u s i n g a m ixture o f g a l a c t o s e (Sigma) and mannose ( C a lb io - chera) in a r a t i o o f 1 :1 by w e ig h t as the sta n d a rd . Roe r e p o r te d th a t t h i s r e a g e n t produced c o lo r w ith hexose and 72 6 - d e o x y h e x o s e , w ith no i n t e r f e r e n c e from h exu ron ic a c id . In com parison w ith g a l a c t o s e , th e approxim ate molar c o lo r y i e l d s are: g a l a c t o s e , 100; mannose, 69; g lu c o s e , 162; and : jfu c o se , 6 0 . 69 S in c e g lu c o s e and fu c o s e are n ot p r e s e n t in j f e t u i n , t h i s method sh ould g iv e th e approxim ate c o n te n t of g a l a c t o s e and mannose. Sodium c h lo r id e was r e p o r te d to i n - ; j 7 3 j j t e r f e r e w ith the c o lo r fo r m a tio n . S in c e a l l the s o l u t i o n s were in w ater or in th e same b u f f e r as th e sta n d a r d s , no e r - j r o r was in tr o d u c e d . Hexosamine was r e p o r te d n ot to r e a c t i jw ith t h i s r e a g e n t , but the c o lo r produced by s i a l i c acid was \~~ ' ' 41 i . 6 9 jeq uivalen t to a p p ro x im a tely 8 % of th e g a l a c t o s e . One i j / I Q jamino acid th a t r e a c t s w ith t h i s r e a g e n t i s try p to p h a n . T h e r e fo r e , i f the p e p tid e p o r t io n o f the g ly c o p r o t e in con t a i n s a la r g e amount of try p to p h a n , i t might g iv e a sm all c o n t r ib u t io n to th e a b so r p tio n at 620 mp. F etu in c o n t a in s i P . 8 6 % tryptophan by w e ig h t and 8.7% s i a l i c a c i d . ^ T h ere- ! f o r e , the num erical v a lu e s o b ta in ed by anthrone r ea g e n t on ! jthe whole f e t u i n must be taken w ith c a u t io n . The amount o f ! i n t e r f e r e n c e from p r o t e i n and s i a l i c acid was su b tr a c te d from the hexose e stim a te d on th e whole f e t u i n . In one e x - I I p erim ent th r ee sam ples of each f e t u i n f r a c t i o n were an alyzed ■ I fo r n e u t r a l h e x o se s and the assumed c o n t r i b u t i o n from t r y p - i i ! jtophan and s i a l i c acid was s u b t r a c t e d . In the second exper-j im en t, the e n t i r e p r o c e s s was r e p e a te d u sin g tw ic e as much ■ i f e t u i n in the sam ple. The r e s u l t i n g v a lu e s of h ex ose con t e n t were very c l o s e to each o th er w ith in e x p e r im e n ta l e r r o r . The average of th e two r e s u l t s were taken as th e e x - ! p e r im e n ta l e s t im a t io n of n e u tr a l h e x o s e s in each f r a c t i o n . A n a ly s is fo r hexosam in e--T h e t o t a l hexosam ine con- | net | jtent was determ ined by the Elson-M organ r e a c t i o n m o d ifie d j ! 76 by G att and Berman f o r m i c r o - s c a le a n a l y s i s . In t h i s pro-! cedure the w hole r e a c t i o n i s c a r r ie d out in a s i n g l e v e s s e l so t h a t the d i l u t i o n o f sam ples at v a r io u s s t a g e s i s avoided! fo m inim ize th e e x p e r im e n ta l e r r o r . T h is method i s so s e n s i t i v e th a t 1 to 2 |ag of hexosam ine can be d e t e c t e d and the i i n t e r f e r e n c e from n e u t r a l su g a rs and p e p t id e s can be 42 d e p r e sse d t o a minimum. I t has been r e p o r te d t h a t n e u t r a l h e x o s e s , g lu c u r o n ic a c id and r ib o s e among o th e r m a t e r i a l s in l i v i n g t i s s u e s i n t e r f e r e w ith th e r e a c t i o n f o r h exo sa m in e, ! i 1 and such i n t e r f e r i n g m a t e r i a l s can be s e p a r a te d from hexosa-j j f i 78 ! mine by Dowex 50 column chrom atography a f t e r h y d r o l y s i s . j I | [But in th e same paper th e p e r c e n ta g e of i n t e r f e r e n c e by man-; n o s e , f u c o s e , f r u c t o s e and g a l a c t o s e was shown to be o n ly ‘ 1.5%, i f th e t o t a l w e ig h t of t h e s e n e u t r a l h e x o s e s was th e j # i jsame as th e w e ig h t o f g lu co sa m in e HC1. F urtherm ore, th e pa-i per showed t h a t hexosam ine c o n t e n t in plasm a (p r o b a b ly humanj plasm a) d e c r e a se d by o n ly 3% a f t e r s e p a r a t in g the i n t e r f e r - ! ; j ;ing m a t e r ia l by Dowex 50 column chrom atography. The 3% d if -j f e r e n c e can be c o n s id e r e d w it h in e x p e r im e n ta l e r r o r . One o f th e most im portant s t e p s in hexosam ine e s t i - ; m ation i s th e r e l e a s e o f hexosam ine by h y d r o l y s i s . The h y- ; d r o l y s i s in t h i s method took p la c e in 2 N HC1 at 100° fo r 10' h o u r s. Hexosamine can be d e s tr o y e d to a s i g n i f i c a n t e x t e n t i under the c o n d i t i o n s o f a c id h y d r o l y s i s u s u a l l y employed fori jamino a c id a n a l y s i s , i . e . , 6 N HC1 at 100° f o r 24 to 36 jhours. I f th e N - a c e t y l group i s h y d ro ly z ed b e f o r e g ly c o s id - j i c l in k a g e i s c le a v e d , th e g l y c o s i d e o f hexosam ine becom es ! f\ Q nore r e s i s t a n t t o a c id h y d r o l y s i s . Use of 4N HC1 f o r 4 - 8 ; t jhours a t 1 0 0 ° h as been employed f o r o p tim a l r e l e a s e o f h e x - | 77 bsamine from s e v e r a l g l y c o p r o t e i n s . Ju d ging from the con d i t i o n s o f h y d r o l y s is f o r o th e r g l y c o p r o t e i n s , th e c o n d itio n j ! j jof h y d r o l y s is employed h ere seems a p p r o p r ia te . However, no I .............................43 exp erim en t to f i n d th e o p tim al h y d r o l y s i s c o n d i t i o n f o r f e - j t u i n was p erfo rm ed . i I t has b een r e p o r t e d t h a t the c o lo r d evelop m en t by jany g iv e n amount o f h exosam ine i s s i g n i f i c a n t l y i n f l u e n c e d ■by th e p r e s e n c e o f NaCl in th e s o l u t i o n . ^ T h e r e f o r e , a l l j th e sam ple s o l u t i o n s fo r th e hexosam ine t e s t were in d i s - i • t i l l e d w a t e r . I f th e t e s t sam ple c o n t a in e d NaC l, an app ro- ; Ipriate amount o f NaCl was added to th e sta n d a r d s o l u t i o n . j : Three sa m p le s were used in each d e t e r m in a t io n u s i n g i D -g lu c o sa m in e HC1 (C a lb io ch em ) as a s ta n d a r d . G att and j ! i ! yA Berman showed th a t th e molar a b s o r p t io n o f g lu c o sa m in e was; ■ I a p p r o x im a te ly 1 0 % la r g e r than t h a t of g a la c t o s a m in e in th e ir ! i a ss a y m ethod. T h e r e f o r e , f o r a c c u r a t e a s s e s s m e n t o f t o t a l \ h e x o sa m in es in f e t u i n f r a c t i o n s an a p p r o p r ia t e m ix tu r e of g lu c o sa m in e and g a la c t o s a m in e must be u sed f o r th e s ta n d a r d . However, th e amount o f g a la c t o s a m in e in f e t u i n was r e p o r t e d t o be o n ly l / 8 of g lu c o s a m in e .^ ^ Thus, th e e r r o r cau sed by ; u s in g g lu c o sa m in e a lo n e would be a p p r o x im a te ly 1 %, w h ich can; be c o n s id e r e d w i t h i n e x p e r im e n ta l e r r o r . A t o t a l o f fo u r | j d e t e r m i n a t i o n s was perform ed and t h e a v e ra g e v a lu e s were | p r e s e n t e d as th e h ex o sa m in e c o n t e n t o f th e f e t u i n f r a c t i o n s . ! } j ! 1(10) T o ta l P e p t id e I ; The p e r c e n t a g e o f p e p t i d e m o ie ty was d eter m in e d by -j o A th e method o f Lowry e t a l ., w hich em ployed F o l i n Ts p h en o l r e a g e n t f o l l o w i n g p r e tr e a tm e n t o f th e p e p t i d e w ith an a l k a - i 1 : > l i n e copper s o l u t i o n . An aqueous b o v in e serum album in i : " .““."". ' ................... ' ....44 (C albiochem ) s o l u t i o n was used as the sta n d a r d . The p r o c e dure i s sim p le and d oes n o t in v o lv e h y d r o l y s i s . The s e n s i - j ; i t i v i t y o f th e r e a c t i o n i s v e r y h ig h , but th e r e a c t i o n has i * . ! [the d is a d v a n ta g e th a t the i n t e n s i t y of the c o lo r v a r i e s f o r j 79 ! d i f f e r e n t p r o t e i n s . The r e a c t i o n seems to in v o lv e n o t | i i ! i 'only th e t y r o s i n e and tryp to p h a n r e s i d u e s , but a ls o th e s e - j jquence o f c e r t a i n amino a cid r e s i d u e s h avin g s id e c h a in j [fu n ction al groups such as h i s t i d i n e , a r g in in e , and g lu ta m ic | i ‘ 80 a c id . B e s id e s th e d i f f e r e n c e s in amino a c id s e q u e n c e s , ! | | th e i n t e r f e r e n c e from th e carb oh yd rate m o ie ty which seems to; [ j be lo c a t e d on the outer s u r fa c e o f the g ly c o p r o t e in m olecule; ; I 81 i must be c o n s id e r e d . G o tt s c h a lk th in k s th a t c a l i b r a t i o n j w ith the p r o t e i n component from th e g ly c o p r o t e in i s n o t j f e a s i b l e . T h e r e fo r e , the num erical v a lu e o f the p e p t id e ob~; t a in e d by t h i s method u s in g bovin e serum albumin as a s t a n d ard are by no means a b s o l u t e . They were e x p r e s s e d in terms o f th e w e ig h t o f bovine serum album in and can be used o n ly f o r com p arative p u rp o se. j The b i u r e t typ e r e a c t i o n fo r e s t i m a t i o n of p e p t id e j | i j Q2 ‘ Was n o t t r i e d becau se o f i t s i n s e n s i t i v i t y . A p p lic a t io n [ i 1 of the n in h y d r in r e a c t i o n to th e h y d r o ly s a te o f p r o t e i n has j a b a s i s fo r q u a n t i t a t i v e e s t i m a t i o n o f p e p t i d e , but g l y c o - i S i . . . . . . ! p r o t e i n c o n t a in s h exosam in es and s i a l i c a c id s b e s i d e s amide j : 831 groups of p e p t id e w hich c o n t r ib u t e to th e c o lo r fo r m a tio n . j i i . j T h e r e fo r e , t h i s method i s u n s u it a b le f o r g l y c o p r o t e i n s . For ithe same r e a s o n , e s t i m a t i o n o f p e p tid e component o f 4 5 ; g l y c o p r o t e i n from n i t r o g e n c o n t e n t by th e m icro K je ld a h l 84 method i s n ot f e a s i b l e u n l e s s th e c o r r e c t i o n f o r n i t r o g e n groups in th e c a r b o h y d r a te m o ie ty and amide i s a c c u r a t e l y j a p p l ie d . T h e r e f o r e , no o th e r method of e s t i m a t i o n o f th e p e p t id e p o r t i o n of f e t u i n f r a c t i o n s was u s e d . However, th e jappr o x im a tio n o f p e p t id e m o ie ty by a d d i t i o n o f amino a c id I 1 • , jre sid u e s was t r i e d . I f a v e ry c a r e f u l amino a c id a n a l y s i s j ! i s perform ed w ith an a c c u r a t e l y d e ter m in e d q u a n t i t y o f p r o - j t t e i n , t h i s method g i v e s an a b s o l u t e v a lu e fo r p e p t i d e . The ! j | jsum o f amino a c id r e s i d u e s p r e s e n t e d in t h i s work, h ow ever, j I s o n ly an a p p ro x im a tio n b e c a u se the amount o f t h r e o n i n e , j s e r i n e , c y s t i n e , and try p to p h a n are e i t h e r r o u g h ly e stim a ted : or ta k en from th e l i t e r a t u r e . ( 1 1 ) Amino A cid A n a ly s is * Amino a c id a n a l y s e s were perform ed by io n -e x c h a n g e chrom atography by t h e t e c h n iq u e o f Moore e t a l . ^ In th e e n t i r e p r o c e s s o f amino a c id a n a l y s i s , th e m ost im p o r ta n t j istep i s th e h y d r o l y s i s where v a r io u s k in d s o f d e s t r u c t i o n of| i 85 ' jamino a c i d s o c c u r . J a c o b so n has p o in te d out t h a t sm a ll j I amounts o f m e t a l s , p r e s e n t as i m p u r i t i e s in th e HC1, a f f e c t th e r a t e o f d e c o m p o s it io n o f amino a c id s d u rin g h y d r o l y s i s due t o c a t a l y s i s o f th e d e a m in a tio n r e a c t i o n . Sm ith and j 86 S t o c k e l l s u g g e s t e d c a r r y in g out th e h y d r o l y s i s in j *The amino a c id a n a l y s i s was k i n d l y perform ed by Mr. N i c h o l a s S a v e d r a . ! i j b o r o s ilic a t e g l a s s tub es which were evacu ated and s e a le d b e - ifore h e a tin g in an oven. In t h i s way, c o l o r l e s s or p a le y e llo w h y d r o ly s a te s o l u t i o n s were o b ta in e d . In our e x p e r i ment P yrex ( b o r o s i l i c a t e ) g l a s s tu b es w ith screw caps were u sed . 2 ml of aqueous f e t u i n s o l u t i o n o f known c o n c en tr a - I jtion was p la c e d in a t e s t tube and l y o p h i l i z e d . Into the i i i it e s t tube th a t co n ta in ed dry f e t u i n 2 ml o f c o n sta n t b o i l i n g jHCl was added, and the air was f lu s h e d out by blow ing w ith n it r o g e n . The t e s t tube was incubated in a c o n s ta n t temper-: | j jature oven at 100° f o r 24, 48 and 72 h o u rs. By th e end of i i 24 hours o f in c u b a tio n , a l l t e s t tu b es showed q u it e e x t e n s i v e humin fo r m a tio n . I t was not c e r t a i n how much o f the j i humin was due t o p o s s i b l e a ir o x id a t io n o f amino a c id s i n s t e a d of carbohydrate d e c o m p o sitio n . The primary cause o f humin fo rm a tio n seems to a r is e from the carbohydrate in the g ly c o p r o t e in in th e h y d r o ly s is ; media. In the o r d in a ry c o n d it io n s of amino a c id h y d r o ly s is ,: i . e . , 6 N HC1 at 100° fo r 24 hou rs, h e x o se s are degraded to ! 1 s ; i ihydroxymethyl f u r f u r a l and some oth er d e g r a d a tiv e prod- j : t ■ 8 7 jucts, which may or may n ot r e a c t w ith amino a c id s l i b e r - | gg ated from p e p tid e c h a in s . According to E a sto e and E astoe e x c e s s i v e d e c o m p o sitio n o f amino a c id s i s n o t l i k e l y t o take, j p la c e . In order to m inim ize the amino a c id d e s t r u c t io n dur-J ;ing acid h y d r o ly s is o f p r o t e in in th e p r e sen ce o f carbohy- i j 79 . • id rate, E astoe s u g g e s t s a high d i l u t i o n o f g ly c o p r o t e in m |the h y d r o ly s a te so th a t th e c o l l i s i o n s betw een the 47 carbohydrate d e g r a d a tiv e p r o d u cts and amino a c id s are m in i- 81 m ized. G o tts c h a lk th in k s 0.1% g ly c o p r o t e in i s p r a c t i c a l , ; i althou gh an even lower c o n c e n tr a tio n o f 0 . 0 1 % g ly c o p r o t e in 89 was s u c c e s s f u l l y employed by P u s t z a i and Morgan. In our ex p e rim en t, f e t u i n c o n c e n tr a t io n s of 0 . 1 to 0 . 2 % in c o n sta n t jb o ilin g HC1 were u sed . No attem pt was made t o d eterm in e the; jammonia, and c o r r e l a t e i t w ith amino acid d e s t r u c t i o n b e - I ! I ; jcause of the u n c e r t a i n t y in the amount o f ammonia c o n t r i b - | ; juted by s i a l i c acid and hexosam ine. However, the amount of ! i j jammonia in e a ch h y d r o l y s is t e s t tube was a p p ro xim ately on e- j I j h a l f o f th e t o t a l e q u i v a l e n t s o f a s p a r t i c a cid and g lu ta m ic j jacid i n d ic a t in g ra th er a l i m i t e d d e s t r u c t i o n . j A fter h y d r o l y s i s , th e s o l u t i o n was d r ie d in a d e s i - j c a to r over 1* 2 ^ 5 311 ^ reduced p r e s s u r e . 2 ml of c i t r a t e b u f f e r , which i s c u sto m a r ily used fo r the amino acid a n a ly z e r , was added, and the s o l u t i o n was f i l t e r e d through a M il l ip o r e f i l t e r , to remove dark c o lo r e d humin p r e c i p i t a t e . A p o r t io n i ;of the f i l t r a t e was used fo r amino acid a n a l y s i s . A Techni-i con au tom atic amino acid a n a ly z e r w ith a column o f 150 cm in! ! ' ! jlength and 0.63 cm in d iam eter was u sed . I t was packed w ith i s ! I uniform s i z e beads of Dowex 50 io n -ex ch a n g e r e s i n c a ll e d ! Chromobeads Type B. The chromatography was run a t 60° fo r 22 hours u s in g a pH g r a d ie n t . The c o lo r d ev e lo p ed by ninhy-l jdrin was read a t 570 mp and 440 mq. The l a t t e r was used in i i I j c a lc u la t in g p r o l i n e . No i n t e r n a l standard was u s e d . The j ; i icolumn was c a l i b r a t e d w ith a standard m ixture of amino acids! ! i 48 o b ta in e d from C albiochem . Assuming l i n e a r p r o p o r t i o n a l i t y o f th e area under th e p eak s w ith the amount of amino a c id , { and c a l c u l a t i n g th e p r o p o r t i o n a l i t y c o n s t a n t s from the sta n d a r d , th e amount of each amino a cid was c a l c u l a t e d by jthe c o n v e n tio n a l method g iv e n in T ech n icon manual. ; i ; S in c e i t has lo n g been known t h a t c y s t i n e , t r y p t o - j 79 |phan and m e th io n in e decompose in acid h y d r o l y s i s , th e y iwere n o t d eter m in e d in t h i s a n a l y s i s . S in c e c y s t i n e i s par-: | 1 i t i a l l y o x id iz e d t o c y s t e i c a c id in th e c o n d i t i o n s o f acid I j h y d r o ly s is , i t i s u s u a l l y c o n v e r te d t o c y s t e i c a c id by p e r - j iformic a c id and th e amount of c y s t e i c a c id i s d eterm in ed by 90 :paper chrom atography a f t e r a c id h y d r o l y s i s . Even in t h i s :p rocess a l o s s of 1 0 % c y s t e i n e du rin g th e o x i d a t i o n o f p r o - 17 ! t e i n must be c o r r e c t e d . M eth io n in e i s o x id iz e d d u rin g hy-| d r o l y s i s , e s p e c i a l l y when i t i s c a r r ie d o u t w ith an a c c e s s o f a ir, to m eth io n in e s u l f o x i d e and e v e n t u a l l y to m ethionine; s u l f o n e .* ^ I t can be c o n v e r te d t o m eth io n in e s u l f o n e by 92 p e r fo r m ic a c id , or to m eth io n in e s u l f o x i d e a lo n e by 92 and c^om a-togra-phic s e p a r a t io n and q u a n t i t a t i o n can j !be f o l lo w e d . Tryptophan i s p a r t i c u l a r l y u n s t a b le in a c id hy-i | d r o l y s i s . In the p r e se n c e o f c a r b o h y d r a te s i t s d e s t r u c t i o n i 94 i s c o m p le te . However, ta k in g advantage o f i t s a b s o r p tio n ; o f u l t r a v i o l e t l i g h t , i t i s u s u a l l y d eterm in ed by s p e c t r o - ; i 114 115 ^photometry w ith o u t h y d r o l y s i s . ’ S in c e th e pu rp ose o f jamino a c id a n a l y s i s was t o l o c a t e d i f f e r e n c e s in amino a c id jco m p osition among f e t u i n f r a c t i o n s , and n o t t o p r e s e n t th e 49 d e t a i l e d c o m p o s itio n s o f them , th e d e t e r m in a t io n o f t h e s e amino a c i d s were n o t p erform ed . i i I ( 1 2 ) P r o t e i n F i n g e r p r i n t i n g j The s o - c a l l e d f i n g e r p r i n t i n g o f p r o t e i n i s the iden-| i I t i f i c a t i o n of v a r io u s p e p t i d e s by a c o m b in a tio n o f paper j I ; |chrom atography and e l e c t r o p h o r e s i s in two d im e n s io n s a f t e r c le a v in g th e p r o t e i n a t c e r t a i n s p e c i f i c p o i n t s by enzym ic j h y d r o l y s i s . In m ost c a s e s t r y p s i n h a s b een em ployed b e c a u se o f i t s narrow s p e c i f i c i t y . T r y p sin a c t s on th e bonds l i n k - j ing the c a r b o x y l group of a b a s i c amino a c i d , i . e . , a r g in in ei I ;and l y s i n e , t o th e amino group of anoth er amino a c id or to j » i Ithe h y d r o x y l group o f an a l c o h o l . ' j The s t u d y o f amino a c id a n a l y s i s o f f e t u i n showed j th a t th e d i f f e r e n c e s in amino a c id c o m p o s itio n among f e t u i n f r a c t i o n s were r a t h e r s m a l l . The method o f f i n g e r p r i n t i n g I ! was a p p lie d t o d e t e c t any o b v io u s d i f f e r e n c e s in t r y p t i c d i-. g e s t i o n m ix tu r e s from two f r a c t i o n s o f f e t u i n in th e hope •that i t m igh t com plim ent or r e i n f o r c e the r e s u l t s o f amino lacid a n a l y s i s . I . - j j The p r o t e i n f i n g e r p r i n t i n g i n v o l v e s two s t e p s r th e I j f i r s t s t e p i s d i g e s t i o n o f p r o t e i n w ith a s p e c i f i c p r o t e o l y t i c enzym e, and th e secon d i s two d im e n s io n a l ch rom atogra-j phy and e l e c t r o p h o r e s i s on p a p e r . For th e d i g e s t i o n o f f e - j i i i 9 7 i t u i n th e p r o c e d u r e d e s c r i b e d by B anaszak e t a l . was prim a r i l y f o l lo w e d u s in g a p H - s t a t . 