A study of protein separation using ion exchange resins

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Content A STUDY OF PROTEIN SEPARATION USING ION EXCHANGE RESINS
A T h e s is
P r e s e n t e d to
th e D epartm ent o f B io c h e m is tr y and N u t r i t i o n
The U n i v e r s i t y o f S o u th e rn C a l i f o r n i a
I n P a r t i a l F u l f i l l m e n t
o f t h e R e q u ire m e n ts f o r th e Degree
M a ste r o f S c ie n c e
by
B u rto n K alim an
F e b ru a ry 1951
B .'o . 15) /cW
This thesis, written by
Burton Kallman
under the guidance of h.ls....Faculty Committee,
and approved by all its members, has been
presented to and accepted by the Council on
Graduate Study and Research in partial fulfill
ment of the requirements for the degree of
Master of Science
Dean
D ate.......
Faculty Committee
Chairman
TABLE OF CONTENTS
PAGE
INTRODUCTION .......................................................................................... I ;
I
HISTORICAL SURVEY ......................... 3-'
A. I o n Exchange Re s i n s . . . . . . . . . . . . . . . . . . . . . . . 3 j
B . Muc opr o te in s ....................................................................... 8
C. P r o t e i n I s o e l e c t r i c P o i n t s . . . . . . . . . . . . . . . . 9j
I
METHODS AND MATERIALS . * . . . . . . . . . . . . . . .................... . 11'
A. I o n Exchange R e s in s . . . . . . . . . 11,
B. P r o t e i n s ................................................................................. 14
G. T y ro sin e D e te r m in a tio n s . . . . . . . . . . . . . . . . . . . 1 4 :
EXPERIMENTAL • » • « » » .. .• • « » • » • » » » » • » . » » • • • » ♦ » • » .* » SO1
A. IRA 400 . . . . .......... . . . . . . .................................................. 20;
I
B . IR 4B ............... . . . . . . . . . . . . . . . . . . 22
C. IRC 30 2 3 1
I
D. IR 120 ...................................................... .. 3 0 j
DISCUSSION AND CONCLUSIONS . ............. ..................................... 33
BIBLIOGRAPHY .................................... ......................................... 36 I
LIST Of TABLES
I . P r o t e i n I s o e l e c t r i c P o i n t s
I I . E x p e r im e n ta l R e s u l t s . . .
LIST OF FIGURES
FIGURE PAGE
1* P r o t e i n P r e p a r a t i o n P ro c e d u re ..................................... 15
2 . T y ro s in e S ta n d a rd Curve ........................ 17
3* Serum Albumin. S ta n d a r d Curve ...................... ............ ** 18
4* Blue o p ro te i n S ta n d a r d Curve . . . . . . . . . . .................* 19
5 . Serum Albumin on IRA 400 Column a t pH 2 .9 . . . 23
6 * . M u e o p ro te in on IRA 400 Column a t pH 2 .6 . . . . . 24
7 . Serum Albumin on IRC 50 Column a t pH 2*5 . . . . 26
8 . B e e f Albumin on IRC 50 Column a t pH 2 .6 28
9 . B e e f Albumin on IRC 50 Column a t pH 2 . 6 ;
Crude Q u a n t i t a t i v e G lucose A n a ly s is . . . . . . . 29
10* Serum Albumin on IR 120 Column a t pH 2 .9 . . . . 51
11* M u e o p ro te in on. IR 120 Column a t pH 1 .5 . . . . . . 32
; INTRODUCTION
l
I
i
i
A v a r i e t y o f p h y s i c a l m ethods a r e u t i l i z e d t o
's e p a r a t e and p u r i f y p r o t e i n s . These in c lu d e s e p a r a t i o n by ;
I ;
( d i f f e r e n t i a l s o l u b i l i t i e s , 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 l a t t e r two m ethods a re t e d i o u s p r o c e s s e s and !
| I
[ in v o lv e e x p e n siv e and i n t r i c a t e a p p a r a t u s . O th er m e th o d s, j
[d e v e lo p e d r e c e n t l y , depend upon th e d i f f e r e n c e s i n p r o t e i n
I
I i s o e l e c t r i c p o i n t s . Putnam and N e u ra th (1) have demon-
j i
; s t r a t e d t h a t d e t e r g e n t io n s p r e c i p i t a t e o p p o s i t e l y c h a rg e d ,
i
p r o t e i n s . A p r o t e i n m ix tu re can th u s be p a r t i a l l y p u r i f i e d |
S
by a d j u s t i n g th e s o l u t i o n t o a d e s i r e d pH and p r e c i p i t a t i n g j
I
: th o s e p r o t e i n m o le c u le s o f e i t h e r c h a rg e by a d d in g a d e t e r -
I ,
ig e n t o f th e o p p o s i t e c h a r g e . F u r t h e r p u r i f i c a t i o n can be ;
I !
j o b ta in e d by r e p e a t i n g th e p r o c e d u r e .
i [
I A second such m ethod, d e v e lo p e d by F ra z e r and c o - \
1 j
w o rk e rs ( 2 ) , u t i l i z e s t h e p r e f e r e n t i a l a d s o r p t i o n o f p r o - ,
t e i n s a t c h a rg e d o i l / w a t e r i n t e r f a c e s . Charged and s t a b i l - j
!
liz e d o i l - i n - w a t e r e m u ls io n s a re f l o c c u l a t e d by p r o t e i n m ole-,
! !