2 .0 ml o f a p p r o x im a te ly 1% f e t u i n s o l u t i o n in w a te r was h e a te d at 85° f o r 15 m in u te s to 50 d e n a tu r e t h e p r o t e i n . The c l e a r s o l u t i o n tu rn ed c lo u d y by t h i s h e a t tr e a t m e n t i n d i c a t i n g d e n a t u r a t i o n of f e t u i n . I t was m ixed w it h 2 . 0 ml o f 0 . 0 2 M C a C ^ s o l u t i o n . C a C ^ was j 98 c la im e d t o p r o t e c t t r y p s i n from a u t o d i g e s t i o n . To p r e v e n t b a c t e r i a l grow th 0 . 0 5 ml o f 5 0 ,0 0 0 u n i t s p er ml of p e n i c i l - i ! i i jlin G p o ta s iu m (S q u ib b ) and d ih y d r o s t r e p t o m y c in ( S q u ib b ) , | 50 m g/m l, s o l u t i o n was added to th e d e n a tu r e d f e t u i n s o l u - j jtio n . The s o l u t i o n was p la c e d in a t i t r a t o r c e l l o f a R a d i- i i jometer T i t r a t o r Type-TTTlc c o n n e c te d w it h T it r a g r a p h Type- |SBRC2. The t i t r a t i o n c e l l was eq u ip p ed w it h a g l a s s j a c k e t | i j 'which was c o n n e c te d t o a t h e r m o s t a t e (H aake) f o r tem peraturej ^ r e g u la tio n . The s o l u t i o n and th e e l e c t r o d e s (R ad iom eter j g l a s s e l e c t r o d e G2222B and R ad iom eter KC1 r e f e r e n c e e l e c tr o d e K 4 1 1 2 ) w ere a d j u s t e d and e q u i l i b r a t e d to pH 8 .2 0 and ! 37° f o r 10 to 15 m in u te s . A f te r e q u i l i b r a t i o n , 0 .1 ml of a f r e s h t r y p s i n C W orthington) s o l u t i o n ( 1 . 5 m g/m l) was added and t h e p H - s t a t was im m e d ia te ly s t a r t e d . As th e d i g e s t i o n p r o c e e d s th e pH of th e d i g e s t i o n m ix tu r e d e c r e a s e s from th e . o p tim a l pH o f 8 . 2 f o r p r o t e o l y t i c a c t i v i t y o f t r y p s i n . i i {T h erefo re, to keep the pH a t 8 . 2 , 0 .0 2 N KOH s o l u t i o n was I ; i : j c o n t in u o u s ly added to th e c e l l . The secon d a l i q u o t o f 0 .1 j ; Iml o f f r e s h t r y p s i n was added 24 h o u r s l a t e r , and th e t h i r d j I | j a liq u o t a t 48 h o u r s l a t e r . T h e < d i g e s t i o n c o n tin u e d f o r 72 I ! i jhours u n t i l th e KOH u p tak e d im in is h e d . The t o t a l amount o f ) ; t r y p s i n added was a p p r o x im a te ly l / 5 0 o f th e f e t u i n by ; i jw e ig h t. A lth o u gh i t was recommended to remove a l l th e s a l t j 51 iin th e d i g e s t i o n m ixtu re b e fo r e a p p l i c a t i o n o f the p e p t id e i . 99 .'mixture on paper f o r chrom atography, d i a l y s i s was n o t per-: formed b ec a u se some s m a lle r p e p t i d e s m ight be l o s t in th e d i a l y s a t e . The h y d r o ly s a t e was l y o p h i l i z e d and th e dry p ep t i d e m ix tu re was s t o r e d at 4° u n t i l f u r t h e r u s e . | The second s te p in t h i s e x p erim en t i n v o l v e s two d i - jmentional chrom atography and e l e c t r o p h o r e s i s . The p roced u re ' 99 i ^described in d e t a i l by Katz e t a l . was p r im a r ily fo llo w e d .! i ; :2 mg o f l y o p h i l i z e d p e p t id e d i s s o l v e d in 0 .0 4 ml o f w ater ; j i Was a p p lie d t o Whatman No. 3M M paper ( 1 8 - 1 / 2 x 2 2 - 1 / 2 i n . ) . j I i A sm a ll a l i q u o t was a p p lie d at a tim e so th a t th e s p o t did j ! I not spread over a l a r g e a r ea . The maximum s i z e o f th e sp o t I ;did not e x c e e d more than 0 . 8 cm in d ia m e te r . A fte r com- ! p l e t e l y d r y in g th e s p o t by b lo w in g c o ld a ir the paper was p la c e d in a chrom atography box and e q u i l i b r a t e d w ith the s o l v e n t fo r 20 to 30 m in u te s . The s o l v e n t was alw ays f r e s h l y made o f th e upper phase o f th e m ix tu re o f N -b u ta n o l- i la c e tic a c id -w a te r ( 4 : 1 : 5 ) . A f te r e q u i l i b r a t i o n of th e paper! i n th e box th e s o l v e n t was poured i n t o th e trou gh through a i jhole in the upper l i d , and d e sc e n d in g chrom atography was run f o r 16 hou rs a t room te m p e r a tu r e . The chrom atographed w et i j ; paper was d r ie d under hood f o r 3 to 4 hou rs and was m o is - i j f itened w ith 0 .0 7 5 M sodium a c e t a t e b u f f e r a t pH 4 . 2 by i sp rea d in g i t w it h a p i p e t t e toward th e o r g in l i n e . The same i b u ff e r was a ls o p la c e d in a l u c i t e e l e c t r o p h o r e s i s tank ' 99 ( S e r v o n u c l e a r ) , s i m i l a r t o the one d e s c r ib e d by Katz e t a l. j 52 The e l e c t r o p h o r e s i s was run a t a p p r o x im a te ly 5° f o r 75 min u t e s a t 2000 v o l t s and 190 m amp. in th e d i r e c t i o n p e r p e n d i c u l a r t o th e ch ro m atogra p h ic d i r e c t i o n . The n e g a t i v e e l e c t r o d e was i n s e r t e d t o th e lo w er tr o u g h . Most p e p t i d e s m ig ra te d toward the n e g a t i v e e l e c t r o d e . The d r ie d paper was isprayed w it h 0.3% n in h y d r in s o l u t i o n in N -b u ta n o l as d e - j j ;sc r ib e d by S t a h l to i d e n t i f y s p o t s f o r v a r io u s p e p t i d e s . | |A ll th e h a n d lin g o f paper was done w ith g l o v e s . i In an attem p t to i d e n t i f y c a rb o h y d r a te s p o t s on th e ; : t p a p er, s e v e r a l d i f f e r e n t c o lo r d e v e lo p in g r e a g e n t s were \ I t r ie d , b u t no s p o t s were i d e n t i f i a b l e due to th e s t r o n g j ^background. j (1 3 ) T i t r a t i o n i The e x p e r im e n ta l p u rp ose o f t i t r a t i o n i s to f i n d the: . inumber o f H+ io n s bound t o a p r o t e i n m o le c u le a t a c e r t a i n pH. The number o f bound H+ io n s can be o b ta in e d by su b- i t r a c t i n g f r e e H io n s from added H io n s in a known volume i |of p r o t e i n s o l u t i o n . The amount o f added H+ io n i s r e a d i l y j o b ta in e d by th e v o lu m e t r ic method u s in g a stan d a rd s o l u t i o n j and a c a l i b r a t e d m i c r o - s y r i n g e . The amount o f f r e e H+ io n j i s o b ta in e d by m easu rin g th e pH o f a p r o t e i n s o l u t i o n w i t h a g l a s s e l e c t r o d e . Thus, th e a c c u r a t e d e t e r m in a t io n o f pH j j becom es v e r y im p o rtan t in t h i s e x p e r im e n t. The c a l c u l a t i o n j i j |o f th e amoutit o f H+ io n from th e pH v a lu e i n v o l v e s th e f o l - j L . .. ioi * I jlow m g e q u a t io n : | 53 pH = - l o g xn + - l o g y H+ + Ej + G ( 9 ) H ( 2 . 303RT/F) where m^* and yh+ r e p r e s e n t the m olar c o n c e n t r a t i o n and a c t i v i t y c o e f f i c i e n t o f H+ i o n . E . r e p r e s e n t s th e d i f f e r e n c e ! i I in th e l i q u i d j u n c t i o n p o t e n t i a l b etw een th e s a l t b r id g e and i th e unknown s o l u t i o n and t h a t b e tw e en th e s a l t b r id g e and | i th e pH s t a n d a r d , R, T and F r e p r e s e n t the gas c o n s t a n t , tem-? p e r a t u r e and F arad y, r e s p e c t i v e l y . G i s th e g l a s s e l e c t r o d e j {e r r o r . I n s t e a d o f d e t e r m in in g i n d i v i d u a l - l o g Y„+, E . and j H J jG, th e y a r e lumped t o g e t h e r f o r p r a c t i c a l p u rp ose as f o l - ; lo w s : j - l o g y V = - l o g v + w * G ( 1 0 ) j I t i s assumed th a t th e v a lu e o f Yt t+ i n a p r o t e i n s o l u t i o n i s H th e same as i t i s i n th e s o l v e n t s o l u t i o n c o n t a i n i n g no pro-i t e i n a t t h e same i o n i c s t r e n g t h and pH. G i s m a in ly p r o du ced by Na+ io n c o n c e n t r a t i o n ( t h e w e ll-k n o w n Na e r r o r in a l k a l i n e s o l u t i o n s ) . S o , i f K+ io n i s u s e d , i n s t e a d o f Na+ io n , G becomes n e g l i g i b l e . By com bining e q u a t i o n s 9 and 10 • {the f o l l o w i n g e q u a t io n i s produced:'*'^'*' pH = - l o g M r + - l o g Y *H+ ( 1 1 ) | With th e u se o f t h e e q u a t i o n 1 1 , - l o g Y’jj+ i s f i r s t d e te r m in e d a t e a c h pH by t i t r a t i n g th e s o l v e n t s o l u t i o n , i . e . , 0.16M KC1. S e c o n d ly , w ith th e u se o f th e exp erim en tal] j v a l u e s o f - l o g y *«+ a t e a ch pH t h e c o n c e n t r a t i o n s o f H+ io n , j W iM H+, i s c a l c u l a t e d a t e a c h pH by t i t r a t i n g th e p r o t e i n s o l u - i t i o n i n t h e same s o l v e n t . The d i f f e r e n c e b e tw e en th e amount I I 54 o f added H+ io n s and the f r e e H+ io n s r e p r e s e n ts the H+ io n s bound t o the p r o te in m o le c u le s . The measurement o f pH and the d e te r m in a tio n of a cid j I or base added were performed w ith Radiometer T it r a t o r Type j TTTlc connected w ith Radiometer T it r ig r a p h Type SBR2c. The j i isyrin ge was c a lib r a t e d by w eighing d i s t i l l e d w ater th a t was ! ^ejected from th e s y r in g e . The pH was measured w ith a g l a s s j j ; jele ctr o d e G2222B and KC1 r e f e r e n c e e le c t r o d e K 4112 in a | ; I g la s s -ja c k e te d t i t r a t i o n c e l l to keep the tem perature con- ; j s t a n t . As standard s o l u t i o n s Beckman and Braun pH standards^ I I land 0.5M KH p a th a la te (Baker and Adamson) d is s o l v e d in d i s - j t i l l e d , d e io n iz e d , b o i l e d water f o r pH 4 .0 0 , Beckman and I iBraun pH stan d ard s for pH 7 .0 0 , and 0.01M sodium t e t r a b o r - j ated (U. S. N a tio n a l Bureau of Stan dards) d i s s o l v e d in the above-m entioned water fo r pH 9 .1 8 were u sed . The t i t r a t i o n s t a r t e d from the i s o i o n i c p o in t of f e t u i n s o l u t i o n in 0.16M KC1. F i r s t the e l e c t r o d e was a d ju ste d w ith a pH standard s o l u t i o n at 2 5 °, and th e n , t i t r a t i o n was begun in e i t h e r d i r e c t i o n using 0.1M HC1 s o l u t i o n or 0.1M KOH s o l u t i o n . After; the co m p letio n of t i t r a t i o n the pH was measured w ith th e | same pH stan d ard . I f the reading was o f f by more than 0 .0 2 ; pH u n i t , the d a ta were d is c a r d e d . The autom atic t i t r a t i o n method d e sc r ib e d in the Radiometer manual did not g iv e con- i s i s t e n t r e s u l t s because o f d r i f t i n g o f the pH meter n e e d le w h ile the s o l u t i o n was s t i r r e d w ith th e m agnetic b a r. T h e r e fo r e , fo r the a c cu ra te work th e sy r in g e was op erated by i 55 i jhand, e q u i l i b r a t e d f o r 3 to 5 m inutes by m ixing the s o l u t i o n i jwith the m agnetic b a r , and pH was measured w h ile th e bar was sto p p e d . Wet n it r o g e n gas was p a ssed over the s u r fa c e of | s o l u t i o n in t i t r a t i o n c e l l when b a s ic r e g io n was t i t r a t e d . P r o t e in s o l u t i o n was made by d i s s o l v i n g l y o p h i l i z e d f e t u i n j jin 0.16M KC1 s o l u t i o n u sin g d i s t i l l e d , d e io n i z e d , b o i l e d wa-i |ter and i t s c o n c e n tr a tio n was determ ined by u sin g a s p e c t r o photom eter assuming th e E i s equal to 4 . 1 0 . ^ i 1cm, 278 mu I | The H+ or OH” io n s bound per p r o t e in m o lec u le was c a l c u l a t e d • i |on the assum ption th a t the m o lecu la r w e ig h t o f f e t u i n was j 4 8 ,5 0 0 . A ll pH measurements were done at 2 5 .0 ° which was ■ i m ain tain ed by a Haake c i r c u l a t i n g th e r m o s ta t. The HC1 solu -l t i o n used fo r t i t r a t i o n was d i l u t e d from c o n c e n tr a te d HC1 (M a llin k r o d t) to a p p ro x im a tely IN and sta n d a r d iz e d by r e a c t i o n w it h sodium t e t r a b o r a t e ( J . T. Baker) to m ethyl red end i n O p o i n t . Three d e te r m in a tio n s were made and th e average v a lu e was taken as th e HC1 c o n c e n t r a t io n . The KOH s o l u t i o n was made from 2N KOH s o l u t i o n (Braun) s a tu r a t e d w ith 102 pa(OH) 2 and sta n d a r d iz e d w ith p o ta ssiu m hydrogen p h tha- jlate (Baker and Adamson) u sin g p h e n o lp h th a le in i n d i c a t o r . jThe C02 f r e e KOH so prepared c o n ta in s at most 1 or 2 mg o f | jcalcium per l i t e r a f t e r d i l u t i o n t o 0.1N KOH. The top of j i ithe b u re t was p r o t e c t e d from atm osp heric C02 by B a r iu lim e , a 1 j CO~ a b so rb er . Three d e te r m in a tio n s were made and the a v e r- i I 2 | age v a lu e was taken as the KOH c o n c e n t r a t io n . j 56 (1 4 ) V i s c o s i t y The d e te r m in a tio n o f v i s c o s i t y i n v o l v e s the m easure ment o f f lo w tim e f o r a d e f i n i t e amount o f s o l u t i o n t h a t j p a s s e s through a v is c o m e te r c a p i l l a r y . The v is c o m e t e r s j :used in t h i s e x p e rim en t were th e O stwald type U -tu b e c a p i l - j j j jlary v is c o m e t e r s c a l l e d th e Cannon-Manning se m i-m icro v i s - ! 103 c o m e te r s. S in c e t h i s typ e of v is c o m e te r r e q u i r e s o n ly j T ml of s o l u t i o n f o r the measurement of v i s c o s i t y , i s sim p le in i t s o p e r a t io n , can be c le a n e d e a s i l y a f t e r each u s e , i s i ' ■ ^ r e la t iv e ly in e x p e n s iv e , and, f i n a l l y , g i v e s s u f f i c i e n t l y ac-; c u r a te r e s u l t s f o r the purpose o f our e x p e r im e n t, i t was chosen in our e x p e r im e n t. The v is c o m e te r was c le a n e d imme- j d i a t e l y b e fo r e ea ch u se w ith d i s t i l l e d w ater and r in s e d nu merous tim e s w ith d i s t i l l e d w ater th a t was f i l t e r e d through medium s i n t e r e d g l a s s . The c a p i l l a r y was d r ie d w ith a ir which had been f i l t e r e d through s i n t e r e d - g l a s s . From tim e to tim e the v is c o m e t e r was c le a n e d w ith H^SO^- ^ 2 0 ^ 2 0 7 s o l u t i o n . The flo w tim e o f each s o l u t i o n was measured s i x to le ig h t tim e s to 0 .0 1 second w ith a s to p w atch (H e s s e r ) and j j th e average v a lu e was ta k e n . The s o l u t i o n in th e v isc o m e te r was a llo w e d to come to the tem p erature e q u ilib r iu m f o r 5 to 7 m in u tes and p a sse d once through the c a p i l l a r y b e fo r e any m easurem ents were made. S in c e th e v i s c o s i t y o f a l i q u i d s o l u t i o n i s trem en- idously in f lu e n c e d by tem p eratu re f l u c t u a t i o n , a h i g h ly s e n s i t i v e th e r m o s ta t was assem bled in our la b o r a t o r y w ith a jmercury column w h ich had f o u r e x t r a arms e x t e n d in g downward 1 ! jfrom t h e s i d e o f t h e main c e n t r a l colum n. I t c o n t a in e d 2 kg jof m ercury and had a l a r g e s u r f a c e area in c o n t a c t w it h the i jwater c i r c u l a t i n g i n the b a t h . With t h i s m ercury column th e 1 jbath te m p e r a tu r e was k e p t c o n s t a n t w i t h i n 0 .0 0 5 ° C. The ! ! -tem perature was k e p t a t 2 5 .0 ° u s in g a therm om eter t h a t was ; ; c a lib r a t e d w i t h a U. S . N a t io n a l Bureau o f S tan d ard c e r t i f i e d th erm om eter. The m ec h a n ica l s t i r r e r was s e c u r e l y a t t a c h e d on a c o m p le t e ly d i f f e r e n t sta n d s o t h a t th e v i b r a t i o n iof th e s t i r r i n g motor d id n o t d i s t u r b th e v is c o m e t e r h o ld e r .' ! The v i s c o m e t e r h o ld e r was a b u r e t clamp t i g h t l y s e c u r e d on 1 i ;an aluminum l a t t i c e above th e w ater b a t h . A g l a s s t u b in g j ■clamped on the h o ld e r was v e r t i c a l l y a l i g n e d w it h a s t r i n g | h a n g in g th r o u g h th e g l a s s t u b i n g . The h o ld e r th u s a l ig n e d was n e v er lo o s e n e d or ta k e n o f f and th e su b s e q u e n t v i s c o s i t y ' d e t e r m i n a t i o n s w ere alw ays done on th e same p o s i t i o n and a lig n m e n t so t h a t th e e r r o r due t o m is a lig n m e n t was m i n i m ize d . S i n c e f e t u i n i s a f a i r l y s m a ll compact m o le c u le o f I 1 5 . . |48,500 m o le c u la r w e i g h t , th e N ew ton ian f l o w o f t h i s s o l u - ; i 1 jtion th ro u g h th e v i s c o m e t e r c a p i l l a r y was assum ed. Newton- ; jian f lo w o f a l i q u i d i s e x p r e s s e d in th e f o l l o w i n g e q u a - j t i o n : ^ ! S = n |a (12) where £ = th e p e r p e n d ic u la r f o r c e per a r e a , c a l l e d s h e a r in g j 58 j . * = the r a t e o f sh ear ( v e l o c i t y g r a d i e n t ) . I f th e p e r p e n d ic u la r f o r c e i n c r e a s e s , th e v e l o c i t y i n c r e a s e s a c c o r d in g ly , but th e v i s c o s i t y r| i s alw ays c o n s t a n t in Newton ia n f lo w . j The v is c o m e te r was c a l i b r a t e d a c c o r d in g to th e f o l - llow ing e q u a t io n 4 ^ * u s in g a s o l u t i o n o f w hich th e v i s c o s i t y i !was a c c u r a t e l y known: I • t i = a p t - (1 3 ) j | Iwhere a = th e v is c o m e te r c o n s t a n t ! j | p = the d e n s i t y o f th e s o l u t i o n i j ( t = the f lo w time j | ' |3 = th e k i n e t i c e n e r g y c o r r e c t i o n f a c t o r j ; j The sta n d a rd s o l u t i o n s were made from c r y s t a l i n e KC1 ( J . T. | Baker) and s u c r o s e ( M a l l i n c k r o d t ) . The v a l u e s o f v i s c o s i - j t i e s and d e n s i t i e s used f o r c a l i b r a t i o n w ere: B P . (c p ) g/m l | w ater 0 .8 9 0 3 00 0.9970® ° 0.16M KC1 0 . 8 9 0 0 ° ’ 1 . 0 0 5 7 ^ 0.02M s u c r o s e 0-^906089 0 .9 9 9 7 8° j ! i |The a v a lu e c a l c u l a t e d from w ater and 0.16M KC1 s o l u t i o n wa4 ! 3 .8 9 5 x lO - - 3 cp ml per g s e c and th e £ 3 v a lu e was n e g l i g i b l e . ; The time w it h w a te r was 2 2 9 .3 se c o n d s . In order t o check | th e e x p e r im e n ta l v a l u e s of v i s c o s i t i e s , th e v i s c o s i t y o f j >0.02M s u c r o s e s o l u t i o n was d eterm in ed by u s in g th e e x p e r i - I i i t i jmental v a lu e o f a and compared w it h the v i s c o s i t y r e p o r te d j Iby J o n e s and T a l l e y . 1 0 4 The v i s c o s i t y found came t o the i l i t e r a t u r e v a lu e w i t h i n 0.11% . T h e r e f o r e , th e e x p e r im e n t a l i v a l u e s o f v i s c o s i t y o b ta in e d w ith t h i s a p p a r a tu s were a s sumed w i t h i n t h a t range o f a c c u r a c y . j ( i The f e t u i n s o l u t i o n s were made by d i s s o l v i n g l y o - j i p h i l i z e d f e t u i n in pH 7 . 5 b u f f e r made o f 0.12M KC1 and ! 0.0152M a t i o n i c s t r e n g t h o f 0 . 1 6 . The f e t u i n c o n - ! c e n t r a t i o n was d e ter m in e d by a b s o r p t io n a t 278 m|i assum ing IE t o be 4 . 1 0 . The c o n c e n t r a t i o n o f f e t u i n d e - 1 cm, 278 mp i s i a l i c i z e d by n e u r a m in id a se was a l s o d e ter m in e d by s p e c t r o - > ip h otom eter, b u t t h e amount o f s i a l i c a c id t h a t was r e l e a s e d j j j Ifrom th e n a t i v e f e t u i n was s u b t r a c t e d in c a l c u l a t i n g th e c o n c e n t r a t i o n . The d i l u t e f e t u i n s o l u t i o n s w ere made by d i — ! i l u t i o n of th e c o n c e n t r a t e d s to c k s o l u t i o n by adding th e same| ;b u ffer w ith a v o l m e t r ic p i p e t t e . A ll th e p r o t e i n s o l u t i o n s j were f i l t e r e d th r o u g h a M i l l i p o r e f i l t e r im m e d ia te ly p r e c e d in g t h e i r i n t r o d u c t i o n i n t o a v i s c o m e t e r . A ll th e w ater u sed f o r making s o l u t i o n s and r i n s i n g th e v is c o m e t e r was d i s t i l l e d , d e i o n i z e d , and f i l t e r e d th ro u gh medium s i n t e r e d - g l a s s . i 1 ; 1 | The d e n s i t i e s o f th e s o l u t i o n s w ere n ot d e ter m in e d • i in t h i s e x p e r im e n t. I n s t e a d , th e c o n v e r s io n o f k in e m a tic v i s c o s i t y to the a b s o l u t e v i s c o s i t y was perform ed u s in g th e j I e q u a t io n g i v e n by T a n fo r d 1 0 5 assum ing t h e p a r t i a l s p e c i f i c 'volume, v , o f f e t u i n and d e s i a l i c i z e d f e t u i n to be 0 .6 9 6 ml iper g" *" ^ and th e d e n s i t y o f th e s o l v e n t 1 . 0 0 3. : 60 jwhere [ v] = the i n t r i n s i c k in e m a t ic v i s c o s i t y w hich i s d e - Ifined as i ; } | Iv ] = lim ( t - t p) C l5 ) | c-*° t oc I 1 Where t = th e f lo w tim e f o r s o l u t i o n ! I j 1 t Q = th e f lo w tim e f o r s o l v e n t I c = the p r o t e i n c o n c e n t r a t i o n j ;Thus, th e k in e m a t ic i n t r i n s i c v i s c o s i t y [ v] i s e x t r a p o l a t e d jfrom th e k in e m a t ic v i s c o s i t i e s of s e v e r a l s o l u t i o n s o f d i f - ! i ife r e n t p r o t e i n c o n c e n t r a t i o n to z e r o c o n c e n t r a t i o n . The in-: i t r i n s i c v i s c o s i t y i s , t h e n , o b ta in e d from th e k in e m a tic v is-; t i i c o s i t y by the u se of e q u a t io n 1 4 . I : | (.15) O p t ic a l R o ta to r y D i s p e r s i o n j O p t i c a l r o t a t i o n o f f e t u i n and d e s i a l i c i z e d f e t u i n was m easured w ith a J a s c o ORD U 5 -5 a u to m a tic r e c o r d in g s p e c t r o p o la r im e t e r at room tem p era tu re over th e wave l e n g t h range o f 210 to 260 m p.* A p p r o x im a te ly 0.3% g l y c o p r o t e i n s o l u t i o n s in the c e l l o f 1 mm p ath l e n g t h ( i n a chamber Mi ere n i t r o g e n was c i r c u l a t e d ) were m easured . The g lycop ro-: jte in s were d i s s o l v e d in 0.0615M fO^PO^ b u f f e r a t pH 7 .5 0 and: i o n i c s t r e n g t h o f 0 . 1 4 . A fte r th e r o t a t o r y m easurem ent th e c o n c e n t r a t i o n of p e p t i d e p o r t i o n o f th e g l y c o p r b t e i n was a l s o e s t im a t e d by th e p h en o l method of Lowry1 2 6 and by th e ! *The o p e r a t io n o f the s p e c t r o p o l a r i m e t e r was p e r - Iformed a t Durram In str u m e n t C o r p ., P a lo A l t o , C a l i f o r n i a . f " " " ' " “ ' 61 74 micro b iu r e t method which agreed w ith the c o n c e n tr a tio n from u l t r a v i o l e t absorbance w ith in 5%. The o p t i c a l r o t a t i o n 1 o f the s o lv e n t was measured fo r each s o l u t i o n as c o n tr o l and; I the d i f f e r e n c e was taken as the o p t i c a l r o t a t i o n caused by j i | the p r o t e in s o l u t i o n . The o p t ic a l r o t a t i o n of th e glycopro-i t t e i n in a d e n a tu r a tin g s o lv e n t was n o t measured, but taken I 3 4 5 3 jfrom the l i t e r a t u r e . ’ The h e l i c a l co n ten t of the p ro- J Itein was e stim a te d by the method of Simmons e t a l . The j 'per cen t h e l i c a l con ten t of f e t u i n and d e s i a l i c i z e d f e t u i n ; j was c a lc u la t e d by sim ple l i n e a r i n t e r p o l a t i o n . j i i i i I i » • i ! i 1 i j RESULTS j | | (1 ) I s o l a t i o n o f f e t u i n j i 1 j The f e t u i n prepared by th e a l c o h o l- m e t a l io n method ' jwas claim ed to be homogeneous by u l t r a c e n t r i f u g a t i o n * and ( e l e c t r o p h o r e s i s over the range of pH 1 .1 to 1 1 . 2 . ^ In o r - j i jder to check th e hom ogen eity o f our f e t u i n p r e p a r a t io n i t ! ; jwas c e n t r i f u g e d in d i s t i l l e d w ater and in an a c e t a t e b u f f e r j ; f at pH 4 .5 0 ( F i g . 2 ) . The pH 4 .5 0 was chosen b e c a u se th e | h e t e r o g e n e i t y i s most c l e a r l y a m p lif ie d at t h i s p o in t by j s t a r c h g e l e l e c t r o p h o r e s i s . ' As th e u l t r a c e n t r i f u g a l p a t t e r n s show, th e f e t u i n ! p r e p a r a tio n at pH 4 .5 0 e x h i b i t s o n ly one peak which appears (sym m etrical. The f e t u i n d i s s o l v e d i n d i s t i l l e d w a te r , how e v e r , shows a s l i g h t l y skewed p e a k , which becom es more obvi-, ous w ith tim e , and c o n s id e r a b le s p r e a d in g . These e f f e c t s m igh t r e s u l t from p r o t e i n - p r o t e i n i n t e r a c t i o n , p o s s i b l y ag- i i g r e g a t i o n , a t z e r o i o n i c s t r e n g t h . ; The f e t u i n s o l u t i o n became s l i g h t l y t u r b id a f t e r p a s s in g through th e d e i o n i z i n g r e s i n a t th e l a s t s t e p s o f j | i *The kind h e lp from Mr. J . DeGroot in o p e r a tin g th e j ( u l t r a c e n t r i f u g e i s d e e p ly a p p r e c ia t e d . j I I j ; F ig* 2 . - - U l t r a c e n t r i f u g a t i o n p a t t e r n of f e t u i n a t j |pH 4 . 5 in a c e t a t e b u f f e r Cthe lo w e r f i g u r e ) and in pu re wa- j ite r Cthe upper f i g u r e ) a t 2 0 ° . The a c e t a t e b u f f e r was made j |o f 0 . 0 8 M KC1 and .0 .0 2 M p o t a s s iu m a c e t a t e a d j u s t e d t o pH j ;4 .5 w ith g l a c i a l a c e t i c a c i d . The p i c t u r e s w ere ta k e n (A) 1 iat 30 ra in ., (B ) 60 m i n ., (C) 90 m i n i , CD)120 m i n ., and CE) '180 m i n ., a f t e r r e a c h in g th e sp eed o f 5 9 ,7 8 0 rpm. j '..~......... ~.64 ithe p r e p a r a tiv e p ro c ed u r e. A lthough no e x p erim en ts were un dertak en to determ ine th e ca u se of t h i s phenomenon, i t was | ; jsu sp ected th a t th e t u r b i d i t y r e s u l t e d from a g g r e g a tio n o f | I s jfe tu in m o le c u le s due to th e high c o n c e n tr a t io n in pure wa- I j I jter . In f a c t , the t u r b i d i t y d isa p p e a r e d when a sm all amount i j jof KC1 was added i n d i c a t i n g th e r e a s o n a b le n e s s o f the a s- | ; jsumption. |(2) S ta r c h g e l v e r t i c a l e l e c t r o p h o r e s i s ! The f e t u i n p r e p a r a tio n was e x t e n s i v e l y s tu d ie d by istarch g e l v e r t i c a l e l e c t r o p h o r e s i s at s e v e r a l pH v a l u e s . j i At pH 8 . 6 in b o r a te b u f f e r a s i n g l e broad band and a sm all i f a s t e r m ig r a tin g band which m ight be a contam inant were ob- I s e r v e d . At pH 6 .3 in c a c o ly d a te b u f f e r and at pH 5 .5 in a c e t a t e b u ffe r s im ila r bands as th o s e at pH 8 . 6 were ob s e r v e d . Although the band was broad, no s e p a r a t io n in t o d i s t i n c t components was o b se r v e d . At pH 4 .5 in a c e t a t e b u f f e r , the s e p a r a t io n i n t o s e v e r a l bands was se en and at pH 4 .2 th e s e p a r a t io n i n t o e ig h t bands was q u it e c le a r ( P ig . j3 ).* At t h i s pH no p r o t e in band m igrated toward the n eg a - | jtiv e e l e c t r o d e . The f a s t e s t and the s lo w e s t bands had l e s s ! i ' ' ibound dye than the bands in th e m iddle p o r t i o n . Bands 3, 4 ,: ! ' I \5 s t a i n most s t r o n g l y . No e l e c t r o p h o r e s i s was performed be-j ! i ilow pH 4 . 2 , where some co n fo rm a tio n a l changes in the j I I I *The s k i l l f u l p h o to g ra p h ic s e r v i c e s were k in d ly _ p r o - jvided by Mr. D. M il l e r of th e S a lk I n s t i t u t e fo r B i o l o g i c a l jS tu d ie s . _________ _ __ ___________ ____________ _ _____________ 65; I I I i : ! ! | F i g . 3 . - - S t a r c h 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 a t pH j |4. 2 ( a c e t a t e b u f f e r ) and 5° f o r 16 h o u r s . j ! i i ! 1 . P o o le d f e t a l c a l f serum 2 . E th a n o l f r a c t i o n a t e d f e t u i n 3. F r a c t io n 6 4 . F r a c t io n 5 5. F r a c t io n 4 6 . F r a c t io n 3 7 . F r a c t io n 2 8 . E th a n o l f r a c t i o n a t e d f e t u i n 9 . Ammonium s u l f a t e p r e c i p i t a t e d f e t u i n ( j } F ig u r e 3 ! 67 i ! - 11 Q ■molecule m ight o c cu r. For i n s t a n c e , Yang and F o ste r have i ireported th a t bovine serum albumin s t a r t s s w e llin g b elow pH j ; |4 .0 , a lth ou gh the albumin i s s t r u c t u r a l l y d i f f e r e n t from fe-j i Q O * |t u i n . Schmid e t a l . r ep o rted the b e s t s e p a r a t io n o f hu- ' I i I {man a - a c id g l y c o p r o t e in components in a pH 2 .9 p h osp hate ! 1 | b u f f e r . They a ls o r e p o r te d th a t th e g ly c o p r o t e in did n o t r e l e a s e any s i a l i c a c id by in c u b a tin g at pH 2 .7 and 4° fo r {24 h o u rs. At 25° o n ly 0.1% o f s i a l i c acid was s p l i t o f f a f te r s i x h o u rs, and 1% a f t e r 24 h o u rs. In com parison w ith {th eir ex p e rim en t, the c o n d it io n s used in t h i s experim en t fori s t a r c h g e l e l e c t r o p h o r e s i s a t pH 4 .2 and a p p ro x im a tely 5 to I j ;10° f o r 16 hours seem v e r y m ild . Thus, i t seems r e a s o n a b le J ' i to assume th a t no chem ical change occu rred during the e l e c - j t r o p h o r e s i s . A sample of f e t u i n prepared by (NH^^SO^ p r e c i p i t a - : t i o n was o b tain ed from a d i f f e r e n t la b o r a t o r y and t e s t e d on s ta r c h g e l e l e c t r o p h o r e s i s under the same c o n d i t i o n s . T h is sample showed th e c h a r a c t e r i s t i c m ic r o h e t e r o g e n e it y o f the 1 f e t u i n prepared in t h i s la b o r a t o r y . S in ce most f e t u i n i s prepared from p ooled s e r a from : s e v e r a l f e t a l c a l v e s , i t may be argued th a t the m ic r o h e te r o g e n e it y i s due to the d i f f e r e n c e s in i n d iv id u a l c a l v e s . As {shown in F ig . 4 , in d iv i d u a l f e t a l s e r a a ls o showed the same i {bands as the f e t u i n prepared from p o o led s e r a . F ive s e r a i | {were c o l l e c t e d from f i v e d i f f e r e n t s i z e d f e t a l c a l v e s ra n g - j I ' | |ing from 30 to 100 cm in le n g t h from head to t a i l . A ll f i v e P ig . 4 . - - S t a r c h g e l e l e c t r o p h o r e s i s p a t te r n at .2 8 ( a c e t a t e b u f f e r ) and 5° fo r 16 h o u rs. 1 . Serum m ixtu re o f f i v e f e t u s e s ( 2 , 3 , 4 , 5 ) 2 . Serum from 100 cm f e t u s 3. Serum from 75 cm f e t u s 4. Serum from 45 cm f e t u s 5. Serum from 30 cm f e t u s 6 . Serum from 30 cm f e t u s 7. E thanol p r e c i p i t a t e d f e t u i n 8 . P o o le d f e t a l c a l f serum (Hyland L ab .) F ig u r e 4 ! 70 jsera showed the e i g h t bands c h a r a c t e r i s t i c of f e t u i n . In jone o f the s e r a , taken from the 1 0 0 cm f e t u s , a f a s t moving component appeared which was ab sen t in the oth er fo u r s e r a , j 1 I This f a s t moving component was a l s o p r e s e n t in th e f e t u i n j I i jprepared in t h i s la b o r a t o r y . Although no fu r th e r exam ina- j Ition of t h i s component was perform ed, t h i s might be a con ta m in a tin g p r o t e i n t h a t a r i s e s in the f e t a l c a l f serum at a jla ter s t a g e o f growth s in c e t h i s component was found on ly in kh e o l d e s t f e t u s . | In e l e c t r o p h o r e s i s the charge d e n s i t y of the m ole- I I c u le i s g e n e r a l ly the d e c i s i v e f a c t o r in m ig r a tio n in the ' e l e c t r i c a l f i e l d . S in c e s i a l i c a cid i s p e r ip h e r a l and has j a n e g a t i v e c h a r g e, i t i n f l u e n c e s the m ig r a tio n of f e t u i n on j e l e c t r o p h o r e s i s . I f th e s i a l i c a c id i s r e s p o n s ib l e fo r the d i f f e r e n t bands of f e t u i n , then d e s i a l i c i z a t i o n would t h e o r e t i c a l l y merge a l l the bands i n t o one band. As shown in P ig . 5, a few bands did d isa p p e a r b u t th r e e d i s t i n c t bands were e v id e n t on s t a r c h g e l e l e c t r o p h o r e s i s a f t e r trea tm en t of f e t u i n w ith n eu ram in id ase which removed 90.6% of th e s i - | ; a lic a c id r e s i d u e s . The n e u r a m in id a s e -tr e a te d f e t u i n b e s t S e p a r a te d at pH 5 .5 0 p rob ab ly b eca u se i t s i s o i o n i c p o in t I s h ifte d upward near pH 5 . 4 . In c o n t r a s t to t h i s f i n d i n g , Ithe t r e a t e d f e t u i n w h ich l o s t 97.2% s i a l i c acid r e s i - ; i ■ I 'dues showed one broad band. The broad band seems to c o r r e - ; ; ! : I ispond to the t h r e e bands o f the n e u r a m in id a s e -tr e a te d f e t u i n las shown in P ig . 6 . For com parison, the ^ S O ^ - t r e a t e d | P i g . 5 . - - S t a r c h 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 o f h e u r a m i n i d a s e - t r e a t e d f e t u i n Cl and 2 ) in a c e t a t e b u f f e r a t pH 5 .5 0 and 5° f o r 16 hou rs a t 7 v o l t s per cm. 90.6% o f th e i s i a l i c a c id was removed by th e enzym e. ! F ig . 6 . - - S t a r c h 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 fo r t r e a t e d f e t u i n (1 and 2) w h ich l o s t 97.2% o f th e s i - la lic a c id and n e u r a m in id a s e - t r e a t e d f e t u i n (3 and 4) w h ich |lo s t 90.6% of th e s i a l i c a c id a t pH 6 .0 ( c a c o l y d a t e b u f f e r ) , 5° and 7 v o l t s p er cm f o r 16 h o u r s . 73 f e t u i n and n e u r a m i n i d a s e - t r e a t e d f e t u i n were exam ined on th e (same s t a r c h g e l a t pH 6 .0 0 ( c a c o l y d a t e b u f f e r ) . A lth o u g h i t h a s b e e n c la im e d t h a t no s t r u c t u r a l ch a n g es occu r by a c id ; 1 5 (tre a tm en t f o r d e s i a l i c i z a t i o n , i t seem s l i k e l y t h a t some j ( i r r e v e r s i b l e c o n f o r m a t io n a l c h a n g e s o ccu r d u r in g th e incuba-j i t io n i n 0 .0 2 5 N H^SO^ at 80° f o r one h o u r, j With f e t u i n d e n a tu r e d in u r e a , w h ich was t r e a t e d (with 2 -m e r c a p to e t h a n o l t o r u p tu r e th e d i s u l f i d e b o n d s, th e ( e l e c t r o p h o r e s i s p a t t e r n showed one lo n g band ( F i g . 7 ) . Sim - Iple u r e a d e n a tu r e d f e t u i n a l s o showed one lo n g band i n a I s t a r c h g e l t h a t c o n ta in e d 6 M u r e a ( F i g . 8 ) . i ((3 ) Immunochemical A n a l y s i s ! The im m unochem ical a n a l y s i s was perform ed w it h th e r a b b i t a n t i - f e t u i n serum o b ta in e d a t th e l a s t b l e e d i n g a f t e r a p p r o x im a te ly s i x w eeks from th e f i r s t i n t r a v e n o u s i n j e c t i o n o f f e t u i n i n s a l i n e s o l u t i o n . A q u a n t i t a t i v e a n a l y s i s was n o t perform ed to f i n d th e t i t e r o f th e serum , b u t th e u n d i - ; ( lu t e d serum produ ced a c l e a r l y d i s t i n g u i s h a b l e p r e c i p i t i n ; i | l i n e a g a i n s t f e t u i n in agar g e l . j j \ \ ; ( a ) Im m u n o d iffu sio n When th e a n t i - f e t u i n serum was a llo w e d t o d i f f u s e ‘ a g a i n s t pure f e t u i n i n 1 % agar g e l , a t h i n l i n e ap p eared onj I th e seco n d d a y . T h is t h i n l i n e g r a d u a l l y became broad and j I a n o th e r t h i n l i n e app eared w h ich became much c l e a r e r i n i it h r e e t o f o u r d a y s ( F i g . 9 ) . I t seem s t h e r e are a t l e a s t 74 i 1 2 3 4 5 I \ j j F ig - 7 . - - S t a r c h 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 f o r 2 - im ercaptoethanol t r e a t e d f e t u i n (1 to 5) a t pH 4 .2 ( a c e t a t e ib u f f e r ) , 5° and 7 v o l t s per cm f o r 16 h o u r s. The s t a r c h g e l jcontained 0 .0 4 m o l e s / l of 2 - m e r c a p to e th a n o l. 75 i w\ 2 _ 3 4 i t t | F ig - 8 . - - 6 M u rea s ta r c h g e l e l e c t r o p h o r e s i s p a tt e r n Ifor 2 - m e c a p t o e t h a n o l- tr e a te d f e t u i n ( 1 and 2 ) and u r e a - jtreated f e t u i n (3 and 4) at pH 4 .2 ( a c e t a t e b u f f e r ) , 5° and 7 v o l t s per cm f o r 15 h ou rs. j F i g . 9 . --I m m u n o d iffu s io n p a t t e r n f o r e th a n o l p r e c i p i t a t e d f e t u i n ( 2 ) , F r a c t io n 2 C l ) , F r a c t io n 3 ( 3 ) , F r a c t io n |4 ( 4 ) , F r a c t io n 5 ( 5 ) , and ammonium p r e c i p i t a t e d f e t u i n ( 6 ) ja g a in st a n t i - f e t u i n r a b b i t serum ( t h e c e n t e r ) . two k in d s o f a n tib o d y formed a g a i n s t th e f e t u i n prepared in jth is l a b o r a t o r y . S im ila r p r e c i p i t i n l i n e s were a l s o found jwhen the a n ti-se r u m was r e a c te d w ith the (N H ^ ^ S O ^ -p r e c ip i- I jtated f e t u i n ( F i g . 9 ) . S in c e no p r e c i p i t i n l i n e d e v e lo p ed | jin th e b u ffe r s o l u t i o n , the two l i n e s seem to r e s u l t from a I i ; gen u in e a n t ig e n - a n t ib o d y r e a c t i o n . (b ) Im m u n o e le ctr o p h o re sis As shown in F ig . 1 0 , th e e l e c t r o p h o r e t i c a l l y s e p a r a te d f e t u i n in s ta r c h g e l was r e a c t e d w ith a n t i-s e r u m . All; the f r a c t i o n s r e a c t w ith a n ti-s e r u m and form e s s e n t i a l l y a s i n g l e p r e c i p i t i n l i n e . T h is r e s u l t i n d i c a t e s th a t ea ch j f r a c t i o n i s im m unochem ically i d e n t i c a l to each oth er and in -l d i s t i n g u i s h a b l e . As shown by th e c o n t r o l w hich was a s i m i - ; la r s t a r c h b lo c k w ith o u t f e t u i n , no p r e c i p i t i n l i n e was formed i n d i c a t i n g t h a t a n o n - s p e c i f i c r e a c t i o n did n o t take p l a c e . S in c e th e r e were a t l e a s t two p r e c i p i t i n l i n e s shown on the d ou b le d i f f u s i o n agar g e l p l a t e , th e r e s u l t of immun- o e l e c t r o p h o r e s i s seems c o n t r a d i c t o r y . The c o n t r a d i c t i o n may be e x p la in e d by the f a c t th a t th e e l e c t r o p h o r e s i s was p e r formed on s t a r c h g e l at pH 4 . 2 . The minor component, r e - I S p o n s ib le f o r the second t h in l i n e , m ight have rem ained in ih e s l o t . A ls o , i t co u ld have m ig ra ted and d i s t r i b u t e d to o t h i n l y to show any v i s i b l e p r e c i p i t i n l i n e . (4 ) P a r t i a l f r a c t i o n a t i o n of f e t u i n on DEAE-Sephadex The f r a c t i o n a t i o n o f p r o t e i n s by DEAE-Sephadex i s 78 i | j j ; j P i g . 1 0 . - - I m m u n o e le c t r o p h o r e s is p a t t e r n f o r f e t u i n j ( 3 ) . The e l e c t r o p h o r e s i s was perform ed i n th e d i r e c t i o n jshown by th e arrow toward th e p o s i t i v e e l e c t r o d e i n s t a r c h igel m edia a t pH 4 . 2 ( a c e t a t e b u f f e r ) . The c o n t r o l s t a r c h b lo c k ( 1 ) c o n t a in e d no f e t u i n . The m id d le tr o u g h ( 2 ) was ' f i l l e d w i t h a n t i - f e t u i n r a b b i t serum. t 79 m o stly dependent upon the charge on th e p r o t e i n m o le c u le s . j |At pH 4 . 5 th e f u n c t io n a l group o f DEAE-Sephadex i s io n iz e d c o m p le te ly and has a p o s i t i v e charge to which n e g a t i v e l y - j i charged p r o t e i n m o le c u le s can be adsorb ed . As th e p h osp h ate I jc o n c e n tr a tio n i s in c r e a s e d th e e l e c t r o s t a t i c i n t e r a c t i o n be-! ! I ; « jtween the p r o t e i n and the DEAE groups i s d im in is h e d . There-! f o r e , th e f i r s t p r o t e in f r a c t i o n to e l u t e w i l l p ro b a b ly have |the h i g h e s t i s o i o n i c p o in t . With DEAE-Sephadex, th e Sepha- dex p a r t i s m erely u sed as the su p p o rtin g media fo r the 'fu n c tio n a l groups ra th er than fo r g e l f i l t r a t i o n . ; The a lc o h o l p r e c i p i t a t e d f e t u i n was f r a c t i o n a t e d on ; DEAE-Sephadex by the method d e s c r ib e d e a r l i e r . The e l u a t e j from th e column was c o l l e c t e d a t the r a t e o f 1 ml per m inute and the u l t r a v i o l e t a b s o r p tio n was measured at 278 mp ( F i g . ; 1 1 ) . The f i r s t peak was e lu t e d from the column w ith 0 .0 9 M NaH2 P04 . At 0 .1 5 M NaH2 P0 4 p r a c t i c a l l y a l l the f e t u i n i n trod uced i n t o the column was e l u t e d . The r e c o v e r y o f the p r o t e i n was 95%. The e lu a t e was im m ed iately l y o p h i l i z e d , d e i o n i z e d , and l y o p h i l i z e d fo r s t o r a g e a t - 2 0 ° . Although o n ly two o b v io u s peaks were o b ta in e d , t h e y ! iwere d iv id e d in to s i x p o r t io n s as i n d ic a t e d in F ig . 