[c u le s o f t h e o p p o s ite c h a r g e . The a d s o rb e d p r o t e i n s can be !
!d e so rb e d from t h e f l o c c u l a t e d m a t e r i a l by c h a n g in g th e pH ■
| j
I to th e o p p o s i t e s id e o f t h e i s o e l e c t r i c p o i n t . I
I i
I I
j A somewhat s i m i l a r s e p a r a t i o n seemed a v a i l a b l e |
th r o u g h th e use o f io n exchange r e s i n s . These r e s i n s a r e [
I
s p e c i f i c f o r io n s o f a p a r t i c u l a r c h a rg e and t h e o r e t i c a l l y j
o f f e r a q u ic k and sim p le method o f s e p a r a t i n g p r o t e i n s . :
I
I f th e exchange r e s i n s were a b le to q u a n t i t a t i v e l y ta k e up ;
i
t
p r o t e i n m o le c u le s o f th e p r o p e r c h arg e th e y c o u ld be u se d |
w ith p r o t e i n s o l u t i o n s a t a pH i n t e r m e d i a t e to th e i s o - j
e l e c t r i c p o i n t s o f two p r o t e i n s i n a m i x t u r e , a s i n th e j
above m e th o d s. I t was th e p u rp o se o f th e p r e s e n t i n v e s t i - j
g a t i o n to d e te rm in e w h e th er such a s e p a r a t i o n o f p r o t e i n s I
i s p o s s i b l e . P a r t i c u l a r em phasis was p l a c e d upon th e
s e p a r a t i o n o f serum m u e o p r o te in , a b lo o d c o n s t i t u e n t o f
|
c l i n i c a l im p o rta n c e , from th e o t h e r serum p r o t e i n s . The
i
p ro b lem was a t t a c k e d by a s tu d y o f th e e f f e c t s o f io n e x - j
change r e s i n s on s o l u t i o n s o f p u re p r o t e i n s a t v a r i o u s pH i
l e v e l s . !
HISTORICAL SURYEY
A. ION EXCHANGE RESINS
Hie phenomenon o f io n exchange h a s "been known f o r j
, more t h a n a c e n tu ry * I t was o r i g i n a l l y d i s c o v e r e d by a
, s o i l e h e m is t, J* T* Way, who was c u r i o u s a s t o why w a te r j
s o l u b l e f e r t i l i z e r s were n o t washed o u t o f s o i l by r a i n f a l l . ;
| E x p e r im e n tin g w i t h s o i l c o lu m n s, he found t h a t on a d d i t i o n J
, o f ammonium s u l f a t e s o l u t i o n s ammonium io n was h e ld b y th e
! column and c a lc iu m s u l f a t e was p r e s e n t i n th e e f f l u e n t wash*
i
| Way c a l l e d t h i s e f f e c t rtc a t i o n e x c h a n g e " . Many io n e x c h a n- 1
I g e r s , b o th c a t i o n and a n io n , a r e now in u s e . These in c lu d e
1 o r g a n ic and i n o r g a n i c , n a t u r a l and s y n t h e t i c m a t e r i a l s *
i
i I o n e x c h a n g e rs may be c o n s i d e r e d , s t r u c t u r a l l y , a s
| ;
: i n s o l u b l e , p o ro u s r e s i d u e s (w hich may be a c i d s , b a s e s o r ;
I ;
1 s a l t s ) i n whose c h a n n e ls a r e l o c a t e d s o l u b l e , e x c h a n g e a b le 1
! I
! io n s* U n t i l . q u i t e r e c e n t l y th e o n ly e x c h a n g e rs i n l a r g e I
; i
. s c a l e u se were th e n a t u r a l and s y n t h e t i c z e o l i t e s , which. J
j
| a r e a l u m i n o s i l i c a t e m i n e r a l s o f th e a p p ro x im ate fo rm u la j
1 J, i
: Na (A lS ig O s) ( 3 ) . These m a t e r i a l s a r e c a t i o n e x - j
j c h a n g e r s , b u t can n o t be u se d i n th e c o m m e rc ia lly im p o r ta n t !