1 1 . i ; S in c e th e f i r s t and l a s t f r a c t i o n s were sm a ll in q u a n t i t y , j the chem ical a n a ly s e s were perform ed m ain ly on f r a c t i o n s 2 , ! 3, 4 and 5. As shown in F ig . 3, each f r a c t i o n c o n ta in e d twcj i !or th r e e bands on s ta r c h g e l e l e c t r o p h o r e s i s . F r a c tio n 2 j jcon tained the slow m ig ra tin g bands, and F r a c tio n 5 th e f a s t 1 i F ig - 1 1 . - - P a r t i a l f r a c t i o n a t i o n o f f e t u i n on DEAE- I jSephadex was perform ed a t pH 4 . 5 , and 4 ° . The o p t i c a l dens-j ;ity was measured at 278 mp, and p l o t t e d a g a i n s t the e l u e n t j jvolume. The c o n c e n t r a t io n of Na^PO^ in c r e a s e d from 0 . 0 4 M j ito 0 .1 5 M as i n d ic a t e d at th e to p o f the f i g u r e . The c o l - ! jle c te d e lu e n t was d iv id e d i n t o s i x p o r t i o n s . j 0.04-0.0^ 0.09-0.15 0.09 0.15 M N o H o P0„ O .D . 0.2 0.1 500 TUBE NUMBER 1500; 1000 Figure 11 m : 82 m ig ra tin g bands. The bands in each f e t u i n f r a c t i o n c o r r e - i isponded to bands in u n f r a c t io n a te d f e t u i n , and in d ic a t e d j th a t the m o lecu lar s t r u c t u r e was not a l t e r e d by the f r a c - I | t i o n a t i o n p r o ced u re. I f the s lo w e s t band i s termed A and | the f a s t e s t H, F r a c tio n 2 i s seen to c o n ta in bands A, B, C, | >and D; F r a c tio n 3 co n ta in e d D and E. F r a c tio n 4 con ta in ed IE and F. F r a c tio n 5 c o n ta in ed F and G, and F r a c tio n 6 con- | j ^ Itained G and H. The bands in F r a c tio n 6 were not c l e a r l y ^segregated, probab ly because the i s o i o n i c p o in t i s s t i l l i j Isomewhat lower than the pH of the b u f f e r . S e p a r a tio n o f the' j i bands i s u s u a l l y most d i s t i n c t near th e i s o i o n i c pH. No j q u a n t i t a t i v e measurement o f th e amount of p r o t e in in each band in th e s e f r a c t i o n s were performed becau se t a i l i n g o f the f a s t moving bands were always ob served . Judging from the area under the curve in F ig . 11 , however, i t appears th a t bands D, E, and F c e r t a i n l y predom inate. F r a c t io n s 2, 3, 4 and 5 (1 and 6 were not a v a ila b le )! were r e a c te d w ith a n t i - f e t u i n r a b b it serum. As shown in F ig . 9, t h e ir p r e c i p i t i n l i n e s merged to g e th e r w ith the ! } : jneighboring l i n e s i n d ic a t in g immunochemical hom ogeneity. I | iHowever , in the u n f r a c t io n a t e d f e t u i n and F r a c tio n 2, an- jother l i n e appeared. F a in t but s im ila r l i n e s were observed j jin F r a c tio n s 3 and 5 in another agar g e l d i f f u s i o n e x p e r i - j ; i ;ment which i s not shown. T h e r e fo r e , i t seems th a t th e r e i s j i . i :at l e a s t another immunochemically d i s t i n g u i s h a b l e p r o t e in j js p e c ie s b e s i d e s the main f r a c t i o n o f f e t u i n . This component |seems to be c o n c e n tr a te d in F r a c tio n 2 more than any o th e r , i jalthough i t was not r e v e a le d by im m u n e le c tr o p h o r e s is. This j p r e c i p i t in l i n e m ight be due to a component o f f e t u i n , b e - I jcause a very c le a r l i n e was a l s o e x h ib it e d by (NH4 ) 2 SC >4- j p r e c ip it a t e d f e t u i n . A l t e r n a t e l y , i t co u ld a r is e from a j | | jcontaminant p r e s e n t in b oth f e t u i n p r e p a r a tio n s . j K5) P h y s ic a l ch em ical p r o p e r t i e s o f f e t u i n f r a c t i o n s (a ) U l t r a c e n t r i f u g a t i o n | The u l t r a c e n t r i f u g a t i o n of 1% s o l u t i o n s of F r a c tio n s ’ i i ;2, 3, 4 and 5 were perform ed at pH 7 . 2 . The c a l c u l a t e d sed-j lim en ta tio n c o n s t a n t s were c o n v erted to the standard e x p r e s - | 1 y I s io n of s . A ll fo u r f r a c t i o n s , as w e ll as u n f r a c t io n - 2 0 , w ’ ated f e t u i n , came w ith in th e e x p e c te d range of 2 .7 t o 3 .0 S,i where S i s th e Svedberg u n it (T a b le 4 ) . No c le a r d i f f e r e n c es among f r a c t i o n s were r e v e a le d by u l t r a c e n t r i f u g a t i o n . ' E v id e n t ly u l t r a c e n t r i f u g a t i o n i s not adequate to show the s u b t le m ic r o h e te r o g e n e it y of f e t u i n . S in c e o n ly one d e t e r - ; m in atio n fo r each f r a c t i o n ( e x c e p t tw ice f o r th e u n f r a c t i o n - jated f e t u i n ) was perform ed, a stan dard e rr o r was n ot c a lc u - j jla te d . But the u l t r a c e n t r i f u g e u s u a l l y g i v e s a 5% stan dard j jerror fo r th e s v a lu e . | (b ) I s o i o n i c p o in t j | The i s o i o n i c p o i n t s of the f e t u i n f r a c t i o n s (0.3% I ic o n c e n tr a tio n ) p r e s e n te d in T able 5 were n ot c o r r e c te d fo r ; ! «t /"V 1 - 9 jexcess o f H+ i o n s . They d e c r e a s e from 4 -3 2 to 4 .0 3 in th e order of e l u t i o n f rom DEAE- S ephadex colum n. When t h e j 84 TABLE 4 SEDIMENTATION COEFFICIENT OF FETUIN FRACTIONS jThe 1% p r o t e i n s o l u t i o n was c e n t r i f u g e d in NaH2 P04 -NaCl buf-j jfer a t pH 7 . 2 . The i o n i c s t r e n g t h was 0 .1 5 o f which 2 /3 was i Jmade o f NaCl. The tem p eratu re was k ep t a t 20 t o 2 0 .5 ° fo r ] j |the e n t i r e e x p e r im e n t. i i i F r a c t io n s s 1 ^ 1 ________________________________2 0 ,w | 2 2 .7 ! 3 2 .7 4 2 .7 5 3 .0 u n f r a c t i o n a t e d f e t u i n 2 .9 u n f r a c t i o n a t e d f e t u i n 2 .7 TABLE 5 8 $ ISOTONIC POINT OF FEIUIN FRACTIONS pH Meter F r a c t io n 2 3 4 5 u n f r a c t i onated TT IT Beckman Z ero m a tic I I (pH) 4 .3 0 4 .2 3 4 .0 4 4 . 0 2 4 .0 5 ) ) 4 .0 1 ) ) 4 .0 3 ) Beckman R ese a rc h 4 .3 3 4 .2 7 4 .0 9 4 .0 5 A v e. 4 .3 2 4 .2 5 4 .0 7 4 .0 3 4 .0 3 8 6 I i 'c o n c e n t r a t io n o f u n f r a c t i o n a t e d f e t u i n was i n c r e a s e d to ap- ! ■ ip r o x im a te ly 1 % th e i s o i o n i c p o i n t d e c r e a s e d to 3 . 6 . | ( c ) U l t r a v i o l e t a b s o r p t io n | The u l t r a v i o l e t a b s o r p t io n o f a p p r o x im a te ly 0.3% ip r o te in s o l u t i o n s was m easured a t room te m p e r a t u r e . The i ip ro tein c o n c e n t r a t i o n was a c c u r a t e l y d e te r m in e d by w e ig h in g ; t ' ; jafter d r y in g under r ed u c ed p r e s s u r e . T ab le 6 shows th e c a l-j 1 j jcu lated r e s u l t s o f th e a b s o r p t io n c o e f f i c i e n t of a 1 % s o l u - i jtio n , E . J u d g in g from the E U ° Frac-j j 1 cm, 278 mp 5 1 cm, 278 mp j jtion s 1 and 6 seem to be c o n ta m in a te d w it h u l t r a v i o l e t ab s o r b in g m a t e r i a l , p r o b a b ly o th e r t y p e s o f p r o t e i n . F r a c tio n j 2 a l s o seem s t o have a l i t t l e h ig h e r a b s o r p t io n c o e f f i c i e n t i | than the o th e r t h r e e f r a c t i o n s w h ich c o m p r ise th e main body o f f e tu in . (d ) C arb oh y d ra te A n a l y s i s C i) S i a l i c Acid C on ten t The a v e r a g e v a lu e o f two d e t e r m i n a t i o n s o f s i a l i c ; l a c id by t h e t h i o b a r b i t u r i c a c id a s s a y seem t o a g r ee w it h thei r e s u l t s o f th e r e s o r c i n o l a s s a y . The s i a l i c a c id c o n t e n t I I i i n c r e a s e s c o n s i d e r a b l y from F r a c t i o n 1 to 5 . S in c e th e d i f - l ! ! irerence b e tw e e n F r a c t i o n s 4 and 6 i s n e g l i g i b l e , as shown byj I : th e l a s t column in T a b le 7 , i t a p p ea r s t h a t th e s i a l i c a c id i c o n t e n t i s n o t th e s o l e c a u se f o r s e p a r a t i o n o f th e f e t u i n ! ; , i f r a c t i o n s on e l e c t r o p h o r e s i s . j J i : - \ | ( i i ) H ex o se C o n ten t j i I j The h e x o s e c o n t e n t in e a c h f r a c t i o n o f f e t u i n i s I TABLE 6 ULTRAVIOLET ABSORPTION OF FETUIN FRACTIONS j F e tu in A b sorption 1% i C V O r * 4 “ A A M P A M A A M 4 - -M 4 - A A M -> 4 - 0 7 0 -rvi I I - E 1 sF raction C o n ce n tr a tio n * at 278 mp 1 cm, 278 m|i I I 1 .3 6 mg/ml 1 .2 8 9 .3 3 2 2 .6 0 1 .1 8 4 .5 0 3 2 .8 3 1 .1 7 4 .1 3 4 3 .6 4 1 .4 8 4.0.5 5 2 .7 8 1 .1 4 4 .1 1 6 2 .7 8 1 .4 7 5 .3 7 U n f r a c t io n a t e d 3 .3 3 1 .3 9 4 .1 7 " 4 .8 2 2 . 0 0 4 .1 5 " 3.41 1 .4 2 4 .1 5 1 ^ A p p ro xim ately 30 mg o f p r o t e in was and d i s s o l v e d in 1 0 . 0 ml of w a te r . weighed •• a f t e r drying F etu in F ra ctio n s Ca) S i a l i c Acid by R esorcin ol TABLE 7 SIALIC ACID CONTENT (a) S i a l i c Acid by T h io b a rb itu ric acid (a ) Ave. 1 1 i j i i (b) S i a l i c Acid! p r o te in j 1 6 . 1 % - 6 . 1 % 6 . 1 % j 9 .6 moles/mole! 2 8.9 8 .4 8.4 8 . 6 t 0 . 1 5 (c ) 1 3 .5 | 3 9 .4 9.0 9 .2 9 .2 1 4 .4 1 4 1 0 . 2 9 .8 9.9 1 0 . 0 1 5 .7 j 5 1 1 . 0 9 .5 1 0 . 1 1 0 . 2 1 6 .0 | 6 1 0 . 2 9 .5 9.9 9 .9 15 .5 u n fra c tio n a ted 9 .6 9.5 9 .7 9.6 1 15.1 1 | i (a ) The s i a l i c acid content was expressed in terms of N -acetylneuram inic i a cid . The j water o f h y d r o ly s is was n ot c o r r e c te d . ; Cb) The m olecular w eight of f e t u in was taken as 4 8 ,5 0 0 . I ( c ) The standard d e v ia tio n of the mean was c a lc u la te d fo r F ra ctio n 2 on ly . 89 'shown in T a b le 8 . The h e x o se c o n t e n t seems t o in c r e a s e - s l i g h t l y in F r a c t i o n s 1 to 5. A lth ou gh h e x o s e s have no ! ' - i i i icharged g r o u p s, t h e y may be r e s p o n s i b l e fo r s t r u c t u r a l d i f - i I j fe r e n c e s in c a r b o h y d r a te m o i e t i e s o f f e t u i n . H ence, th e y I jmight i n f l u e n c e th e e l e c t r o p h o r e t i c m ig r a t io n in s t a r c h g e l . ! I The e x t e n t of the i n f l u e n c e on the m ig r a tio n r a t e by th e j s t r u c t u r e o f c a r b o h y d r a te m o ie ty i s unknown. ( i i i ) H exosam ine C on ten t T ab le 9 p r e s e n t s th e hexosam in e c o n t e n t in each ^ fr a ctio n o f f e t u i n . A lth ou gh hexosam ine a n a l y s i s i s d i f f i - ! | i c u l t , th e r e s u l t i n g v a l u e s are v a l i d fo r c o m p arativ e p u r- i j Eposes. The d i f f e r e n c e s in hexosam in e c o n t e n t among f e t u i n | ; 1 F r a c t io n s 1 to 5 i s r o u g h ly p a r a l l e l to t h a t f o r the o th e r ! c a r b o h y d r a te com p on en ts. ( e ) P e p t id e C on ten t The p e p t i d e c o n te n t o f e a ch f r a c t i o n d eterm in ed by 126 the method of Lowry e t a l . i s p r e s e n t e d in T able 1 0 . A d e f i n i t e d e c r e a s e in p e p t i d e from 77.5% to 69.9% in F r a c t i o n s 2 to 5 was c l e a r l y shown. The d e c r e a s e in th e peptide; -co n ten t i s r e a s o n a b l e , b e c a u se th e c a r b o h y d r a te c o n t e n t i n - ic r e a s e s c o r r e s p o n d i n g l y . The c a r b o h y d r a te and p e p t id e p o r - I ; jtio n s accou nt f o r 95-100% o f t h e t o t a l (T a b le 1 1 ) . The maini isource o f e r r o r i s p r o b a b ly in th e p e p t i d e d e t e r m in a t io n | i ! 1 O £ \ jsin c e th e a s s a y o f Lowry e t a l . g i v e s o n ly an approxim a- I jtio n due to th e d i f f e r e n c e s in c o m p o s it io n betw een b o v in e 'serum a lb u m in , th e s ta n d a r d , and f e t u i n . ! TABLE 8 HEXOSE CONTENT F e tu in (a) (a ) Hexose (m ole) F r a c tio n Hexose_____ Hexose Ave. P r o t e in (m o le) 1 6.9% 6.9% 6.9% 1 8 .6 2 7 .9 8 . 0 7 .9 t 0 .0 6 1 9 .1 3 8 .3 8 .3 8 .3 2 0 . 2 4 8 . 6 - 8 . 6 2 0 .7 5 8 .7 9 .1 8 .9 2 1 .5 6 8 .3 8 . 8 8 .5 2 0 .5 u n f r a c t i onated 8 . 6 8 . 6 8 . 6 2 0 .7 ( a ) The hexose c o n te n t i s e x p r e ss e d in term s o f a g a l a c t o s e and mannose m ixture in the r a t i o o f 1 : 1 by w e ig h t . The water o f h y d r o l y s is was n o t c o r r e c t e d . TABLE 9 HEXOSAMINE CONTENT (a) Ca) F etu in F ra ctio n Hexosamine Hexosamine Ave. N -a c ety l- hexosamine Hexosamine p r o te in 1 - 5 .2 5 .4 5.1% 5.2% 6.4% 1 4 . i moles mole 2 7 .8 7.0 6 .7 7 .3 7.2 8 .9 + 0 .1 7 1 9 .5 3 - 7 .3 7 .1 7 .7 7 .4 9.1 2 0 . 0 | 4 8 . 0 7.0 7 .4 - 7 .5 9.3 20.3 5 - 8 . 0 7 .7 7 .7 7 .8 9.6 2 1 . 1 6 7 .5 - 6 .9 7 .9 7 .4 9.1 2 0 . 0 u n fra c tio n a ted 7.7 7 .2 7 .4 7 .6 7 .5 9 .3 20.3 (a ) Hexosamine conten t was expressed in terms of glucosam ine. The water of h y d r o ly sis was not c o r r e c t e d . TABLE 10 PEPTIDE CONTENT ( a ) (a ) F e tu in P e p t id e by Lowry P e p tid e Ave. 1 7 6 .6 % 76.6% 2 7 8 .8 7 6 .1 7 7 .5 3 7 2 .7 7 4 .1 7 3 .4 4 7 2 .0 7 3 .3 7 2 .6 5 6 9 .5 7 0 .3 6 9 .9 6 6 8 . 1 6 8 . 1 u n fr a c t io n a te d 7 4 .8 7 7 .4 7 5 .5 ( a ) P e p t id e c o n t e n t was e x p r e s s e d in term s o f e q u i v a l e n t t o b o v in e serum album in. TABLE 11 PERCENTAGE OF CARBOHYDRATE AND PEPTIDE COMPONENTS F etu in F ra c tio n s . ________1_________2_________3_________4_________5__________6__________u n fra c tio n a ted S i a l i c a c id (a ) 5.7% 8.1 8 .7 9 .4 9.6 9 .3 9.0 Hexose(a ) 6 .2 7 .1 7 .5 7 .7 8.0 7 .6 7 .7 N~Acetylhexosamine^a^ 5 .8 8.0 8 .2 8 .4 8 . 6 8 .2 8 .4 P ep tid e by Lowry 76.6 7 7 .5 7 3 .4 72.6 69.9 68.1 75.5 ! T otal 9 4.3 100.7. 9 7 .8 98.1 96.1 9 3 .2 1 0 0 . 6 (a ) Corrected fo r the water o f h y d r o ly s is . 94 ( f ) Amino Acid A n a ly s is | | The r e s u l t s o f amino a c id a n a l y s i s o f F r a c t io n s 2, i 3, 4 and 5 are t a b u la te d in Table 12 . The o r i g i n a l d a ta j are g iv e n in Table 1 2 ( a ) , ( b ) , ( c ) and ( d ) . The average w a s j | c a l c u l a t e d from th r e e sam ples which were h y d roly zed fo r 24 , I | j 48 and 72 h ou rs, r e s p e c t i v e l y . Threonine and s e r i n e were ' i * I e stim a te d from the e x t r a p o r t a t io n to z e r o tim e h y d r o l y s i s . H y d r o ly s is o f i s o l e u c i n e and v a l i n e were n o t com p lete in 24 fiours. T h e r e fo r e , t h e i r v a lu e s were the average of two sam-i p i e s h y d ro lyzed fo r 48 and 72 h o u rs. S in c e h a l f - c y s t i n e andj ; i try to p h a n are known to be d e str o y e d d u rin g th e h y d r o l y s i s , ! 17 ! t h e i r v a lu e s were taken from the l i t e r a t u r e . No d e t e c t - j 'able amount o f m e th io n in e was p r e s e n t in any of the sam p les j a n a ly z e d . G lucosam ine and g a la c to sa m in e were a ls o d e t e c t e d in a l l o f the sa m p le s. From t h e s e o r i g i n a l d a ta the amounts: o f amino a c id s per mg of p r o t e i n were c a l c u l a t e d . As shown in Table 1 2 ( e ) , the combined sum o f amino a c id s and carb oh yd rate i s 9 9 .7 to 103.4%. The amino acid c o m p o s itio n s o f F r a c t io n s 3, 4 and 5 f a l l c l o s e to 6 ach oth-| i : ;er, c e r t a i n l y w i t h i n ex p e rim en ta l e r r o r . F r a c tio n 2, how- j j lever, d i f f e r s s i g n i f i c a n t l y from th e o t h e r s . A lthough the j r e l a t i v e d i s t r i b u t i o n i s s i m i l a r to the o t h e r s , th e t o t a l ; i * i ! number o f amino a c id r e s i d u e s per mole of p r o t e in i s d e f i n - j i t e l y g r e a t e r in F r a c t io n 2 (T able 1 2 [ f ] ) . The t h r e o n in e , | : I Iserine and p r o l i n e r e s i d u e s in F r a c tio n 2 exceed the other j i i I | jf r a c tio n s by 3 - 4 . j ! i 95 TABLE 12(a) AMINO ACID ANALYSIS OP FETUIN FRACTIONS F r a c tio n 2 1 i 24 hr. 48 h r . 72 hr. Ave. Asp 0 .8 9 4 \i m oles 1 .3 0 0 mg 0 .9 6 7 0 .8 9 9 0 .9 2 0 Thr 0 .6 1 9 0 .5 9 6 0 .5 5 8 0 . 6 6 _ Ser 0 .7 7 2 0 .7 2 5 0 .6 6 3 0 .8 4 Glu 1 .0 3 0 1 .0 6 0 1 . 0 2 6 1 .0 3 7 Pro 1 .2 6 4 1 .1 8 9 1 .2 0 5 1 .2 1 9 61 y 0 .6 8 1 0 .7 0 4 0 .677 0 .6 8 7 Ala 0 .9 5 6 0 .9 5 2 0 .9 3 4 0 .9 4 7 Val 0 .5 1 9 1 .0 7 4 1 .1 0 5 1 .0 8 9 I s o l 0 .3 4 7 0 .4 1 0 0 .3 9 1 0 .3 9 6 Leu 0 .8 2 2 0 .8 4 2 0 .8 6 3 0 .8 4 2 Tyr 0 .2 4 5 0 .2 4 4 0 .2 3 6 0 .2 4 2 Phe 0 .3 4 6 0 .3 4 7 0 .3 2 1 0 .3 3 8 ILys 0 .473 0 .4 6 6 0 .4 6 5 0 .4 6 8 His 0 .2 9 2 0 .3 1 4 0 .3 3 4 0 .3 1 3 Arg 0 .3 9 3 0 .4 0 8 0 .4 5 1 0 .4 1 7 nh3 i i 1 i i 1 ________ 0 .7 3 2 0 .8 4 2 0 .7 2 7 96 TABLE 12(b) F r a c t io n 3 24 h r. 48 h r . 72 h r . A ve. Asp 0 .7 3 5 jj m o les 0 .7 5 4 0 .8 1 3 0 .7 6 7 1 .1 3 1 mg Thr 0 .4 6 3 0 .4 5 9 0 .4 6 5 0 .4 7 Ser 0 .5 5 8 0 .5 4 6 0 .5 0 0 0 .6 1 Glu ' 0 .8 1 0 0 .8 5 4 0 .9 1 2 0 .8 5 9 Pro 0 .9 6 5 0 .9 96 1 .0 2 4 0 .9 9 5 Gly 0 .5 1 3 0 .5 4 3 0 .5 9 7 0 .5 5 1 A la 0 . 7 2 2 0 .7 2 4 0 .7 8 6 0 .7 4 4 Val 0 .7 30 0 .8 6 5 0 .9 7 3 0 .9 1 9 I s o l 0 .2 6 1 0 .3 2 2 0 .3 7 0 0 . 346 Leu 0 . 6 6 6 0 .6 7 2 0 .7 3 5 0 .6 9 1 Tyr 0 .1 9 4 0 .1 8 9 0 .2 0 4 0 .1 9 6 Phe 0 .2 6 7 0 .2 8 7 0 .3 1 3 0 .2 8 9 Lys 0 .3 5 7 0 .3 9 4 0 .4 6 8 0 .4 0 6 H is 0 .2 3 3 0 .2 5 5 0 .2 7 6 0 .2 5 5 Arg 0 . 3 1 8 0 .3 3 7 0 .3 5 9 0 .3 3 8 nh3 0 .5 6 3 0 .6 5 6 0 .8 3 8 97 F r a c t i o n 4 Asp Thr Ser 1 jGlu I jPr o ;Gly I 'Ala ;Val I s o l Leu Tyr | Phe Lys |H is | Arg nh3 i I ! TABLE 12(c) 24 h r . 48 h r . 72 h r . A v e . 1 .0 3 8 0 .6 1 5 0 .6 9 5 1 .1 3 8 1 . 3 5 4 0 .6 8 1 1 .0 0 8 1 .0 5 6 .3 8 8 0 .9 0 4 0 .2 7 4 0 .3 8 3 0 .5 2 5 0 .2 9 5 0 .4 3 6 0 .7 8 5 (U m ol) 1.4f>4 mg 1 .0 0 9 0 .6 2 8 0.688 1 .1 4 3 1 .4 4 2 0 .6 8 5 0 .9 9 3 1.210 .4 2 9 0 .9 4 7 0 . 2 8 2 0 .3 9 6 0 .5 6 3 0 .3 3 6 0 .4 6 5 0 .9 2 6 0 .9 3 8 0 .5 7 9 0 .6 1 6 1 .0 9 7 1 .1 7 8 0 .6 1 2 0 .9 3 3 1 .1 7 1 .4 2 5 0 .8 4 8 0 .2 4 7 0 .3 7 8 0 .5 2 5 0 .3 1 7 0 .4 4 2 1 .0 7 7 0 . 9 9 5 0.66 0 . 7 7 1 .1 2 6 1 . 325 0 .6 5 9 0 . 9 7 8 1 .1 9 0 .4 2 7 0 .9 0 0 0 . 2 6 8 0 .3 8 6 0 . 5 3 8 0 .3 1 6 0 .4 4 8 98 Fraction 5 Asp Thr iSer Glu Pro Gly i Ala Val I s o l Leu Tyr Phe Lys ;His j |Arg | !NH3 0 .8 5 2 0 .7 1 9 0 .7 2 7 0 .7 2 1 0 .4 5 4 0 .4 3 7 0 .4 8 ( 0 .4 9 4 0 .4 7 9 i 0 .5 6 j 0 .8 2 8 0 .8 6 7 0 .8 2 9 j 0 .9 0 3 1 .0 5 1 0 .9 4 3 | 0 .5 2 3 0 .5 0 9 0 .5 1 0 j 0 .7 5 3 0 .7 1 9 0 .7 5 4 j 0 .9 0 5 0 .9 1 6 0 .9 1 0 0 .3 2 2 0 . 330 0 .3 2 6 ; 0 .6 9 3 0 .6 5 7 0 .6 6 4 0 .2 0 9 0 .1 8 8 0 .1 9 6 0 .2 8 5 0 .2 7 0 0 .2 7 7 0 .3 9 5 0.39.5 0 .389 0 .2 3 8 0 .2 2 4 0 . 2 2 2 0 .3 1 4 0 .3 2 7 0 .3 1 3 0 .7 2 0 1 .0 7 3 TABLE 12(d) 24 h r . 48 h r . 72 h r. A ve. 0 .7 1 8 u mol 1 . 1 1 0 mg 0 .4 4 9 0 .5 2 8 0 .7 8 9 0 .8 7 4 0 .4 9 8 0 .7 9 1 0 .7 2 9 0 .2 6 1 0.6 4 1 0 .1 9 0 0 .2 7 7 0 .3 7 6 0 .2 0 5 0 .2 9 9 99 F r a c tio n TABLE 2 1 2 ( e ) — ------ 1 0 0 mg 3 4 5 ; Asp 7 .3 2 7 .0 2 7 .0 7 6 .7 2 Thr 4 .6 3 3 .8 2 4 .0 9 3.91 Ser 5 .0 8 4 .2 3 4 .1 5 4 .0 0 Glu 9 .2 5 8 .8 4 9 .0 0 8 . 6 8 Pro 8.17 7 .6 9 7 .9 6 7 .3 4 Gly i 2 .7 1 2 .5 0 2. 32 2 .3 7 | jAla 4 .6 5 4 .2 0 4 .2 9 4 .3 5 ! i Val 7 .4 7 7 .2 4 7 .2 9 7 .3 1 j T s o i 3 .1 0 3 .1 2 2 .9 9 3 . 0 0 j •Leu 6 .5 9 6 .2 3 6 .3 0 6 . 1 0 | Tyr 2 .7 4 2 .5 4 2 .7 0 2 .5 8 ; Phe 3 .4 5 3 .3 8 3 .5 1 3 .3 1 ; ;Lys 4 .1 6 4 .1 4 4 .2 7 4 .0 5 H is 2 .9 8 2 .7 8 2 . 6 8 2 .4 7 j Arg 4 .5 2 4 .2 0 4 .3 3 3 .9 7 1 /2 Cys* j 2 .5 5 2 .5 5 2 .5 5 2 .5 5 ; Try* 1 0 . 8 6 0 . 8 6 0 . 8 6 0 . 8 6 ! j I 8 0 .2 3 7 5 .3 4 76 .3 6 7 3 .5 7 j ' Carbohydrate 2 3 .2 2 4 .4 2 5 .5 2 6 .2 1 0 3 .4 3 9 9 .7 1 0 1 .9 9 9 .8 Taken from the l i t e r a t u r e . 100 TABLE 12(f) 48500 gm F r a c t io n 2 3 4 5 S p i r o f ' S p ir o & F i s h e r x e t a l Asp 3 0 .9 2 9 .6 2 9 .9 2 8 .4 3 3 .0 2 9 .1 Thr 2 2 18 19 19 2 4 .9 1 6 .8 Ser 28 24 23 2 2 2 6 .2 2 2 .4 Glu 3 4 .8 3 3 .2 3 3 .8 3 2 .6 3 3 .9 3 3 .5 Pro 4 0 .9 3 8 .4 3 9 .8 36. 8 3 3 .7 3 7 .9 G ly 2 3 .0 2 1 . 2 1 9 .8 2 0 . 1 2 4 .2 1 9 .9 Ala 31 . 8 2 8 .7 29. 3 2 9 .7 3 3 .4 3 0 .0 Val 3 6 .6 3 5 .0 3 5 .2 3 5 .8 4 0 . 3 3 5 .4 I s o l 1 3 .3 1 2 .5 1 2 . 8 1 2 .9 1 4 .9 1 3 .1 Leu 2 8 .2 2 6 .7 2 7 .0 2 6 .1 2 6 .8 3 0 .1 Tyr 8 . 1 0 7 .5 7 8 .0 5 7 .6 6 6 . 8 8 . 2 Phe 1 1 .4 1 1 . 1 1 1 .5 1 0 .9 1 0 . 8 1 0 . 2 Lys 1 5 .7 1 5 .7 1 6 .1 1 5 .3 1 6 .5 1 6 .9 H is 10 .5 9 .7 0 9 .4 7 8 .7 4 1 0 . 2 1 1 . 6 |Arg I 1 4 .0 1 3 .0 1 3 .4 1 2 .3 1 1 . 8 1 6 .9 J j l /2 Cys* 1 2 . 1 1 2 . 1 1 2 . 1 1 2 . 1 1 2 . 1 — b r y * 2 . 2 2 . 2 2 . 2 2 . 2 2 . 2 1 . 9 j i j* Taken from th e l i t e r a t u r e . I S i o i i | The amino a c id c o m p o s it io n o f a p o o le d f e t u i n r e - 17 10 8 p o r t e d by S p ir o and S p ir o and F is h e r e t a l . are ta b u - i l a t e d in T ab le 1 2 ( f ) f o r co m p a riso n . j j ( g ) P r o t e i n f i n g e r p r i n t i n g ; ] 1 | S in c e o b v io u s d i f f e r e n c e s in amino a c id c o m p o s it io n [ jof th e f e t u i n f r a c t i o n s w ere n o t a p p a r e n t, f i n g e r p r i n t i n g o f 'F r a c t io n s 2 and 5 was perfo rm ed to d i s t i n g u i s h d i f f e r e n c e s i jin se q u en ce in t h e i r r e s p e c t i v e p e p t i d e c h a i n s . As shown i n F i g . 1 1 , m ost o f th e p e p t i d e s found f o r F r a c t io n 2 c o r r e - | ispond t o t h o s e in F r a c t i o n 5. P e p t i d e s 7 and 1 7 , h o w ev er, ; d i f f e r in t h e i r r e s p e c t i v e p o s i t i o n s . There are two a d d i- | i t i o n a l s p o t s , 29 and 30, from F r a c t i o n 2 w hich do n o t c o r r e - t :spond to any s p o t s from F r a c t io n 5. A c co r d in g to the amino a c id a n a l y s i s , F r a c t io n 2 c o n t a i n s 16 l y s i n e and 14 a r g i n i n e r e s i d u e s , and F r a c t io n 5 c o n t a i n s 15 l y s i n e and 12 a r g in in e r e s i d u e s . T h e r e f o r e , F r a c t io n 2 sh o u ld t h e o r e t i c a l l y be c l e a v e d by t r y p s i n i n t o 31 p e p t i d e s , and F r a c t io n 5 i n t o 28 p e p t i d e c h a i n s . In t h e I ! i f i n g e r p r i n t s , F r a c t i o n 2 showed 30 s p o t s and F r a c t i o n 5 j jshowed 28 s p o t s . The m is s in g p e p t i d e c h a in s c o u ld have j s t a y e d a t th e o r i g i n b e c a u s e t h e t h r e e b u lk y g l y c o p e p t i d e s i jmay n o t r e s o l v e in the c h r o m a to g r a p h ic and e l e c t r o p h o r e t i c ! Isystem s u s e d . The d e t e c t i o n o f the c a r b o h y d r a te g ro u p s was i u n s u c c e s s f u l . 1(6) T i t r a t i o n j i | | In T a b le 13 th e o r i g i n a l d a t a f o r t i t r a t i o n o f j j Fig* 1 2 . - - P e p t id e map o f t r y p t i c d i g e s t of f e t u i n f r a c t i o n s 2 and 5 s ta in e d w ith n in h y d r in . D escen ding chrom-j atography was c a r r ie d out as shown by the o r d in a t e , and j e l e c t r o p h o r e s i s was run su b s e q u e n tly in th e d i r e c t i o n of the! icathode. The c o n d it io n s f o r chromatography and electro p h or-J ;esis were d e s c r ib e d in M ethods. The shaded s p o t s were addedi from the p e p tid e map fo r F r a c tio n 2 . ! CHROMATOGRAPHY 9 O 15 0 1 4 0 13 0 • 2 n © 0 ' 7 < ? ° \ ? 0 \ t 1 6 ( j 18 10 19 30 n 2.0 .& < 5r*?2 9 0 0 29 0 24 ^ 28 26 25 23 0 Of o V ^ 0 4 7p 6 o 2< ~ ^ 3 _____________ 5 ___________ a Q » ELECTROPHORESIS pj FRACTIO N 5 F igu re 12 TABLE 1 3 (a ) Acid t i t r a t i o n (b la n k ) .00 ml c f 0 .1 6 M KC1 was t i t r a t e d by 0 .1 1 8 0 N HC1 pH V o l. o f HC1 - l o g y T H+ 3 .1 7 0 .0 1 9 8 5 (m l) 0 .0 6 2 .5 0 0 .0 9 9 2 8 0 .0 8 2 .1 4 0 .2 3 8 2 7 0 .0 7 2 .0 2 0 .3 1 7 6 9 0 .0 7 1 .8 5 0 .4 9 6 4 0 0 .0 7 105 TABLE 13(b) Acid titration of neuraminidase-treated a^-acid glycoprotein 3 .0 0 ml o f 3.19% g l y c o p r o t e i n in 0 .1 6 M KC1 was t i t r a t e d by 0 .1 1 8 0 N HC1 and .042 0 N KC1 s o l u t i o n a t 25° H+ pH + lo g 7 *17+ v o l . HC1 added p r o t e i n 4 .7 7 0 0 (m l) i J4.62 .0 3 9 7 1 .9 5 j 14.46 .0 7 9 4 3 .9 3 i 4 .2 8 .119 0 5 .8 9 |4.1 4 .1 5 8 7 7 .8 5 i 4 .0 0 .1 9 8 5 9 .7 9 ;3 . 8 6 .2380 1 1 .7 2 3 .7 4 0 .2 7 8 0 1 3 .6 4 3 .6 0 - 0 . 0 6 .31 7 0 1 5 .5 3 3 .4 7 - 0 . 0 7 .35 7 0 1 7 .3 9 3 .3 3 .3 9 7 0 1 9 .1 8 :3.20 .4 3 7 0 2 0 .8 2 .9 7 .4 9 6 0 2 3 .0 2 .8 4 .5 3 6 0 2 4 .2 i |2.70 .5 7 5 0 2 5 .3 2 .5 8 .6 1 5 0 2 6 .0 2 .4 0 .6 2 4 0 2 6 .4 2 .2 4 .7 5 4 0 2 6 .9 |2.1 3 .8 3 3 0 2 8 .4 ‘ 1 .9 6 - 0 . 0 7 .9 9 3 0 2 7 .8 ! 1 .1 5 1 2 6 .3 106 TABLE 1 3 ( c ) Acid t i t r a t i o n ( F e t u i n I) 3 .0 0 ml o f 1.680% f e t u i n i n 0 .1 6 M KC1 was t i t r a t e d by 0 .1 1 8 0 N HC1 a t 25° H+ pH + lo g YT tr+ V o l . HC1 added P r o t e i n 4 .0 2 0 .0 4 6 8 6 4 .2 3 3 .8 7 .0 7 0 2 9 6 .3 6 3 .7 5 .0 9 3 7 2 8 .4 7 3 .6 4 .1 1 7 1 5 1 0 .5 6 i 13 .5 1 .1 4 0 5 8 1 2 .5 7 ;3 .3 9 .16 401 1 4 .5 2 3 .2 7 .1 8 7 4 4 1 6 .3 8 13.16 0 .2 1 0 8 7 1 8 .1 5 3 .0 4 - 0 . 0 6 .2 3 4 3 1 9 .2 9 2 .9 3 - 0 . 0 7 .2 5 7 7 2 0 .4 9 2 .8 4 .2 8 1 2 2 1 .7 3 2 .7 5 .3 0 4 6 2 2 .7 2 2 .6 0 .3 5 1 5 2 4 .3 7 2 .4 7 .3 9 8 3 2 5 .4 9 2 . 3 8 .4 4 5 2 2 6 .8 6 2 .2 6 .5 1 5 5 2 8 .2 2 .1 6 .5 8 5 8 2 8 .9 12.03 .7 0 2 9 2 9 .6 ! | l . 9 4 .82 0 1 2 9 .1 ! l .87 .9 3 7 1 3 2 .3 107 TABLE 1 3 ( c ) (C o n t.) Acid t i t r a t i o n ( F e tu in I) + lo g Y ' + V o l. HC1 added H 1 .1 7 1 5 1 .4 0 5 8 - 0 .0 7 1 .7 5 7 3 P r o te in 3 4 .4 3 6 .3 I 3 8 .5 | I 108 TABLE 1 3 ( d ) Acid t i t r a t i o n ( F e t u i n I I ) - * pH +lo g Y1tt+ V o l. HC1 added H 4 .0 6 0 .03971 (m l) 13.48 0 .1 4 8 9 j2.96 - 0 . 0 7 .2 4 8 2 12.42 - 0 . 0 7 .39 7 1 2 .2 7 - 0 . 0 7 .4 9 6 4 ;1.9 3 - 0 . 0 7 .7 4 4 6 i ;*A cid t i t r a t i o n ( F e t u i n I ) was r e p e a t e d . P r o t e i n i i 3 .6 7 1 3 .7 2 2 0 .8 3 2 6 .3 1 3 0 .2 9 j 3 0 .1 2 | 109 TABLE 13(e) Acid titration (acid-desialicized Fetuin I) 3.00 ml of 1 .1 1 3 x 10 “^ d e s i a l i c i z e d f e t u i n in 0 .1 6 M KC1 t i t r a t e d by 0 .1 1 8 0 N HC1 at 25° j I pH =fclog VT jj+ V o l. HC1 added H+ P rotein! 5 .4 5 0 0 0 !5.35 .00928 (m l) 1 .0 4 I i 15.19 .019856 2 .0 0 j |5.08 .029784 3 .1 4 I ! ! 14.96 .039712 4 .1 9 I : i 4 .8 7 .04964 5 .2 3 j 4 .7 7 .05957 6 .2 7 j i ' i 4 .5 8 .07942 8 .3 5 j 4 .4 2 .09928 1 0 .4 2 ! 4 .2 7 .11914 1 2 .4 8 j 4 .1 1 .13899 1 4 .5 2 | i 3 .9 7 .15885 1 6 .5 3 I 3 .8 4 .17870 1 8 .5 4 j 3 .6 6 .20849 2 2 .0 4 j i 3 .4 7 .23827 2 4 .2 8 I 13.28 0 .26806 2 6 .8 7 ! j I 13.09 - .06 .29784 2 8.81 | L .9 2 - .07 .32762 30.51 j ! | 2 .7 7 - .07 .35741 3 1 .8 8 ! i I 2 .6 0 - .07 .3 9712 3 3.10 j pH TABLE 1 3 ( e ) (C o n t .) Acid t i t r a t i o n ( a c i d - d e s i a l i c i z e d F e tu in I) ± log y ’ + V o l. HC1 added 1 1 0 ! I \ \ i + H n P r o te in 2 .4 6 - .43683 3 3 .7 5 2 .2 9 - .07 .49640 3 4 .6 2 1 2 .1 7 - .07 .55597 3 3 .5 8 ! j 2 . 0 2 ' - .07 .65525 i 3 2 .6 ! 2 .9 1 - .07 .75453 3 1 .2 1 .8 2 - .07 .89352 3 2 .5 1 .7 0 - .07 1 .0 9 2 1 2 9 .6 1 .6 4 - .07 1 .2 9 0 6 3 3 .2 1 .6 0 - .07 4 .4 8 9 2 3 8 .9 i j tr it ' ~ . ~~ '.."" ..' ."... Ill TABLE 1 3 ( f ) A c tu a l pH and o b serv ed pH w ith th e g l a s s e l e c t r o d e a t 25° pH 9 .1 8 t o b a s i c pH pH r ea d in g A c tu a l pH 9 .1 8 0 9 .1 8 0 Borax b u f fe r 9 .6 1 9 .6 0 9 .9 7 1 0 .0 0 1 0 .3 2 1 0 .4 0 " 1 0 . 6 8 1 0 .8 0 " 1 1 .0 1 1 1 .1 0 Na2 HP04 1 1 .4 1 1 1 .5 0 " 1 1 .8 0 1 1 .9 0 " 1 2 .3 6 1 2 .5 0 . " 1 2 .7 6 1 2 .9 0 ” pH 9 .1 8 t o a c i d i c pH 9 .1 8 0 9 .1 8 0 Borax b u f f e r 8 .7 9 8 .8 0 tt 8 .3 8 8 .4 0 tr 8 . 0 1 8 . 0 0 TT 7 .7 0 7 .7 0 T r is 7 .3 9 7 .4 0 tt 7 .0 1 7 .0 0 (Braun) 5 .8 0 5 .8 0 P h t h a la t e 4 .9 3 4 .9 0 tt 4 .0 5 4 .0 0 tr | TABLE 13(g) | Base t i t r a t i o n (Blank) 3 . 0 0 ml of 0.16M KC1 was t i t r a t e d by 0.100 M KOH and 0.06M KC1 s o lu tio n at 25°. pH reading ( 1 ) ( 2 ) (3 ) (4) (5) Ave. V ol. of KOH - lo g 9.97 2 10.050 10.080 10.060 10.04 .00595 (ml) 10.149 1 0 . 2 0 1 1 0 . 2 2 0 10 .208 10.233 1 0 . 2 0 .00794 10.276 10.315 10.327 10.313 10.340 10.32 .00992 10.456 10.490 10.497 10.469 10.4 8 .01390 10.636 10.663 10.670 10.641 10.677 1 0 . 6 6 .01986 0 .1 4 10 .762 10.787 10.794 10.760 10.800 10 .78 .02580 10.861 10.880 1 0 . 8 8 8 10.851 10.894 10.87 .03175 10.963 10.979 10.990 10.947 10.994 10.97 .0395 0 .1 4 1 1.067 11.072 11.090 11.050 11.088 1 1 .0 8 .0496 11.230 11.241 1 1 . 2 0 0 11.233 11.23 .0695 11.373 11.385 11 .398 11.360 11.387 11.38 .0992 11.548 11.560 11.576 11.536 11.560 11.56 .1489 0.11 112 TABLE 13(g) (Cont.) Base t i t r a t i o n (Blank) Vol. of (1 ) ( 2 ) (3) (4) (5) Ave. KOH - lo g Y’ + 11.760 11.770 11.788 11.751 11.772 11.77 .2480 n 11.903 11 .922 1 1 . 8 8 8 11.905 11.91 .347 1 2.03 2 12.040 11 .058 12.041 12.04 .496 0 . 1 1 12.156 1 2.158 11.181 12 .149 12.160 12.16 .695 12.273 12.277 11.302 12.267 12.279 12.28 .992 G lass e le c tr o d e i adjustment b efo re and a fte r t i t r a t i o n by pH 9.180 Borax standard (U. S. N ational Bureau of S tan dards). Before 9.180 9.180 9.180 9.180 9.180 A fter 9.195 9.180 9.191 9.164 9.207 113 ~ '........ ' .......... 114- TABLE 1 3 (h ) Base t i t r a t i o n (b o v in e serum album in) 3 ml o f 0 .9 2 4 mg per ml b o v in e serum albumin in 0 .1 6 M KC1 s o l u t i o n t i t r a t e d by 0 .1 0 0 M KOH and 0 .0 6 M KC1 s o l u t i o n I ; V o l. o f KOH added OH~ j pH Vo! • o f KOH added _______to b lan k p r o t e i n I ! 0 0 (m l) 0 5 .7 9 .0 1588 (m l) 3 .7 9 5 .9 8 .02184 5 .1 9 6 . 2 0 .02780 6 . 6 2 6 .4 4 .03376 8 .0 5 6 .6 0 .03773 8 .9 8 6 .8 4 .04 368 1 0 .4 0 6 .9 8 .04765 1 1 .3 6 7 .2 1 .05371 1 2 .7 9 7 .4 1 .05957 1 4 .1 9 7 .6 1 .06 552 1 5 .6 0 • 00 .07148 1 7 .0 2 8 . 0 0 ■ .07744 1 8 .4 3 8 . 2 2 .08340 1 9 .8 6 8 .4 3 .08935 2 1 .2 9 8 .6 7 .09531 2 2 .6 7 8 .8 2 .09 928 2 3 .6 0 8 .9 8 j .1 0 3 3 2 4 .5 2 19.18 .1 0 9 2 2 5 .9 0 9 .3 7 .1 1 5 2 2 7 .2 6 i 9 .5 7 .1231 0 2 9 .0 2 115 TABLE 13(h) (Cont.) Base t i t r a t i o n ( b o v in e serum alb u m in ) pH V o l . o f KOH added 9 . 7 8 .1330 jlO .0 2 .1 4 6 9 i 11 0 . 2 2 .1 6 0 8 (m l) i 1 0 .3 7 .17 6 7 1 0 .5 8 .1 9 8 6 jlO. 81 .2 2 8 3 1 1 . 0 0 .26 8 1 111.22 .3 3 7 6 j l l . 42 .4 0 7 0 j 111.63 .4 9 6 8 1 1 .8 6 .6 3 5 4 1 2 .0 3 .7 9 4 2 ,1 2 .1 7 .9 9 2 8 1 2 .2 7 1 .1 9 1 4 I l 2 .3 8 1 .4 8 9 2 V o l . o f KOH added OH~ _to b la n k p r o t e i n 3 1 .2 1 .0 0 5 1 9 3 3 3 .7 8 .0 0 8 3 3 7 (m l) 3 6 .3 1 .0 1 0 4 8 3 9 .5 7 .0 1 6 8 2 4 3 .2 9 .0 2 7 5 0 4 7 .8 1 .0 4 0 5 7 5 4 .1 7 .0 6 5 0 8 6 4 .8 8 .1 0 4 8 7 1 .9 5 .1 6 1 6 7 9 .7 1 .2 7 2 0 8 6 .4 8 .3 9 9 7 9 3 .9 5 .5 8 2 5 9 7 .6 9 .7 8 6 7 9 8 .7 4 1 .0 4 9 1 0 4 .7 6 116 TABLE 13(i) Base titration (Fetuin I) 3 .0 0 ml o f 0.1776% f e t u i n in 0.16M KC1 by 0.100N KOH and 0 .0 6 M KC1 s o l u t i o n V ol. of KOH added V ol. o f KOH OH“ bound pH to f e t u i n s o l u t i o n added to b la n k p r o t e i n |4 .3 4 .02978 (m l) 0 (m l) 2 .7 0 i t 14.45 .06950 6.3 1 k . 6 5 .07942 7 .2 1 1 i |4.79 .09928 9 .0 2 |5.02 .1291 1 1 .7 3 ■5.19 .1489 1 3 .5 2 5 .3 9 .1688 1 5 .3 3 5 .6 1 .1886 1 7 .1 3 5 .8 5 .2085 1 8 .9 4 6 .1 1 .2283 2 0 .7 4 6 .3 4 .2432 2 2 .0 9 6 .6 0 .2581 2 3 .4 4 6 .7 9 .2681 2 4 .3 5 16.99 .2780 2 5 .2 5 7 .2 1 .2879 2 6 .1 5 7 .4 4 .2978 2 7 .0 5 7 .6 9 .3078 2 7 .9 6 7 .9 6 .3177 2 8 .8 6 i 8 .2 9 .3276 2 9 .7 5 18.52 .3336 30.29 i i 1 8 . 6 8 .3376 3 0 .6 4 117 TABLE 1 3 ( i ) (C o n t.) Base t i t r a t i o n (F e t u in I) pH V o l. o f KOH added t o f e t u i n s o l u t i o n V o l. o f KOH added to blan k OH“ bound p r o t e i n 8.90 .3425 (m l) (m l) 3 1 .0 8 9.1 1 : .3475 31 .5 3 ! 9 .2 9 I .3524 3 1 .9 5 9 .4 5 .3574 3 2 .3 8 i 9 .7 2 .3673 33 .2 0 j 9 .9 1 .3773 0 34 .0 1 1 0 .0 5 .3872 .0056 34.66 1 0 .1 7 .3971 .0067 35.4 6 j l0 .3 5 .4170 .0113 3 6 .8 4 1 0 .4 9 .4368 .0159 3 8 .2 3 1 0 .6 5 .4666 .0205 40 .52 1 0 .7 7 .4964 .02 7 5 4 2 .5 9 1 0 .9 5 .5460 .0405 4 5 .9 1 1 1 . 1 1 .5957 .058 5 4 8 .7 9 1 1 .3 2 .6354 .0892 4 9 .6 1 1 1 .5 4 .7049 .1460 5 0 .7 6 1 1 .7 3 .7942 .2 1 3 2 5 2 .7 7 1 2 . 0 0 j .9928 .398 5 5 3 .9 8 1 2 .1 6 1 .1 9 1 4 .5952 5 4 .1 5 112.30 1 .4 8 9 2 .8961 5 8 .8 7 i 118 TABLE 13(j) Base Nitration (Fetuin II) 3 .0 0 ml o f 0.1776% f e t u i n in 0 .1 6 M KC1 t i t r a t e d by 0 .1 0 0 N KOH and 0 .0 6 M KC1 s o l u t i o n V o l . o f KOH added V o l. o f KOH OH~ bound pH to f e t u i n s o l u t i o n added t o blank p r o t e i n - 4 .1 8 0 4 .4 0 .0 3 9 7 (m l) 0 (m l) 3 .6 1 4 .6 7 .0 6 9 5 6 .3 1 |4 .8 2 .0 9 9 3 9 .0 2 ! |5 .0 6 .1 291 1 1 .7 3 '5 .2 4 .14 8 9 1 3 .5 2 5 .4 4 .1 6 8 8 1 5 .3 3 ! !5 .6 7 .18 8 6 1 7 .1 3 5 .9 1 .20 8 5 1 8 .9 4 6 .1 9 .2 2 8 3 2 0 .7 4 6 .4 3 .2 4 3 2 2 2 .0 9 6 .7 1 .2581 2 3 .4 4 6 .9 0 .26 87 2 4 .3 5 ;7 .1 2 .2 7 8 0 2 5 .2 5 i 17.34 .288 0 2 6 .1 6 7 . 5 8 .2 9 7 8 2 7 .0 5 7 .8 5 .3 0 7 8 2 7 .9 6 8 .0 0 .31 2 7 2 8 .4 0 8 .1 7 .31 77 2 8 .8 6 8 .3 4 .32 27 2 9 .3 1 8 .5 5 .3 2 7 6 2 9 .7 5 119 TABLE 13(j) (Cont.) Base N i t r a t i o n ( F e t u i n I I ) V o l. o f KOH added V o l . - o f KOH OH~ bound pH to f e t u i n s o l u t i o n added to b la n k p r o t e i n 8 .7 7 .33 2 6 (m l) (m l) 30 .1 9 9 .0 0 . 3376 3 0 .6 4 9 .2 1 i .3 4 2 5 3 1 .0 6 t i 9 .3 8 | .3 4 7 5 3 1 .4 9 1 9 .5 3 | i .3 5 2 4 3 1 .9 1 1 9 .7 6 ! .3 6 2 4 3 2 .7 7 1 9 .9 3 .3 7 2 3 0 3 3 .5 5 1 0 .1 5 .3 8 7 2 .0 0 6 7 3 4 .5 6 1 0 . 33 .4 0 7 0 . 0 1 1 2 3 5 .9 5 1 0 .4 8 .42 6 9 .01580 3 7 .3 4 1 0 .6 3 .4 5 6 7 .0 1 8 2 3 9 .8 3 1 0 .7 9 .4 9 6 4 .0 2 8 7 4 2 .4 8 1 0 .9 5 .5361 .0 4 0 5 4 5 .0 1 1 1 . 1 2 .5 7 5 8 .0 5 5 9 4 7 .2 2 | 1 1 . 33 .6 3 5 4 .0 9 1 5 4 9 .4 0 1 1 .5 2 ( .7 0 4 9 .1 3 7 0 5 1 .5 8 ! 1 1 .7 6 .8141 .230 0 5 3 .0 5 1 1 .9 8 1 .9 9 1 8 .3 8 0 3 5 5 .6 3 1 2 .1 3 1 .1 9 1 4 .5 5 3 5 5 7 .9 4 1 2 .2 8 1 .4 8 9 2 .8591 5 7 .2 3 120 TABLE 1 3(k) Base t i t r a t i o n ( a c i d - d e s i a l i c i z e d f e t u i n I) 3 .0 0 of 4 .1 2 x 1 0 “4 rooles d e s i a l i c i z e d f e t u i n was t i t r a t e d by 0 .1 0 0 N KOH and 0 .0 6 KC1 s o l u t i o n V ol. o f KOH added t o V ol. o f KOH QH~ bound O ■ ! « 1 > i /i -i > - 7 « / 4 o+>n i m m / > ! / 4 l* v * 1 ’ m .a a i. _ • pH d e s i a l i c i z e d f e t u i n added to b l ank p r o t e in 5 .5 7 0 ; 5 .7 2 .01489 (ml) 0 (ml) 1 . 2 1 : 5 .9 4 .03475 2 .8 2 I 6 .1 4 .05460 4 .4 2 i 6 .3 5 .0695 5 .6 2 | 6 .5 9 .08935 7 .2 3 6 .79 .10424 8 .4 3 6 .9 9 .11914 9 .6 4 j 7 .2 2 .13403 1 0 .8 4 7 .4 6 .14892 1 2 .0 5 7 .6 4 .15885 ... 1 2 . 8 6 ; 7 .8 5 .16877 1 3 .6 6 ■ 8 .0 8 .17870 1 4 .4 6 8 .3 4 .18863 i 1 5 .2 6 8.61 .19856 1 6 .0 5 8 . 8 8 ! .20849 1 6 .8 5 ! 9 .1 3 .21842 1 7 .6 4 ! 9 .3 3 .2283 1 8 .4 1 ! 9 .5 0 .2383 1 9 .2 0 i 9.71 i | .2532 2 0 .3 5 121 TABLE 13(k) (Cont.) Base titration (acid-desialicized fetuin I) V o l . o f KOH added to V o l. o f KOH added OH~bound pH d e s i a l i c i z e d f e t u i n t o b lan k______ p r o t e i n 9 .8 7 . 2 6 8 1 (m l) 0 (m l) 21 .5 1 1 0 .1 5 i 1 .2978 .00 6 5 2 3 .5 7 ;10.43 j .3376 .0 1 3 2 2 6 .2 5 1 0 .6 5 j .377 3 . 0 2 0 0 2 8 .9 1 1 0 .8 1 .4170 .0291 3 1 .3 8 j l l . 0 0 .4567 .042 9 3 3 .4 8 I 1 1 .1 7 .4964 .0 614 3 5 .1 9 1 1 .4 3 j .5758 .11 2 4 3 7 .4 9 1 1 1 . 6 6 .6751 .1783 4 0 .1 9 1 1 . 8 6 .8141 .2 8 4 8 4 2 .8 2 1 2 .0 4 .99 2 8 .429 3 4 5 .5 9 1 2 .1 8 1 .1 9 1 8 .625 7 4 5 .7 7 1 2 . 30 1 .4 8 9 2 . 8940 4 8 .1 2 1 2 .3 8 1 .7 8 7 0 1 .1 1 4 0 5 4 .4 5 j I i 1 i i i t i i I 122 TABLE 1 3 ( 1 ) Base t i t r a t i o n ( a c i d - d e s i a l i c i z e d f e t u i n I I ) 3 .0 0 ml o f 4 .1 2 x 1 0 “^ d e s i a l i c i z e d - f e t u i n was t i t r a t e d by 0 .1 0 0 N KOH and 0 .0 6 M KC1 s o l u t i o n . pH V o l . o f KOH added to d e s i a l i c i z e d f e t u i n V o l. o f KOH added t o b la n k OH" p r o t e i n 5 .5 7 0 (m l) 0 (m l) 0 5 .7 8 .0 198 56 1 .6 1 ; j 6 . 0 0 .03971 2 .7 5 | 6 .2 4 .059 57 4 .8 1 j 6 .4 9 .0 7 9 4 2 6 .4 2 | ; 6 .7 4 .0 9 9 2 8 8 .0 3 1 i ; 7 .0 1 .11910 | 9 .6 4 j i i ■ 7 .2 1 .1340 1 0 .8 4 i 7 .4 8 .15 3 9 1 2 .4 5 | i 7 .6 7 .1 5 8 8 1 2 .8 5 : 7 .8 5 .1 6 8 8 1 3 .6 6 8 .0 8 .1 7 8 7 1 4 .4 6 j 8 .3 4 .1886 1 5 .2 6 I i 8 .6 3 | .1986 1 6 .0 2 | 8 .9 0 .2 0 8 5 1 6 .8 4 | i | 9 .1 4 .2184 1 7 .6 3 9 .3 4 .2283 1 8 .4 1 9 .5 1 .2383 1 9 .1 9 ■ ! 9 .7 1 .2 5 3 2 2 0 .3 5 j 9 .8 9 .2681 0 2 1 .4 8 1 0 .0 8 .2879 .0065 2 2 .7 7 123 TABLE 13(1) (Cont.) Base titration (acid-desialicized fetuin II) j j e S ___ [10.31 | l 0 .50 jlO . 71 i jlO .8 9 i | l l . 18 i jll .41 i i l l . 63 i l l . 83 112.05 1 2 .1 8 1 2 .3 0 1 2 .3 9 V o l. o f KOH added t o d e s i a l i c i z e d f e t u i n V o l. o f KOH added to b la n k .3177 .3475 .387 2 .4269 .4964 .5659 .655 2 .774 4 .9 9 2 8 1 .1 9 1 4 1 .4 8 9 2 1 .7 8 7 0 (m l) .0 0 8 8 .01 5 4 .0245 .0 3 4 8 .0 6 1 4 .1 0 8 8 .1 6 7 5 .2 6 2 8 .4 3 3 5 .6 2 3 3 . 8944 1 .1 3 5 7 (m l) OH~ pro te ii? 2 4 .9 9 2 6 .0 6 j j 2 9 .3 4 j 3 1 .7 2 | 3 5 .1 9 | i 3 6 .9 8 | 3 9 .4 6 4 1 .3 9 4 5 .2 5 i j 4 5 .9 6 | 4 8 .1 2 | 5 2 .6 9 I j 1 2 4 i jn ativ e and a c i d - d e s i a l i c i z e d f e t u i n , b o v in e serum album in, j jand th e oc^-acid g ly c o p r o t e in are t a b u la t e d . Bovine serum t I I jalbumin and a ^ -a c id g ly c o p r o t e in were used as sta n d a r d s . I jBovine serum albumin was t i t r a t e d in the b a s i c r e g io n , and i ; jthe oc^-acid g l y c o p r o t e in was t i t r a t e d in the a c i d i c r e g io n . j S in c e both of them t i t r a t e d as e x p e cted from l i t e r a t u r e j jvalues ( w it h in e x p erim en ta l e rr o r of t 2 H+ io n s per p r o t e i n j | 107 109 ! im o le cu le ), ’ the r e s u l t s ob ta in ed w it h t h i s apparatus I i jean be c o n sid e r e d s u i t a b l e . | The e x p e r im e n ta l v a lu e of - l o g YT t t+ of e q u a tio n 10 ! I H ! was found t o be 0 .0 6 at pH 3 .0 0 and in c r e a s e s to 0 .0 7 at pH j ; ( i n i s 2 .5 0 to pH 1 .8 5 (T ab le 1 0 a ) . Tanford r e p o r te d t h a t j i — 1 og Y,tt+ i *1 th e same pH r e g io n was 0 . 0 8 . In b a s i c r e g io n I the e x p e r im e n ta l v a lu e s of - l o g YfQj|- were 0 .1 4 at pH 1 0 .5 but d e c r e a se d t o 0 .1 1 at pH 1 1 .5 (T ab le l O g ) . The c o r r e - 101 sponding v a lu e s r e p o r te d by Tanford i s 0 . 1 7 . In c a l c u l a t i n g H+ io n s bound per mole o f p r o t e i n , 0 .0 7 was used fo r i - l o g y 't t+ in the a c i d i c r e g io n . In the b a s i c r e g io n the rl lamount of b a se added to the blan k s o l u t i o n was su b tr a c te d j d ir e c t ly from th e amount of base added to th e p r o t e i n s o l u - j I I jtion . The e x p e r im e n ta l v a lu e s of H+ io n s bound per mole o f j | . ! p r o t e i n are shown in F ig . 13. Some o f the p o i n t s o b ta in ed are o m itte d in the f i g u r e , b u t recorded in Table 13. i i ! i i In order to o b ta in a d d i t io n a l in fo r m a tio n , the l o g - | a r ith m ic graph ( F i g . 1 4 ) , was drawn to o b ta in the e l e c t r o - ; ! is t a t i c i n t e r a c t i o n f a c t o r w from the e x p e r im e n ta l t i t r a t i o n j 125 40 20 !U J z p !o C D 2 0 r !<O ]Z O Q j T i g . 1 3 . - - T i t r a t i o n o f N a tiv e F e t u in and A cid- i D e s i a l i c i z e d F e t u in . The b la c k d o t s r e p r e s e n t n a t i v e f e t u i n land th e open d o t s r e p r e s e n t a c i d - d e s i a l i c i z e d f e t u i n . T i t r a t i o n was c a r r ie d out from th e i s o i o n i c p o in t in b o th d i r e c t i o n s o f pH. The i o n i c s t r e n g t h was 0 .1 6 in KC1 and the (tem perature was kep t a t 2 5 ° . I ; P i g . 1 4 . - - T i t r a t i o n d a ta f o r c a r b o x y l g rou p s o f a c id j d e s i a l i c i z e d f e t u i n , a t 25° and i o n i c s t r e n g t h 0-sl6 p l o t t e d jaccord in g to e q u a t io n 10 w it h s l i g h t m o d i f i c a t i o n . N r e p r e s e n t s th e t o t a l number o f c a r b o x y l g r o u p s, and N" r e p r e s e n t s ;the number o f i o n i z e d c a r b o x y l g r o u p s . Z i s th e t o t a l num ber o f c h a n g e s. p H — Log 4 4 4 .2 4.0 3.8 3 6 0 Q 10 i i 20 30 127; " 128 c u r v e. The l i n e showed a s l i g h t c u r v a tu re w hich m ight i n d i c a te a change in shape or volume o f the p r o t e i n m o le c u le as i i | th e pH i s lo w e r e d . The e x p e r im e n ta l p K ^ ^ was e s t im a t e d to be 4 .4 2 , and th e w f a c t o r 0 .0 2 9 . The t h e o r e t i c a l w f a c t o r , c a l c u l a t e d a cco r d in g to e q u a tio n 2, was 0 .0 3 1 and i s v ery jc lo s e to the e x p e r im e n ta l v a lu e c o n s id e r in g th e v a r io u s a s - j isum ptions in v o lv e d in th e c a l c u l a t i o n . The r a d iu s o f th e I ' d e s i a l i c i z e d f e t u i n m o le c u le was c a l c u l a t e d to be 2 4 .9 A as-- jsuming 20% bound w ater and ta k in g 4 8 ,7 0 0 fo r the m o le c u la r I j ! jw eight, and 0 .6 9 6 ml per g fo r th e p a r t i a l s p e c i f i c volum e. j iCSee e q u a tio n s 2 and 3 . ) The e x p e r im e n ta l w f a c t o r of na- j : t iv e f e t u i n was not d eterm in ed b eca u se th e t i t r a t i o n o f the ! i 1 l ia s p a r t y l and g lu ta m y lc a r b o x y l groups and th e s i a l i c a cid ca rb o x y l groups p a r t i a l l y o v e r la p . An attem p t to o b t a in a j t i t r a t i o n curve of s i a l i c a c id c a rb o x y l groups by s u b t r a c t in g th e H+ io n s bound to d e s ia lic iz e d f e t u i n from th o s e o f n a t i v e f e t u i n did n o t appear f e a s i b l e . | r(7) V i s c o s i t y ! i . i j The r e s u l t s o f v i s c o s i t y m easurem ents are shown in | i j F i g . 1 5 . From t h e s e m easurem ents o f s p e c i f i c k in e m a tic vis-j c o s i t i e s , the i n t r i n s i c k in e m a tic v i s c o s i t i e s f o r n a t i v e and n e u r a m in id a s e - t r e a t e d f e t u i n were determ ined by e x t r a p o l a - ! | jtio n t o z e r o c o n c e n t r a t i o n . The i n t r i n s i c k in e m a tic v isc o s-j i i t i e s were c o n v e r te d to i n t r i n s i c v i s c o s i t i e s by em ploying j ^equation (1 4 ) assuming th e p a r t i a l s p e c i f i c volum es of both j n a t i v e a n d n e u r a m in id a s e - t r e a t e d f e t u i n to b e 0 .6 9 6 ml p e r j ° 10- c o |V J CONCEN TRAT I ON F i g . 1 5 . - - V i s c o s i t y o f N a tiv e and N eu ra m in id a se- t r e a t e d f e t u i n . The p r o t e i n was d i s s o l v e d in KC1 and I KH2 PO4 s o l u t i o n a t pH 7 . 5 and th e i o n i c s t r e n g t h 0 . 1 6 . The j v i s c o s i t y was measured a t 2 5 . 0 ° , and th e tem p eratu re flu c tu -: a t i o n of th e water b a th was k ep t w i t h i n 0 . 0 0 4 ° . The p r o t e in c o n c e n t r a t io n was m easured by u l t r a v i o l e t a b s o r p tio n at j278 mp. The amount of s i a l i c a c id removed from n a t i v e f e - jtu in (6.7% of th e t o t a l w e ig h t) was s u b t r a c t e d in c a l c u l a t i n g the n e u r a m in id a s e - t r e a t e d f e t u i n c o n c e n t r a t i o n . The iblack d o t s r e p r e s e n t n e u r a m in id a s e - t r e a t e d f e t u i n and th e jempty d o t s r e p r e s e n t n a t i v e f e t u i n . 15 g . From the i n t r i n s i c v i s c o s i t i e s the v i s c o s i t y i n c r e ments;. which are p a ram eters o f asymmetry, were c a l c u l a t e d A /L a c c o r d in g to the f o l l o w i n g e q u a tio n . _ In] 100 I Vh v + — I p jwhere [ r)] = i n t r i n s i c v i s c o s i t y j i v = p a r t i a l s p e c i f i c volume o f p r o t e i n j j I ! w = g o f water absorbed per g of p r o t e i n which i s a p p ro x im a te ly 0 . 2 g • i p = d e n s i t y of s o l v e n t w hich i s a p p r o x im a te ly 1 . 0 0 g | per m l. | ! |The a x i a l r a t i o s of th e e l l i p s o i d s o f r e v o l u t i o n were e s t i - j 4 * 6 jmated from t h e . t a b l e s adapted by Young from p a p ers report-j ; i ed by many i n v e s t i g a t o r s . The c a l c u l a t e d r e s u l t s are com- j p i l e d in Table 14 . i(8) O p tic a l R otatory D is p e r s io n The o p t i c a l r o t a t o r y d i s p e r s i o n o f n a t i v e f e t u i n and; n e u r a m in id a s e - t r e a t e d f e t u i n are shown i n F ig . 1 6 . In c a l - ; jc u la tin g th e reduced mean r e s id u e r o t a t i o n th e con cen tratio n ! • j o f f e t u i n and d e s i a l i c i z e d f e t u i n was e x p r e s s e d in term s o f | i | the w e ig h t of p e p t id e per volum e, e x c lu d in g the carbohydrate! | g r o u p s, so th a t the h e l i c a l c o n t e n t s were e x p r e ss e d in terms! j o f per c e n t o f the p e p t id e p o r t i o n o n ly . The e x p e r im e n ta l j jdata are shown in T able 1 5 . In c a l c u l a t i n g h e l i c a l c o n te n t j l i t was assumed t h a t th e mean r e s i d u e r o t a t i o n o f d en atu red j ip r o te in was -2 0 0 0 d e g . T h is assu m p tion was b ased upon th e | TABLE 14 | N ative f e t u in Neuram inidase- tre a ted f e t u i n VISCOSITY DATA POR NATIVE FETUIN AND NEURAMINIDASE-TREATED FETUIN Kinematic I n t r i n s i c I n t r i n s i c V is c o s it y A xial R atio V is c o s it y V is c o s it y increment P r o la te Oblate 5 .2 5 .5 ~ 6.1 5 .3 6 . 8 g g 4 .9 5 .2 5 .8 5.0 6 . 6 4000 210 220 230 240 250 260 m /x W AVE LENGTH F ig . 1 6 . --O p tic a l r o ta to r y d is p e r s io n between 210 and 260 m\i fo r n a tiv e (b lack ) d o ts ) and neuramini d a s e - t r eated (open d o ts ) f e t u in a.t pH 7 .5 and room tem perature. TABLE 15 F etuin Neur am inidase- tr e a te d f e t u i n OPTICAL ROTATION AT 233 m|i AND HELICAL CONTENTS FOR NATIVE AND NEURAMINIDASE-TREATED FETUIN a233 mu ^233 mu ° /o h e l ix 0.69 0 .6 8 0 .7 4 0 .0 3 2 deg -3600 25 0 .4 4 0 .4 7 0 .4 2 0.0 15 -3000 21 C oncentrati on (26) - 4*1 by by by P .P . Lowryrs B iuret 133 134 53 r e p o r t s by B lo u t e t a l . on p o l y - l - g l u t a m a t e and on th e 34 f i n d i n g by V e rp o o rte e t a l . on h e a t d en a tu red f e t u i n . DISCUSSION Cl) A l c o h o l - m e t a l io n f r a c t i o n a t i o n o f f e t u i n ! | The f r a c t i o n a t i o n o f f e t u i n from f e t a l c a l f serum in! 1 ! I jthe p r e s e n c e o f e t h a n o l and z i n c i o n s at low te m p e r a tu r e i s > 1 ; Q :the a d a p t a t io n o f Method 10 of Cohn e t a l . d e v e lo p e d f o r i ' . j human serum p r o t e i n s . W a t e r - m i s c i c i b l e o r g a n ic s o l v e n t s , j, ' _ | su ch as e t h a n o l , low er th e d i e l e c t r i c c o n s t a n t o f th e m e d i- j ; 1 1 0 ! um, r e d u c in g t h e s o l u b i l i t y o f p r o t e i n s . P r o t e i n com- j < | 4 A ! p l e x e s are form ed w h ich e v e n t u a l l y l e a d to p r e c i p i t a t i o n . j s G e n e r a l l y , th e l e s s p o la r th e o r g a n ic s o l v e n t , th e g r e a t e r ;its p r e c i p i t a t i n g a c t i o n on p r o t e i n s . The p r e c i p i t a t i o n i s enhan ced by t h e s p e c i f i c c o m b in a tio n o f th e m e ta l io n s w it h th e p lasm a p r o t e i n s . M etal io n s combine w it h th e s i d e c h a in f u n c t i o n a l g r o u p s t h a t a t t r a c t p r o t o n s , i . e . , c a r b o x y l, im- i i d a z o l , am ino, p h e n o l, and g u a n id in o g r o u p s , and make th e i I i 1 1 0 * 'com plexes l e s s s o l u b l e i n aq u eou s m ed ia . A ll t h e s e p roc-i | | e s s e s are c a r r i e d out a t - 5 to - 1 0 ° w h ich c o m p le te s p r e c i p i - i S “ : j t a t i o n and p r e v e n t s d e n a t u r a t i o n o f t h e p r o t e i n . There have! been s u g g e s t i o n s o f d e n a t u r a t i o n of p r o t e i n s by th e e t h a n o l j i ; p r e c i p i t a t i o n m ethod. Thus f a r , h o w ev e r, no one h as u n d e r- ! : i I I f a k e n a s y s t e m a t i c s tu d y o f th e e x t e n t o f d e n a t u r a t i o n 1 1 1 1 1 2 p au sed by th e e t h a n o l . In t h i s r e g a r d , G reen and Kay ’ ! 135 136 compared p r o p e r t ie s of s a l t - p r e c i p i t a t e d and e t h a n o l- f r a c t i o n a t e d f e t u i n . According to thenu, the e th a h o l- j p r e c i p i t a t e d f e t u i n showed a g r e a te r s u s c e p t i b i l i t y to p ro t e o l y t i c d i g e s t i o n by t r y p s in and lower h e l i c a l c o n te n t. They su g g ested th at the e th a n o l f r a c t i o n a t i o n produces i r - i | j r e v e r s ib le s t r u c t u r a l changes in f e t u i n m o le c u le s . Although! i s t r u c t u r a l changes might occur during th e e th a n o l treatm ent,; | ! |they must be r a th e r s u b t le because our f e t u i n p r e p a r a tio n , d i s s o l v e d in a pH 4 .5 b u f f e r , showed a sym m etric u l t r a c e n - i t r i f u g a l p a tt e r n ( F ig . 2 ) . Furtherm ore, over a la r g e pH j i Irange the f e t u i n prepared by a lc o h o l f r a c t i o n a t i o n appears 15 isymmetric by u l t r a c e n t r i f u g a t i o n and e l e c t r o p h o r e s i s . The Isame p r e p a r a tio n , however, showed a skewed p a tte r n i f d i s s o l v e d in pure w a te r . This phenomenon can be a t t r ib u t e d to a g g r e g a tio n o f th e f e t u i n m o le c u le s . T h is c o n c lu s io n seems to agree w ith the o b s e r v a tio n o f a s l i g h t t u r b i d i t y found when th e f e t u i n s o l u t i o n was c o m p lete ly d e io n iz e d . The ten dency to ag greg a te at low pH, even a t high io n ic * 3 4 .str e n g th , was a ls o r ep o rted by V erpoorte e t a l . w ith sa lt-' p r e c i p i t a t e d as w e ll as e t h a n o l - f r a c t i o n a t e d f e t u i n . j i ; l | D e sp ite the i n d i c a t i o n of hom ogeneity by u l t r a c e n - I ; j t r i f u g a t i o n , two d i s t i n c t peaks appeared when our f e t u i n was j ; Ifr a c tio n a te d on DEAE-Sephadex ( F ig . 1 1 ) . When our f e t u i n j 'was r e a c te d w ith a n t i - f e t u i n serum, two p r e c i p i t i n l i n e s j Iwere a ls o found ( F i g . 9 ) , although the im m unoelectrop horesiS j | jrevealed o n ly a s i n g l e l i n e , i n d i c a t i v e o f immunochemical j 137 f (homogeneity ( F ig . 1 0 ) . The second l i n e in the double d i f f u - i s io n experim en t might have r e s u l t e d from d im e r iz a t io n of f e - i t u in . There seems to be a sm all amount of m a te r ia l contam i n a t i n g our f e t u i n p r e p a r a tio n . Judging from th e u l t r a v i o - t ! ( jlet a b s o r p tio n at 278 mu, F r a c tio n s 1 and 6 c o n ta in a d d i- i ; jtio n a l u l t r a v i o l e t absorb ing m a t e r i a l, p rob ab ly some contam- i iin atin g serum p r o t e i n . The con ta m in a tio n seems very s m a ll, ; how ever, l e s s than 1 % judging from the change in the absorp-j jtion c o n s t a n t , and the amount o f f e t u i n in t h e s e f r a c t i o n s I j ((Table 6 ) . There was a l s o a very fa st-m o v in g component (found on s ta r c h g e l e l e c t r o p h o r e s i s , which appears q u it e i ' S ( d if f e r e n t from th e r eg u la r e l e c t r o p h o r e t i c components o f fe-j ituin ( F ig . 4 ) . I t could p o s s i b l y be co n ta m in a tin g m a te r ia l.; ( 2 ) M ic r o h e te r o g e n e ity o f f e t u i n I t seems th a t th e e t h a n o l - f r a c t i o n a t e d f e t u i n i s homogenous by a l l of the c l a s s i c a l c r i t e r i a o f hom ogen eity, j jbut some i n d i c a t i o n s of h e t e r o g e n e i t y was a lso shown by im- j jm unodiffusion and io n -ex ch a n g e chromatography. As shown in j ! f i g . 3 and 4 th e e x i s t e n c e o f m ic r o h e t e r o g e n e it y in f e t u i n I i 1 jis q u it e c l e a r . | The f e t u i n prepared by a lc o h o l f r a c t i o n a t i o n c o u ld , j iof c o u r s e , have undergone some co n fo rm a tio n a l m o d if ic a t io n Iduring the p r e p a r a tio n . T h is was su g g e s te d by Green and i i 111 112 'Kay ’ in com parison w ith the f e t u i n p r e c i p i t a t e d by am- I • I (monium s u l f a t e . E v id e n t ly , th e m ic r o h e te r o g e n e it y o f | | 138 I J e t h a n o l- p r e c ip it ated f e t u i n d o e s n o t a r i s e from p a r t i a l d e - i jn a tu r a tio n , how ever, b e c a u se th e u n t r e a t e d f e t a l c a l f serum, from which f e t u i n was p rep a red , a l s o e x h i b i t e d th e c o r r e - j sp ond ing bands on e l e c t r o p h o r e s i s ( F i g . 3 ) . Thus, i t seems i jsafe to assume t h a t f e t u i n i s p r e s e n t in th e f e t a l c a l f s e - j jrum in s e v e r a l form s w hich can be f r a c t i o n a t e d by e th a n o l I p r e c i p it a t i o n at low tem p era tu re w ith o u t g r e a t l y m od ify in g j ; the p h y s ic a l and ch em ical s t r u c t u r e . F u rth erm ore, th e cleari Idem onstration o f m ic r o h e t e r o g e n e it y fo r th e (NH^^SO^- j p r e c ip it a t e d f e t u i n ( F i g . 3) l e a d s to the r e a s o n a b le assump-j |tio n th a t th e s e p a r a t io n i n t o s e v e r a l bands i s n o t th e r e - ; \ s u i t of the method of p r e p a r a tio n . F e tu in was p r e v i o u s l y ! ; i I i Iconsid ered homogeneous sim p ly b e c a u se i t had n o t been exam- I ined w ith a method as s e n s i t i v e as s t a r c h g e l e l e c t r o p h o r e - ; s i s . The s u s p i c i o n th a t th e m ic r o h e t e r o g e n e it y m ight r e s u l t from p o o lin g s e r a from many f e t a l c a l v e s was c o n v in c - ; i n g l y e li m in a t e d by t h e d e m o n s tr a tio n of s e v e r a l bands on j e le c t r ophor e s i s f o r e a c h of s e r a c o l l e c t e d from f i v e f e t a l j jca lv e s ( F i g . 4 ) . A m ix tu re of t h e s e f i v e s e r a a l s o e x h ib it - j j i jed th e e i g h t c o r r e sp o n d in g bands c h a r a c t e r i s t i c o f f e t u i n , ! ^ in d icatin g t h a t each band in th e d i f f e r e n t f e t a l c a l f s e r a 1 !is p r o b a b ly i d e n t i c a l . ; : ! The r e d u c tio n of th e number o f bands on s t a r c h g e l I i ” i e l e c t r o p h o r e s i s from e i g h t to th r e e when f e t u i n was d e s i a l i - j i I icized i n d i c a t e s the im portance o f the s i a l i c a c id r e s i d u e s j i 139; in th e m i c r o h e t e r o g e n e i t y i n f e t u i n ( F i g . 5 ) . S in c e s i a l i c a c id c a r b o x y l g r o u p s c o n t r i b u t e s u b s t a n t i a l l y t o th e ch a rg e ] on th e p r o t e i n , t h e y l i k e w i s e i n f l u e n c e th e r a t e o f e l e c t r o p h o r e t i c m i g r a t i o n . S in c e t h r e e d i s t i n c t bands s t i l l r e m ained a f t e r rem oving 90% o f th e s i a l i c a c id by n e u r a m in i d a s e , o th e r f a c t o r s must p la y a r o l e in c r e a t i n g th e m ic r o - j ' h e t e r o g e n e i t y . A p p a r e n t ly , tr e a tm e n t o f f e t u i n w it h 0 .0 2 5 N i ; iH^SO^ a t 80° f o r one hour c a u s e s d e n a t u r a t i o n of th e g l y c o p r o t e i n , in a d d i t i o n t o the c le a v a g e o f th e s i a l i c a c id r e s - ! ] i jid u es, making th e s t a r c h 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 a d i f - j f u s e , e x te n d e d band ( F i g . 6 ) . ! ; i The e x i s t e n c e o f m i c r o h e t e r o g e n e i t y in human a^ -acid! 38 [ g l y c o p r o t e in h a s been known f o r some t im e . The m ic r o h e t - : e r o g e n e i t y o f f e t u i n seem s v e r y s i m i l a r to t h a t o f oc^-acid g l y c o p r o t e i n , b e c a u s e t h r e e bands on s t a r c h g e l e l e c t r o p h o r e s i s w ere a l s o foun d a f t e r rem oval o f th e s i a l i c a c id r e s i d u es from th e g l y c o p r o t e i n . D i f f e r e n c e s in t h e amount o f i s i a l i c a c i d , t h e r e f o r e , are n o t t h e o n l y c a u se f o r th e s e p - | a r a t i o n on e l e c t r o p h o r e s i s f o r a , - a c i d g l y c o p r o t e i n . T h is j I 1 • ! I g ly c o p r o t e in can a l s o be s e p a r a t e d , h ow ev er, i n t o s e v e r a l j i I jcom ponents by e l e c t r o p h o r e s i s i n c e l l u l o s e s u p p o r t in g m edia I 1 OR in d e n a tu r e d form i n 8 M u r e a . In t h i s r e s p e c t , f e t u i n l ■ i d i f f e r s from th e a c id g l y c o p r o t e i n , b e c a u s e th e f e t u i n dena-i i i i | itu red i n u r e a or u r e a p l u s 2 - m e r c a p t o e t h a n o l , showed no j ; . ( I c l e a r s e p a r a t i o n on e l e c t r o p h o r e s i s ( F i g . 7 and 8 ) . I t was,j [ t h e r e f o r e , c o n c lu d e d th a t a d i s t i n c t c o n fo r m a tio n o f e a ch j 140 e l e c t r o p h o r e t i c component must be m a in ta in e d f o r s e p a r a t i o n . A lth o u gh th e charge d i f f e r e n c e i s im p ortan t a s s u g g e s t e d by | th e DEAE-Sephadex chrom atography and e f f e c t o f d e s i a l i c i z a - t i o n , i t i s n o t th e o n ly b a s i s f o r m ic r o h e t e r o g e n e i t y . | The p o s s i b i l i t y t h a t the m i c r o h e t e r o g e n e it y a r i s e s jfrom th e a c t i o n by p r o t e o l y t i c enzymes in the serum i s un- i l i k e l y b ec a u se th e s e d im e n ta tio n c o n s t a n t s o f p a r t i a l l y j { fr a c tio n a t e d f e t u i n are a l l a l i k e and th e amino and th e c a r - 5 | |boxyl te r m in a l amino a c i d s o f f e t u i n have been shown e x c l u - I I ; I 3.6 j s i v e l y t o be i s o l e u c i n e . The p o s s i b i l i t y t h a t the e l e c - j jt r o p h o r e t ic bands m ight r e s u l t from a g g r e g a t io n o f f e t u i n I i m o le c u le s i s a l s o u n l i k e l y . The fo r m a tio n o f e i g h t d i f f e r - I lent bands i m p l ie s th a t th e b i g g e s t a g g r e g a t e s be made o f s e v e r a l s u b u n it s w hich would be d i s t i n g u i s h e d by u l t r a c e n t r i f u g a t i o n . At pH 4 . 5 th e u l t r a c e n t r i f u g a l p a t t e r n showed | o n ly one sym m etric peak ( F i g . 2 ) . A lth ou gh no d e f i n i t e ca u se fo r s e p a r a t i o n i n t o elec-! i it r o p h o r e t ic com ponents o f g l y c o p r o t e i n s has been d is c o v e r e d ,! j ithere are s e v e r a l p o s s i b i l i t i e s . R e c e n t ly , F o s te r e t j : j j a i.1 1 3 ,1 1 4 ,1 1 5 r e p o r te d th e e x i s t e n c e o f th e m ic r o h e te r o g - j i e n e i t y o f human serum album in w hich h i t h e r t o had been con - I i s id e r e d e x tr e m e ly hom ogeneous. Though human serum album in jis n o t a g l y c o p r o t e i n , t h e i r c o n c e p t o f m i c r o h e t e r o g e n e it y jof p r o t e i n " a r is i n g from a p o p u la tio n o f g r o s s l y s i m i la r in-l 'd iv id u a l p r o t e i n s w hich may d i f f e r e i t h e r d i s c r e t e l y or con- j tin u o u s ly i n a number o f p r o p e r t i e s " i s i n t e r e s t i n g . They 141 s u g g e s t t h a t 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 and s e q u e n c e s , in th e number or arran gem en t o f amide g r o u p s , i n |s e c o n d a r y and t e r t i a r y s t r u c t u r e , and in d i s u l f i d e c r o s s l i n k s are r e s p o n s i b l e . In ord er to p i n p o i n t th e b a s i s f o r d i f f e r e n c e s in | f e t u i n , i t was hoped t h a t th e i n d i v i d u a l e l e c t r o p h o r e t i c ! |com p on en ts c o u ld be i s o l a t e d . P r e p a r a t iv e f r a c t i o n a t i o n o f; i f e t u i n by s t a r c h g e l e l e c t r o p h o r e s i s , h o w ev e r, was i m p r a c t i - I | i j c a l b e c a u se o f th e l i m i t e d q u a n t i t i e s t h a t can be a p p lie d !t o th e s t a r c h g e l and i n e f f i c i e n t e x t r a c t i o n p r o c e d u r e s . S i ! i f 'The a l t e r n a t i v e , DEAE-Sephadex ch rom ato grap h y, r e s u l t e d i n j I i ‘p a r t i a l f r a c t i o n a t i o n o f th e f e t u i n form s ( F i g . 3 ) . W ith ! ' t h e s e f r a c t i o n s th e d i f f e r e n c e s in c o m p o s it io n and p h y s i c a l ; c h e m ic a l p r o p e r t i e s c o u ld be e l u c i d a t e d . S in c e no s p e c i f i c p h y s i o l o g i c a l f u n c t i o n o f f e t u i n has b een d i s c o v e r e d , i t i s n o t p o s s i b l e t o c la im t h a t a l l th e com ponents a re f u n c t i o n a l l y s i m i l a r . F ree and S on en - ! 116 b erg r e p o r t e d t h a t t h e r e w ere t h r e e com ponents o f b o v in e I ;growth hormone e x h i b i t e d by s t a r c h g e l e l e c t r o p h o r e s i s and each o f them was shown t o p o s s e s s th e b i o l o g i c a l a c t i v i t y as grow th hormone. W hatever th e o r i g i n o f th e e l e c t r o p h o r - : e t i c s e p a r a t i o n , one can c la im t h a t e a ch com ponent e x h i b i t s , ! j la s i m i l a r b i o l o g i c p r o p e r t y . The grow th p ro m o tin g p r o p e r ty ! I i i o f f e t u i n f r a c t i o n s can be t e s t e d i n th e f u t u r e a s a com- j j p a r i s o n o f b i o l o g i c a l p r o p e r t i e s . j 142 ( 3 ) Comparison o f p a r t i a l l y - s e p a r a t e d f e t u i n f r a c t i o n s ; As shown in F ig . 3, the se p a r a tio n o f the e l e c t r o - I p h o r e t ic components by DEAE-Sephadex chromatography was not com p lete. Each f r a c t i o n con ta in ed two or th r e e bands, one or two of which o v e r la p w ith th e n eig h b o rin g f r a c t i o n . A l though th e se f r a c t i o n s were not id e a l fo r stu d y in g sm all | ! j d if fe r e n c e s in p h y s ic a l and chem ical p r o p e r t i e s , i t was j jhoped th a t a p p ro p ria te com parisons could be made, j (a ) Gross s i m i l a r i t i e s j The im m unodiffusion p a tt e r n ( F ig . 9) r e v e a ls th a t {all f r a c t i o n s r e a c t w ith th e a n t i - f e t u i n serum; a continuous! ! | l i n e i s formed. The second p r e c i p i t i n l i n e was most p ro - i inounced w ith F r a c tio n 2, in d ic a t in g th a t t h i s p a r t ic u la r an-j jtigen i s lo c a t e d pred om in an tly in t h i s f r a c t i o n . F r a c tio n s 3, 4 and 5 e x h ib it e d v e ry f a i n t th in l i n e s . The a n t i g e n t i c s i m i l a r i t y was more c l e a r l y dem onstrated w ith im m unelectro- ; p h o r e s is ( F ig . 1 0 ) , which showed a s i n g l e con tin u ou s l i n e i a g a in s t a n t i - f e t u i n serum. i i As shown in T a b les 4 and 6 , the s e d im e n ta tio n con- j I [ jsta n ts and th e u l t r a v i o l e t a b s o r p tio n c o e f f i c i e n t s r e v e a l noj s i g n i f i c a n t d i f f e r e n c e s e x ce p t fo r the p r e se n c e of s l i g h t j jcontam ination in F r a c tio n s 1 and 6 as su g g e ste d by u ltr a v io -; i I |le t a b so r p tio n . I : Cb) D if f e r e n c e s A d e f i n i t e sm all d i f f e r e n c e was observed in the I 143 i s o i o n i c points'*" o f the f r a c t i o n s d eterm in ed a t a p p roxim ate l y 0.3% in d i s t i l l e d w a te r . The d e c r e a s e from pH 4 .3 2 to i 4 .0 3 p a r a l l e l s th e order o f e l u t i o n from the DEAE-Sephadex I column. S in ce the f r a c t i o n s e l u t e in order o f in c r e a s in g i i ( e l e c t r o s t a t i c i n t e r a c t i o n w ith the DEAE m a t e r ia l ( a s the I ; I lio n ic s t r e n g t h o f the e lu a n t i s i n c r e a s e d ) , o r, in o th er | jwords, as t h e i r a c i d i t y i n c r e a s e s , the l a t t e r f r a c t i o n s iwould be e x p e c te d to have a lower i s o i o n i c p o i n t . T h is a s - . 1 ; [sumption i s born out by the e l e c t r o p h o r e t i c m ig r a tio n w hich I [in c r e a s e s from F r a c t io n s 2 to 6 . The combined r e s u l t s o f j i i i J the i s o i o n i c p o in t d a t a , column chrom atography, and e le c tr o -j I | jp h o re sis show d e f i n i t e l y t h a t the n e t n e g a t iv e charge i n - | c r e a s e s from F r a c t io n s 2 to 6 . T h is s l i g h t in c r e a s e o f n e g -: a t i v e charge a l s o c o in c i d e s w ith the s m a ll in c r e a s e in the s i a l i c a c id c o n te n t from F r a c t io n s 1 t o 5 (T ab le 7 ) . I t ap p e a r s p l a u s i b l e th a t th e n e g a t iv e charge in c r e a s e i s a t l e a s t p a r t i a l l y a c c o u n ta b le by the in c r e a s e in s i a l i c a c id c o n t e n t . However, th e i n c r e a s e o f s i a l i c acid i s o n ly 2 .5 ; [moles per mole o f f e t u i n , and i s n o t th e s o l e cause fo r | | i e l e c t r o p h o r e t i c s e p a r a t io n b ec a u se th e r e are e i g h t bands j I formed on e l e c t r o p h o r e s i s . ; N e u tr a l h ex ose c o n te n t a l s o in c r e a s e s s l i g h t l y in i jthe order o f e l u t i o n (T ab le 8 ) . A lthou gh th e r e was no ! *The i s o i o n i c p o in t i s d e f in e d as th e pH o f d e io n - jized p r o t e in s o l u t i o n . | 144 a p p r e c ia b le i n c r e a s e in hexosam ine c o n te n t (T a b le 9 ) , th e jhexosamine a n a l y s i s o f f e r s more d i f f i c u l t i e s than the o th e r s u g a r s , and sm a ll d i f f e r e n c e s m ight be o b scu re d . N e v e r th e l e s s , th e carb o h yd ra te c o n t e n t i n c r e a s e s due t o th e s i a l i c a c id and n e u tr a l h e x o se . C o r r e s p o n d in g ly , a d e c r e a s e in the i jpeptide c o n te n t was found (T able 1 0 ) . Thus, the d i f f e r e n c e s I j |in th e ca rb oh yd rate m o iety may be a s s o c i a t e d w ith the e l e c - i 't r o p h o r e tic s e p a r a t io n over and above th e s i a l i c a c id d i f - i ■ j fe r e n c e s . The th r e e bands s t i l l app earing a f t e r d e s i a l i c i - j jza tio n may o r i g i n a t e from d i f f e r e n c e s in the ca rb o h y d ra te m o iety y e t r e m a in in g , o r , of c o u r se in th e p e p t id e p o r t i o n , i I " " i As a n oth er p o s s i b i l i t y , th e one or two s i a l i c a c id r e s i d u e s j ! per p r o t e i n rem ain in g in th e d e s i a l i c i z e d f e t u i n , b ec a u se the n eu ram in id ase tr e a tm e n t was n ot c o m p le te , may be r e s p o n s i b l e f o r the appearance o f t h r e e b a n d s. The carb o h y d ra te c o m p o sitio n o f f e t u i n has not been e s t a b l i s h e d u n e q u iv o c a lly to d a te (T able 1 6 ) . The r e a so n f o r the f l u c t u a t i o n in th e c o m p o sitio n i s unknown, but su g - j - I . 1 ig e s t iv e o f h e t e r o g e n e i t y in the ca rb oh yd rate m o ie ty . The j i , | icarbohydrate c o m p o s itio n and t h e sequ en ce m ight vary w it h ! | jthe g e n e t i c ty p e or age o f th e f e t u s . Some s u b m a x illa r y g l y c o p r o t e i n s seem to show very d i f f e r e n t c o m p o s itio n d e - 27 i p end ing on th e ty p e and b reed o f th e a n im al. | The r e s u l t s o f amino a c id a n a l y s i s show a marked 17 j d i f f e r e n c e from th e r e s u l t o f S p ir o and S p ir o . There are ; labout one t o f i v e r e s i d u e s o f d i f f e r e n c e s in ev ery amino j TABLE 16 CARBOHYDRATE COMPOSITION OF FETUIN Schulze 117 S i a l i c (a) i acid 7.0 TeUg i e 1 7 1 1 o IDS " 3A This Spiro ’ Graham Fisher Verpoorte Laboratory 8 .7 8.1 5 .8 6.1 & 7.1 (c ) 10.2 IHexose 7 .4 8.3 6 .7 8.0 8.6 iHe? o s ( b ) i amine ^ 4 .5 6.3 5 .4 5 .4 7.5 (a ) Expressed in terms of N -acetylneuram in ic acid (b) Expressed in terms o f fr e e base Cc) Two d i f f e r e n t sou rces of f e t u i n i-* 4* 146 10 8 a c i d . However, the r e s u l t s of F is h e r e t a l . show a v ery c l o s e agreem ent w it h our r e s u l t s . E xclu d in g th r e o n in e and s e r i n e w hich are d i f f i c u l t to a s s e s s , o n ly fo u r amino a c i d s , l e u c i n e , l y s i n e , h i s t i d i n e , and a r g i n i n e , d i f f e r by more than one r e s i d u e . In t h e s e amino a c i d s , how ever, th e y made la c o r r e c t i o n in w hich the d e c r e a s e d amount o f r e s i d u e , d u r- j 1 i ; ling th e h y d r o l y s i s p e r io d o f 16 to 24 h o u r s, was added to jthe maximum v a l u e . I f t h i s c o r r e c t i o n was n o t made, t h e i r v a l u e s th en become i d e n t i c a l w ith o u r s . The d i f f e r e n c e s be-; tween our r e s u l t s and th o s e of S p ir o and S p ir o appear o u t - I I i Isid e o f e x p e r im e n ta l e r r o r . The r e a so n fo r th e d is c r e p a n c y j i i s unknown. j The amino a c id c o m p o sitio n o f th e fo u r f r a c t i o n s an a ly z e d are s i m i l a r . F r a c t io n 2 seems to c o n t a in one or two | r e s i d u e s more o f each amino a c i d , and F r a c t io n 5 seem s to c o n ta in a p p r o x im a te ly one r e s id u e l e s s than F r a c t io n s 3 and ; 4 w hich are v e r y c l o s e in c o m p o s itio n . I t can be r o u g h ly i f i n f e r r e d t h a t th e r e l a t i v e c o m p o s itio n s o f th e fou r f r a c - I j Itio n s are s i m i l a r b u t th e amount o f amino a c id seem s to d e - j ; I Icrease somewhat from F r a c t io n s 2 to 5. The d e c r e a s e in the j i i j \ ip ep tid e p o r t i o n a g r e e s w ith the f i n d i n g by Lowry’ s p r o t e i n ! t e s t . The p e p t id e p o r t io n was more c a r e f u l l y a n a ly zed by t I f in g e r p r in t in g o f F r a c t io n s 2 and 5 ( F i g . 1 2 ) . There were lonly two p e p t i d e s , 7 and 1 7 , w hich moved d i f f e r e n t l y , and Itwo a d d i t i o n a l s p o t s in F r a c t io n 2 w h ich were n ot d e t e c t e d j '.... 147: Jin F r a c t io n 5 . A c t u a l l y , t h e r e seem s to be o n ly s l i g h t d i f - j ; ifer en ce s in the p e p t i d e p o r t i o n , but the d i f f e r e n c e may be j enough to accou n t f o r v a r i a t i o n in e l e c t r o p h o r e t i c m ig ra - I i t i o n . As m en tioned p r e v i o u s l y , th e d i f f e r e n c e i n th e number jand arrangem ent o f amide groups in th e p e p t id e c h a in may r e s u l t in the d i f f e r e n c e in th e f i n g e r p r i n t s , but no f u r t h e r j (experim ents to e x p l o r e t h i s a s p e c t o f p e p t i d e was p erform ed .! I ; j(4) M o le c u la r s t r u c t u r e of f e t u i n in r e l a t i o n to m ic r o - i | h e t e r o g e n e i t y . j i ! There are s e v e r a l p o s s i b i l i t i e s f o r the ca u se o f thej j ( e le c t r o p h o r e t ic s e p a r a t i o n o f f e t u i n : I ! (a ) Charge d i f f e r e n c e s w h ich a re cau sed by the d i f f e r - J en ce in s i a l i c a c id c o n t e n t or the p e p t id e p o r t i o n , p o s s ib ly ! due to th e number or seq u en ce o f amide groups o f a s p a r t i c and g lu ta m ic a c i d , or o th e r c h a r g e d ^ sid e c h a in s ; (b ) Shape and s i z e d i f f e r e n c e s w h ich m ight be c a u sed by th e d i f f e r e n c e s in th e c o m p o s itio n and seq u en ce in th e p e p - ! jtide p o r t io n or c a r b o h y d r a te . The f i r s t o b v io u s d i f f e r e n c e among th e com ponents is! ; i jthe i s o i o n i c p o i n t . T h is charge d i f f e r e n c e i s a l s o presumed jto r e s u l t in p a r t i a l f r a c t i o n a t i o n on io n -e x c h a n g e chroma- jtography. F u r t h e r , th e s e p a r a t i o n , b e s t shown near th e i s o - i jionic p o i n t , i n d i c a t e s t h a t th e charge d i f f e r e n c e i s more \ \ jpronounced t h e r e . At th e i s o i o n i c p o i n t th e charge d i f f e r en ce i s a p p r o x im a te ly 6 p r o to n u n i t s per 0 . 3 pH u n i t s (th e ipH d i f f e r e n c e b e tw een i s o i o n i c p o i n t s o f F r a c t io n s 2 & 5 ) , 148 w h ereas a t pH 8 .0 i t i s a p p r o x im a te ly 1 p r o to n u n it per 0 . 3 pH u n i t s a c c o r d in g to th e t i t r a t i o n curve ( F i g . 1 3 ) . 18 A c co rd in g to S p ir o th e f e t u i n m o le c u le i s composed o f t h r e e s t r u c t u r a l l y s i m i l a r o l i g o s a c c h a r i d e groups a t ta ch ed to a s i n g l e c h a in o f p e p t i d e . T h is c o n c l u s i o n was jbased on p a p a in d i g e s t i o n o f f e t u i n w h ich gave g ly c o p e p t id e ^ jwith 4300 m o le c u la r w e i g h t . F r a c t i o n a t i o n o f th e d i g e s t on! j ! ID E A E -c e llu lo se , how ever, showed s e v e r a l g l y c o p e p t i d e p e a k s , j iwhich w ere a t t r i b u t e d to p e p t i d e c h a in d i f f e r e n c e s . The jmolar r a t i o o f the s i a l i c a c id to h e x o se to h exosam ine in i | t h e s e p e p t i d e s was found to be 1 .0 : ( 1 . 4 7 t o 1 . 7 9 ) : ( 0 . 9 3 j :t o 1 . 1 9 ) . The a v e ra g e v a l u e s came c l o s e to t h o s e o f n a t i v e ! ; i j f e t u i n , but th e f l u c t u a t i o n cou ld be s i g n i f i c a n t , in d icatin g* i d i f f e r e n c e s in c a r b o h y d r a te c o m p o s it io n . The n e x t s t e p o f i n v e s t i g a t i o n would be to l o c a t e any d i f f e r e n c e s in th e ca rb o h y d r a te c o m p o s it io n o f the g l y c o p e p t i d e s . A f t e r a l l , S p ir o o f f e r e d no c o n v in c in g d e m o n s tr a tio n o f h om o g en eity f o f h i s g l y c o p e p t i d e s . The d i f f e r e n c e s in c a r b o h y d r a te c o m p o s it io n , and th e j j j e f f e c t o f d e s i a l i c i z a t i o n , can a f f e c t b oth ch arge and sh a p e ! i - ! or s i z e o f th e f e t u i n m o le c u le . The d i f f e r e n c e i n e l e c t r o p h o r e t i c com ponents co u ld j j ; jeven r e s i d e e x c l u s i v e l y in the ca rb o h y d r a te g r o u p s, s i n c e I jthree o l i g o s a c c h a r i d e u n i t s o f e a c h e l e c t r o p h o r e t i c corapo- j I I Inent c o u ld d i f f e r from each o t h e r . T h is v ie w i s su p p o r te d I |by th e c o m p o s it io n o f th e s i a l i c a c id where o n ly one o u t o f j i 149 \ j 1 C |tw e lv e i s N - g ly c o l y l n e u r a m in i c a c id . No one h a s a c t u a l l y i jstu d ie d i t s d i s t r i b u t i o n , b u t i f i t i s d i s t r i b u t e d e q u a l l y among m o le c u le s , a t l e a s t one o l i g o s a c c h a r i d e u n i t w i t h i n i i th e m o le c u le must be d i f f e r e n t from th e o th er two u n i t s . i A lth ou gh N - g ly c o l y l n e u r a m in i c a c id h a s a s i n g l e c a r b o x y l j j ; Igroup l i k e N - a c e ty ln e u r a m in ic a c i d , th e m i c r o h e t e r o g e n e i t y ! i. . I i s a p p a re n t. j | The amino a c id a n a l y s i s showed minor d i f f e r e n c e s in jco m p o sitio n and the f i n g e r p r i n t i n g d e f i n i t e l y showed some t d i f f e r e n c e s in the p e p t i d e s . The p e p t id e m o ie t y , t h e r e f o r e ,, may a l s o p la y a r o l e in th e m i c r o h e t e r o g e n e i t y by a f f e c t i n g I e i t h e r t h e charge or sh a p e. Immunochemical s t u d i e s s u g g e s t j jthat th e a n t i g e n i c s i t e s o f a l l th e com ponents are s i m i l a r , j H o w e v e r , th e a n t i g e n - a n t i b o d y r e a c t i o n i s n o t v e r y s e n s i t i v e to d e m o n str a te sm a ll d i f f e r e n c e s in th e s t r u c t u r e o f a n tig e n m o l e c u l e s . The s e p a r a t i o n by DEAE-Sephadex chrom atography i n t o j i i e l e c t r o p h o r e t i c com ponents d e f i n i t e l y d e m o n s tr a te s the shape i i i .and s i z e are n o t th e prim ary d i f f e r e n c e s . H owever, th e j i i jshape ( t e r t i a r y c o n fo r m a tio n ) must e v i d e n t l y be m a in ta in e d | j i | i jin order to d i s t i n g u i s h the ch arge d i f f e r e n c e s , b e c a u se no 1 : I f ^sep aration o c cu rred when f e t u i n was d en a tu re d in u r e a or th e i ; i d i s u l f i d e bonds w ere b r o k e n . j ; i ; i ; I |(5 ) I n t e r p r e t a t i o n o f t i t r a t i o n d a ta j The d i r e c t in fo r m a tio n we g e t from a t i t r a t i o n curve i i j i s th e number and t y pe of th e t i t r a t a b l e groups i n t h e _______ i 150 I ] p r o t e in m o le c u le . To compare w ith the ex p ected number, which can be o b tain ed from amino acid a n a l y s i s , the t i t r a - i i i t i o n curve i s rou gh ly d iv id e d in to th r e e p a r t s : (a ) the \ a c id ic r e g io n from pH 2 .0 0 to 6 .3 0 where the carboxyl groups t i t r a t e ; (b) the n e u t r a l r e g io n from pH 6.30 to 9 .3 0 where i Ithe im id a zo le and a-amino groups t i t r a t e ; and ( c ) the b a s ic | jregion from pH 9 .3 0 to 1 2 .0 0 where the e-amino and p h e n o lic i i i jgroups t i t r a t e (T able 17:) . In c o u n tin g the a c i d i c groups, |the maximum number fo r f e t u i n from th e i s o i o n i c p o in t was counted as 36, alth ou gh i t had on ly reached 30 groups at pH ; i i 2 . 0 . For d e s i a l i c i z e d f e t u i n , however, 36 groups had t i - I tr a te d and the number should be the same fo r f e t u i n assuming! |the number of the p o s i t i v e ch arges are the same. As shown in Table 17, the e x p erim en ta l and the ex p e cted number o f t i - t r a t a b l e groups a g r ee s w ith in e x p erim en ta l e r r o r . The change of the i s o i o n i c p o in t from pH 4 .2 3 to 5 .5 7 in 0 .1 6 M KC1 upon removal o f the s i a l i c acid from f e t u i n i n d i c a t e s j the a c id ic natu re o f f e t u i n i s p r im a r ily due to i t s c o n te n t ! i : .of s i a l i c a c id . The charge d i f f e r e n c e between the n a t iv e ! | and th e d e s i a l i c i z e d f e t u i n at pH 7 .0 0 i s 15 which c o in c id e sj jwith the number o f s i a l i c a cid r e s id u e s l o s t as measured | ; jchem ically. The t o t a l number of p o s i t i v e charges at the | iis o io n ic p o in t i s the sum o f the l y s i n e , h i s t i d i n e , and ar- j g in in e r e s id u e s and a-am ino group. I t i s ex p ected t o be I j near 40 from the amino a cid a n a l y s i s . However, the number j i | bf maximum p o s i t i v e ch arg es at pH 2 .0 was 36 fo r 151 TABLE 17 THE TITRATABLE GROUPS OF FETUIN F e tu in D e s i a l i c i z e d F e tu in ' e x p e c te d * found ex p ected found (a) 58 57 42 41 Cb) 1 1 1 0 1 1 13 ( c ) i 24 25 24 25 1 i 93 92 77 79 ; - * 1 ( a ) The a c id r e g io n pH 2 . 0 - - 6 . 3 where g lu ta m ic a c id , aspar-| ; ' ‘ j t i c a c id , a -c a r b o x y l group, and s i a l i c a c id are e x - j | I j p e c te d to d i s s o c i a t e . | j i j(b) The n e u tr a l r e g io n pH 6 . 3 - - 9 . 3 where h i s t i d i n e and j i a-am ino group are e x p e cted to d i s s o c i a t e . - - - Kc) The b a s i c r e g io n pH 9 . 3 - - 1 2 . 0 where l y s i n e and tyrosine^ are e x p e c te d to d i s s o c i a t e . G uanidine groups may d i s - j i : s o c i a t e at or near pH 1 2 .0 but t h e y are n o t counted I | ■ | j h e r e . *In d e ter m in in g the e x p e c te d number, th e r e s u l t s o f amino a c id a n a l y s is f o r th e two f e t u i n f r a c t i o n s , i . e . , F r a c t io n s 3 and 4 , were u sed . The number o f amid n itr o g e n which must be su b tr a c te d from th e number o f a s p a r t ic and g lu ta m ic a c id s was ta k en from S p ir o and S p i r o . ^ 152 d e s i a l i c i z e d f e t u i n , and 30 f o r f e t u i n . For d e s i a l i c i z e d f e t u i n e x t r a p o l a t i o n To a p la t e a u l e a d s t o a v a lu e o f 37 i f which f a l l s c l o s e to th e 40 e x p e c te d from amino a c id a n a ly s i s . I t i s u n d e r s ta n d a b le t h a t f e t u i n would g iv e a low er v a lu e a t pH 2 .0 b e c a u se o f th e low pK o f the s i a l i c a c id . lit i s n o t l i k e l y t h a t some o f the r e s i d u e s are i n v o lv e d in j ! ; Icovalen t l in k a g e in such a way t h a t th e p o s i t i v e ch arge i s I j ; jnot e x p r e s s e d . S in c e i t i s known t h a t f e t u i n m o le c u le s a s - ! 3 a js o c ia te a t low pH, some o f th e g ro u p s may p o s s i b l y be j jburied or in v o l v e d in th e a s s o c i a t e d com p lex, and t h e r e f o r e I i j ' t i t r a t e b elow pH 2 . 0 0 . j i I The e l e c t r o s t a t i c i n t e r a c t i o n f a c t o r w, 0 . 0 2 9 , ob- i i : I t a in e d from th e l o g a r i t h m i c t i t r a t i o n curve ( F i g . 14) comes j c l o s e to th e c a l c u l a t e d w f a c t o r , 0 . 0 3 1 , on th e a ssu m p tio n I t h a t th e d e s i a l i c i z e d f e t u i n i s a compact s p h e r e . T h is agreem ent in th e w f a c t o r i n d i c a t e s t h a t th e d e s i a l i c i z e d f e t u i n m o le c u le i s r a t h e r com pact, b u t n o t n e c e s s a r i l y a ;sphere. I f th e m o le c u le i s a random c o i l , th e e x p e r im e n ta l ' 1 . iw f a c t o r co u ld be as l i t t l e as l / l O o f th e c a l c u l a t e d w v a l-| AO jue. The asymmetry o f th e f e t u i n m o le c u le , how ever, i s I | i j d i f f i c u l t to a s c e r t a i n by t h i s approach s i n c e th e e x p e r i - I ; Imental e r r o r in d e te r m in in g w may be as much as 2 0 %. I ' The l o g a r i t h m i c p l o t o f t i t r a t i o n d a t a showed a m i- j jnor d e c r e a s e in s l o p e as th e pH d e c r e a s e d s u g g e s t i v e o f a j I s lig h t change in th e t e r t i a r y s t r u c t u r e . The w f a c t o r d e - | i jc r e a se s i f th e volum e o f th e p r o t e i n m o le c u le i n c r e a s e s due j ! 153 to u n fo ld in g o f the p e p tid e a ccord in g to e q u a tio n 2. The burvatu re of a p l o t of th e carb o x y l t i t r a t i o n of human s e r - ! urn albumin was i n t e r p r e t e d as the e x p a n sio n o f th e m olecu le lu r in g the t i t r a t i o n . T h e d e t a i l e d in fo r m a tio n on a con fo rm a tio n a l change which i n v o l v e s a change in the number o f i f t it r a t a b le groups, or in the pK^n t of t h e s e g rou p s, in s te a d ! o f m o lecu la r e x p a n sio n , i s n o t a v a i la b l e from the t i t r a t i o n 1 i curve a lo n e . Although the a s s o c i a t i o n o f m o le c u le s at low pH was a n t i c i p a t e d , t h e o r e t i c a l trea tm en t o f such an e f f e c t j jon t i t r a t i o n curve i s l a c k i n g . On the assum ption th a t the j I A O ^ a s s o c i a t i o n a f f e c t s the m olecu lar w e ig h t, Tanford ex p la in s! ! th e in c r e a s e o f the w f a c t o r th a t f o l l o w s a s s o c i a t i o n . j i ■ ■ ! S in c e most t i t r a t a b l e groups are on th e su r fa c e of the m ole-| c u l e , a c e r t a i n number o f t h e s e s id e ch a in groups would be a f f e c t e d by a s s o c i a t i o n , and th e b a s i c assum ption th a t the charge i s spread on the s u r fa c e o f th e a s s o c ia t e d la r g e m o lecu le would n o t h o ld . T h e r e fo r e , when a s s o c i a t i o n i s in-i y o lv e d , the r e s u l t s o f t i t r a t i o n must be taken w ith c a u tio n . In F ig . 14 the t o t a l number o f ch a rg es was equated | ; w ith th e ch arges c r e a te d by a s s o c i a t i o n or d i s s o c i a t i o n of jprotons, and no c o n s i d e r a t io n was a llo w ed f o r the s a l t io n s p o s s i b l y absorbed to th e m o le c u le . S in c e the i s o i o n i c pointj i i jof f e t u i n i n c r e a s e s from pH 4 .0 3 in pure water to 4 .2 3 in j ; ! 0 .1 6 M KC1, some d egree of anion a b s o r p tio n seems to o c c u r , j I But due to the l a c k of a proper method of a ssessm e n t of the ) number o f a b s o r b e d C l“ i o n s , i t was n o t measured and sim p ly 154 treated as an experimental error. One drawback o f t h i s exp erim en t was th e u n c e r t a in t y i o f th e s t r u c t u r a l s t a b i l i t y when f e t u i n was t r e a t e d fo r d e- s i a l i c i z a t i o n w ith 0 .0 2 5 N H^SO^ at 80° f o r one hour. This trea tm en t could have caused some i r r e v e r s i b l e con fo rm a tio n a l i i ! jchanges w hich were in d ic a t e d by s ta r c h g e l e l e c t r o p h o r e s i s . | A lthou gh n euram inidase trea tm en t d oes not remove a l l th e si-; ! ' I ja lic acid r e s i d u e s , as the a cid trea tm en t d o e s , i t would be i i ; jad visab le t o check w hether th e n e u r a m in id a s e -tr e a te d f e t u i n ; I i jis t i t r a t e d s i m i l a r l y . j i i j ; i ( 6 ) I n f lu e n c e o f s i a l i c a c id on the conform ation of f e t u in .i As se en from T able 14 , the f e t u i n m o lec u le d o es not | behave as a sp h e r e . I t may be somewhat e lo n g a te d w ith an | ia x ia l r a t i o o f r e v o l u t i o n o f 5 . 3 , or i t may be a f l a t m ole c u le w ith the a x i a l r a t i o o f 6 . 8 . T h is f i n d in g i s s t i l l com p a tib le w ith the sm a ll d i f f e r e n c e observed betw een the t h e o r e t i c a l and the e x p e r im e n ta l w f a c t o r s . j There appears no change in th e shape or con form ation t ; o f the f e t u i n m olecu le by c le a v in g o f f th e s i a l i c a c id r esi-j i dues by n eu ram in id ase tr e a tm e n t. The d i f f e r e n c e in th e i n - ! t r i n s i c v i s c o s i t y o f 5 .5 m l/g f o r f e t u i n to 5 .2 m l/g fo r d e- ! s i a l i c i z e d f e t u i n i s very s m a ll. Human serum album in, fo r lexample, was shown t o d en a tu re at low pH and form an expand-! jed m o lecu le w hich accom panies a t w o -fo ld in c r e a s e in i n t r i n - : • -a. 1 1 9 ,1 2 0 A - , . . . _ _ iSic v i s c o s i t y . A t e n - f o l d in c r e a s e m reduced 155 v i s c o s i t y was r e p o r te d when human serum albumin was d en a- 121 tu red i n 8 M u r e a . S in c e no such d r a s t i c i n c r e a s e in i n t r i n s i c v i s c o s i t y f o r d e s i a l i c i z e d f e t u i n was o b s e r v e d , i t would be s a f e to assume th a t no a p p r e c ia b le change in con fo r m a tio n r e s u l t e d from removing the s i a l i c a c id r e s i d u e s from f e t u i n . The sm all change may be a r e f l e c t i o n of th e j j : iform ation of a s l i g h t l y sh o r te r m o le c u le by rem oving th e i j | {term inal sugar from th e o l i g o s a c c h a r i d e u n i t s . i | The i n t r i n s i c v i s c o s i t y o f f e t u i n r e p o r te d by j i I *j j * {Spiro of 7 . 8 m l/g i s c o n s id e r a b ly g r e a t e r than the 5 .5 j 1 ' : m l/g w hich was found in t h i s la b o r a t o r y . The cau se o f d i s - j crep an cy may be due t o the d i f f e r e n t c o n d i t i o n s used in d e - j • | te r m in in g the i n t r i n s i c v i s c o s i t y . He used p o ta ssiu m p h o s- ' ph ate b u f f e r , a t pH 6 . 5 , 0 .1 i o n i c s t r e n g t h w ith sodium c h l o r i d e making up 80% o f the i o n i c s t r e n g t h a t 2 0 ° and m easured the d e n s i t y o f each s o l u t i o n . W e u sed p o ta ssiu m ph o sp h ate b u f f e r , pH 7 . 5 , 0 .1 6 i o n i c s t r e n g t h w ith p o ta ssiu m c h l o r i d e making up 75% o f the i o n i c s t r e n g t h at 25° and th e ; j . j i i n t r i n s i c v i s c o s i t y was c o n v e r te d from the i n t r i n s i c k i n e m a tic v i s c o s i t y u s in g 0 .6 9 6 m l/g f o r the p a r t i a l s p e c i f i c j I volum e. A lthou gh tem p eratu re sh ou ld n o t a f f e c t the i n t r i n - | 1 P7 s i c v i s c o s i t y g r e a t l y , th e e f f e c t o f i o n i c s t r e n g t h may i l l ■ be a p p r e c ia b le . Green and Kay r e p o r te d t h a t the i n t r i n - i jsic k in e m a tic v i s c o s i t y o f e t h a n o l - f r a c t i o n a t e d f e t u i n was i j6 .6 5 m l/g in p h osp hate b u ffe r and 7 .9 5 m l/g in b o r a te bufferj ! I jat n e u t r a l pH and 0 .1 6 i o n i c s t r e n g t h . They s u g g e s t e d the | 156 j j v i s c o s i t y o f f e t u i n i s s e n s i t i v e to ch a n g es in th e i o n i c I i : jco m p o sitio n o f the b u f f e r . S in c e p h o sp h a te b u f f e r s were used in t h r e e l a b o r a t o r i e s , th e d i f f e r e n c e in th e b u f f e r io n s can n ot be th e r a t i o n a l e o f th e d i f f e r e n c e s in i n t r in s i c j v i s c o s i t y . I i } ; I ! Ju d g in g from th e mean r e s i d u e r o t a t i o n a t 233 mp, I | I jthe h e l i c a l c o n t e n t o f f e t u i n i s n o t changed a p p r e c ia b ly by ; jthe e n z y m a tic rem oval o f s i a l i c a c id (T a b le 1 7 ) . A lthough ! ; j f e t u in i s n o t h i g h l y h e l i c a l , the r e s u l t s i n d i c a t e th e s e c - j jondary s t r u c t u r e i s not d i s t u r b e d , and th e o v e r a l l s t r u c t u r a l c o n fo r m a tio n u n a l t e r e d . T h is c o n c lu s i o n i s c o m p a tib le w ith th e r e s u l t s of v i s c o s i t y s t u d i e s . I t d i s a g r e e s , how- ! j i ;e v e r , w i t h the r e s u l t s o b ta in e d from o p t i c a l r o t a t o r y s t u d - : 34 i e s by V erp o o rte e t a l . , who r e p o r t e d th a t the tr e a tm e n t o f f e t u i n w ith n e u r a m in id a se d e c r e a s e d th e h e l i c a l c o n te n t from 15% t o 0%. They s u g g e s t th a t s i a l i c a c id r e s i d u e s are in some way n e c e s s a r y f o r th e m a in te n a n c e o f th e seco n d a ry s t r u c t u r e . The c a u se f o r the d is c r e p a n c y i s unknown. I The h e l i c a l c o n t e n t was e s t im a t e d by Kay and c o - j i w ork ers by com p arison w it h th e h e l i c a l form o f p o ly - L - j 5 3 34 ’ g lu ta m a te and h e a t -d e n a t u r e d f e t u i n . ’ S in c e th e c a r b o - i h y d ra te i s n o t d i s r u p t e d by h e a t d e n a t u r a t i o n , th e y assumed j t h a t th e o p t i c a l r o t a t o r y change was in d ep en d en t o f th e car-; I j ibohydrate and e n t i r e l y due to c h a n g es m th e h e l i c a l p ep - I I - tid e . In s p i t e o f t h e s e d a t a , h o w ever, Beychok and j ; i IKabat r e p o r te d t h a t th e amide l i n k a g e in 1 N - a c e ty lh e x o s a m in e g i v e s a s i m i l a r o p t i c a l r o t a t o r y d i s p e r - ; s i o n cu rve to p r o t e i n s . They have shown t h a t th e P- p y r a n o s id e s ( b u t n o t the a - p y r a n o s i d e s ) o f N- a c e t y l g lu c o s a m in e and N - a c e t y l g a l a c t o s a m i n e have t h e i r m i n i mum p o i n t s a t 230 m|i. They have a l s o shown t h a t pneum ococ- | j leal p o l y s a c c h a r i d e , a b ran ch ed polym er c o n s i s t i n g o f D- | i ; g l u c o s e , D - g a l a c t o s e , and N - a c e t y l - D - g l u c o s a m i n e , i n aqueous; i [ ' s o l u t i o n h a s a tr o u g h a t 230 mp. S in c e f e t u i n c o n t a i n s •amide g rou p s in s i a l i c a c id and h e x o sa m in e , t h e i r c o n t r i b u - : t i o n to o p t i c a l r o t a t i o n a t 233 mu c o u ld be s u b s t a n t i a l . No' ! | one h as y e t shown i f the a or P p y r a n o s i d e s o f s i a l i c a c id j g i v e o p t i c a l r o t a t i o n a t 233 mp. T h e r e f o r e , th e e s t i m a t e d 1 lvalu es o f h e l i c a l c o n t e n t o f f e t u i n , or any o th e r g l y c o p r o - j t e i n must be ta k e n w ith r e s e r v a t i o n . In t h i s r e g a r d , th e s l i g h t d e c r e a s e in th e p a r a m e te r s f o r h e l i c a l c o n t e n t o f de-; s i a l i c i z e d f e t u i n m ight be due to th e l o s s o f th e amide g rou p s o f s i a l i c a c id r a th e r than the h e l i c a l c o n t e n t . This! : ! i n t e r p r e t a t i o n seems t o a g r e e w ith t h e r e s u l t s o b ser v ed with! th e d e s i a l i c i z e d f e t u i n in t h i s l a b o r a t o r y , s i n c e , when f e - I jtuin was d e s i a l i c i z e d , th e tro u g h changed from 229 to 233 mp; CFig. 1 6 ) . The maximum d e c r e a s e in n e g a t i v e mean r e s i d u e ! r o t a t i o n a t 229 mp c o u l d , t h e r e f o r e , i n d i c a t e th e l o s s o f ; j c a r b o h y d r a te amide grou p s r a t h e r th a n change in h e l i c a l c o n -| } < ! j t e n t . I 123 • ■ Schmid and Kamiyama s t u d i e d th e e f f e c t o f d e s i - j ^ l i c i z a t i o n on the s t r u c t u r e o f a ^ - a c i d g l y c o p r o t e i n . They j 158 concluded t h a t removal of a r e l a t i v e l y l a r g e group of n e g a t i v e l y charged s i a l i c a c id r e s id u e s produced l i t t l e a l t e r a - i t i o n in th e secon dary s t r u c t u r e o f the g l y c o p r o t e in b eca u se the o p t i c a l r o t a t i o n o f th e sodium D l i n e changes s i m i l a r l y when observed in w ater and 8 M u rea b e fo r e and a f t e r removal Of s i a l i c a c id . This c o n c lu s io n was a ls o supported by th e ; j s im ila r it y in th e pH p r o f i l e of the s p e c i f i c o p t i c a l r o t a t i o n of both g l y c o p r o t e i n s . F u r th e r, the s o l u b i l i t y p r o p e r t i e s were a l s o i d e n t i c a l . T heir d ata i n d i c a t e s th a t the ! I i O lig o s a c c h a r id e u n i t s do n ot c o n tr ib u t e s i g n i f i c a n t l y to thej | i i i con form ation o f t h i s g l y c o p r o t e in , which i s o f a n o n -h e lic a l! } Itype and e x tr e m e ly s t a b l e as judged by slo w h e a t d e n a tu ra - j itio n and r e n a tu r a t io n a f t e r exposure to r e a g e n t s such as ; jurea, g u a n id in e HC1, and p h e n o l. In c o n t r a s t to the above f i n d i n g s are s t u d i e s on f e t u in by Green and K a y ^ ^ and t h e i r c o l l e a g u e s . ^ They su g g e s t e d the h i g h ly charged s i a l i c a c id groups c o n t r ib u t e ap p r e c i a b l y to the m aintenance of th e seco n d ary and t e r t i a r y I s t r u c t u r e o f f e t u i n , b ecau se d e s i a l i c i z e d f e t u i n was more |l ia b le to t r y p t i c a t t a c k , and th e change in o p t i c a l rotatory! d i s p e r s i o n in d ic a t e d the l o s s o f h e l i c a l c o n te n t. As observed by t i t r a t i o n , v i s c o s i t y and o p t i c a l r o - j Itatory d i s p e r s i o n s t u d i e s performed in t h i s la b o r a t o r y , r e - j ; _ i moval of the s i a l i c a cid from f e t u i n d oes n o t le a d t o d r a s - j I i it ic changes in the s t r u c t u r e o f the g ly c o p r o t e in a lth o u g h itj j ] io b v io u s ly red u c es the m o lecu lar w eig h t by ap p roxim ately 6 %. j ! I B eing a t the p e r ip h e r y o f th e o l i g o s a c c h a r i d e u n i t s , and u n d o u b te d ly e x i s t i n g a t the s u r f a c e o f th e m o le c u le r a th e r than i n s i d e where i t would i n t e r a c t w it h hydrophobic s i d e c h a in s o f the p e p t i d e , s i a l i c a c id can be e lim in a t e d w i t h out a l t e r i n g th e o v e r a l l s t r u c t u r e o f th e m o le c u le . SUMMARY AND CONCLUSION F e t u i n , a f e t a l c a l f serum g l y c o p r o t e i n , was i s o l a t e d by a l c o h o l - m e t a l io n f r a c t i o n a t i o n . The h o m o g en eity j jof the p r e p a r a t io n h as been d em o n stra ted by c o n v e n t io n a l ( te c h n iq u e s . A m i c r o h e t e r o g e n e i t y , how ever, was c l e a r l y • ! ' jdem onstrated by r e s o l u t i o n i n t o e i g h t com ponents on s t a r c h j | |g el e l e c t r o p h o r e s i s at pH 4 . 2 . I t was shown t h a t th e micro-: ' | ( h e t e r o g e n e it y did n o t r e s u l t from th e method o f a l c o h o l - ; j i m etal io n f r a c t i o n a t i o n b e c a u se u n f r a c t i o n a t e d serum and the! t f e t u i n , p r e c i p i t a t e d by ammonium s u l f a t e , a l s o showed th e ; same e l e c t r o p h o r e t i c b a n d s. F u rth erm o re, th e m ic r o h e t e r o g - j e n e i t y d id n o t r e s u l t from p o o l i n g o f s e r a from many f e t a l c a l v e s b e c a u s e th e i d e n t i c a l b a n d s , c h a r a c t e r i s t i c o f f e - . t t u i n , were p r e s e n t in s e r a c o l l e c t e d from f i v e f e t a l c a lv e s .! | The d e s i a l i c i z a t i o n red u ced th e number o f bands from I : I ) I j 'e ig h t to t h r e e w hich i n d i c a t e d th e s i a l i c a c id i s p a r t i a l l y | I j ( r e s p o n s ib le f o r th e m i c r o h e t e r o g e n e i t y . The f e t u i n d e n a - ! ! ■ 1 {tured in u r e a as w e l l as f e t u i n t r e a t e d by 2 -m ercap to eth an o li j f a i l e d t o show d e f i n i t e e l e c t r o p h o r e t i c bands and em p hasized j jthe im p o rta n ce o f t e r t i a r y s t r u c t u r e . i F e t u in was p a r t i a l l y r e s o l v e d by DEAE-Sephadex, and ! jthe p h y s i c a l and c h em ica l p r o p e r t i e s o f th e f r a c t i o n s were j I 160 (compared. The s i m i l a r i t y in the immunochemical p r o p e r t i e s Iwere shown by im m u n o d iffu sio n and im m u n o e le c t r o p h o r e s is . j ; The s e d im e n t a t io n c o n s t a n t s and th e a b s o r p t io n c o e f f i c i e n t s a t 278 mp were a l s o s i m i l a r . The i s o i o n i c p o i n t s o f th e I ( f r a c t io n s d e c r e a s e d in th e order o f e l u t i o n . The s i a l i c i ' i : 1 jacid and h e x o se c o n t e n t in c r e a s e d s l i g h t l y , b u t the h e x o s a - j jmine was th e sam e. T h is was su pp lem en ted by a sm a ll d e - i c r e a s e in th e p e p t id e c o n t e n t . The r e s u l t s of amino acid i : .a n a l y s i s r e v e a le d r a t h e r a s i m i l a r i t y among F r a c t io n s 3 - 5 , jbut minor d i f f e r e n c e s from F r a c t io n 2 . F in g e r p r in t i n g r e v e a le d a d e f i n i t e d i f f e r e n c e in two p e p t i d e s betw een F r a c - j t i o n s 2 and 5. ; ! ! i I t was c o n c lu d e d th a t the ca u se o f m ic r o h e te r o g e n e - : (ity r e s i d e s p r i m a r i ly in d i f f e r e n c e s in c h a r g e s . However, m o le c u la r c o n fo r m a tio n must be m a in ta in e d to r e v e a l th e charge d i f f e r e n c e s . The s i a l i c a c id i s a t l e a s t p a r t i a l l y r e s p o n s i b l e f o r th e charge d i f f e r e n c e s , and the p e p t i d e por-; t i o n as w e l l may c o n t r i b u t e b e c a u s e d i f f e r e n c e s in th e p e p - j i # S (tid e c h a in s were r e v e a l e d by f i n g e r p r i n t i n g . i ! ; i The t i t r a t i o n s t u d i e s c a r r ie d ou t on a l c o h o l - i f r a c t i o n a t e d f e t u i n showed th a t th e number o f t i t r a t a b l e groups in a c i d i c , n e u t r a l and b a s i c r e g i o n s agreed w ith th e j | number b a sed on amino a c id a n a l y s i s . There was no d r a s t i c | I 1 ( d if f e r e n c e b etw een th e t h e o r e t i c a l e l e c t r o s t a t i c i n t e r a c t i o n (fa c to r w ( u s in g th e c a r b o x y l g ro u p s) o f d e s i a l i c i z e d f e t u i n , jO.0 3 1 , and th e e x p e r im e n ta l v a l u e , 0 . 0 2 9 . T h is r e s u l t j 1 i s u g g e s t s th a t th e m o lec u le i s r a th e r compact. There was l i t t l e d i f f e r e n c e in the i n t r i n s i c v is c o s -i i t y between n a t i v e f e t u i n ( 5 . 5 m l/g ) and d e s i a l i c i z e d f e t u i n ( 5 . 2 m l / g ) , and i n d i c a t e s no o v e r a l l shape change took p la c e on l o s s o f s i a l i c a c id . | The h e l i c a l c o n t e n t s f o r n a t i v e f e t u i n , 25%, and de-| s i a l i c i z e d f e t u i n , 2 1 % , were c a lc u l a t e d from the mean r e s i - I due r o t a t i o n at 233 mp. Such a sm a ll d i f f e r e n c e s u g g e s t s , i i l i k e the oth er d a ta , no a p p r e c ia b le change in the secon dary j i t s t r u c t u r e o f the g l y c o p r o t e in by removing the s i a l i c a c id . I t | j ! 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This dissertation has been microfilmed exactly as received 67— 13,037 OSHIRO, Yuki, 1935- MICROHETEROGENEITY OF FETUIN. University of Southern California, Ph.D., 1967 Biochemistry University Microfilms, Inc., Ann Arbor, Michigan

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Asset Metadata

Creator Oshiro, Yuki, 1935- (author)

Core Title Microheterogeneity of fetuin

School Graduate School

Degree Doctor of Philosophy

Degree Program Biochemistry

Degree Conferral Date 1967-04

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 Eylar, Edwin H. (committee chair), [illegible] (committee member), Mehl, John W. (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c18-142453

Unique identifier UC11360280

Identifier 6713037.pdf (filename),usctheses-c18-142453 (legacy record id)

Legacy Identifier 6713037.pdf

Dmrecord 142453

Document Type Dissertation

Rights Oshiro, Yuki

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