, h y d ro g en c y c le ( p r o d u c t i o n o f d e io n i z e d w a te r by th e r e -
; p la c e m e n t o f m e t a l l i c c a t i o n s w ith h y d ro g e n io n s and su b -
, s e q u e n t a c i d a b s o r p t i o n on an a n io n e x c h a n g e r) s in c e th e y ;
i
, a r e d e s t r o y e d i n a c i d s o l u t i o n w i t h th e fo r m a tio n o f s i l i c a .;
I
4
In 1935 p h e n o l-fo rm a ld e h y d e r e s i n s were r e c o g n iz e d
a s e f f i c i e n t c a t i o n e x c h a n g ers* These r e s i n s a r e p ro d u ce d
■ by m ix in g c o n c e n t r a t e d s o l u t i o n s o f p h e n o l o r p h e n o li c
a c i d s and fo rm aldehy de i n th e p r e s e n c e o f an a c i d c a t a l y s t .
M ethylene b r i d g e s a r e formed a t random on th e o r t h o and
p a r a p o s i t i o n s , p ro d u c in g a t h r e e d im e n s io n a l n e tw o rk :
! The p h e n o lic h y d ro g e n , and th o s e o f a n y s u b s t i t u t e d a c i d
group on th e p h e n o l, a r e e x c h a n g e a b le . These s y n t h e t i c
■ r e s i n s a r e more e f f i c i e n t e x c h a n g e rs th a n th e z e o l i t e s a s
t h e y a r e more r a p i d , have a h i g h e r c a p a c i t y and do n o t
c o n ta m in a te th e e f f l u e n t w ith t r a c e s o f s i l i c a t e .
Anion e x c h a n g e r s i n com m ercial u se a re m a in ly s y n
t h e t i c o r g a n ic r e s i n s p ro d u ce d by th e c o n d e n s a t io n o f
p o ly a m in e s w ith fo rm a ld e h y d e , fo rm in g i n s o l u b l e b a s e s o f
v e r y h ig h m o le c u la r w e i g h t.
Io n exchange may be d e fin e d a s wth e r e v e r s i b l e i n t e r
change o f io n s betw een a l i q u i d and a s o l i d i n v o l v i n g no
a p p r e c i a b l e change i n th e s t r u c t u r e o f th e s o l i d ” ( 4 ) . A
t y p i c a l c a t i o n ex change r e a c t i o n i s :
where R r e p r e s e n t s th e i n s o l u b l e p o r t i o n o f t h e resin*.
OH
2 NaR * Ca+* i . y CaRg * 2 Ha*
5
T h is r e a c t i o n i s a t y p i c a l r e v e r s i b l e r e a c t i o n , i h s o f a r
a s a l k a l i m e ta l c a t i o n s a r e c o n c e rn e d . Some h e a v y m e ta l
i o n s , h ow ever, a p p e a r t o u n d e rg o s p e c i f i c , n o t r e a d i l y r e -
v e r s i b l e r e a c t i o n s a t t h e r e s i n s u r f a c e . Such r e a c t i o n s
a r e a n a la g o u s t o th e b e h a v io r o f th e s e m u l t i v a l e n t and
r e a d i l y p o l a r i z e d io n s i n s o l u t i o n , in w hich th e y form
s t a b l e com plexes w ith n e u t r a l m o le c u le s o r a n i o n s . Such
io n s a r e b e s t removed from th e r e s i n by w ashin g w ith a r e
a g e n t w ith w hich t h e y form s t a b l e a n io n s ( 5 ) ,
The m echanism o f a n io n ( a c id ) exchange i s somewhat
more com plex. I t was f o r m e r ly th o u g h t t h a t t r u e a n io n ex
change d id n o t ta k e p l a c e , and t h a t " a n io n e x c h a n g e rs "
were a c t u a l l y a c i d a d s o r b e n t s a c t i n g by s a l t fo rm a tio n ( 6 ):
R * BHg * HCL RHHg , HCL
K unin ( 7 ) , how ever, p r e s e n t s e v id e n c e f o r t r u e a n io n ex
c h a n g e , s u g g e s t i n g a r e a c t i o n o f th e f o l lo w in g t y p e :
R.HHg * H3 0* * GET , . . > RHHg (H3 O )* * q h ~
RNHg (H3 O}* 4 * O H "* 4 0 1 “ . . . . > R m g (K 3 Q}* * C l" * OH"
OH" 4 HgO4 , -------> JSHgO
R e a c ti o n s o f b o t h ty p e s a p p e a r t o o c c u r . The i n i t i a l s t e p
o f a n io n exchange i s p r o b a b ly an a c id a d s o r p t i o n , b u t t r u e
a n io n exchange does o c c u r b etw een th e a h io n o f an a c i d ex
h a u s te d r e s i n and a second a c i d :
6
R = N * H G 1 > R s N. HC1
a R £ K1HC1 * NagSO^ . . . .> (R = N££)g S04 * 2NaCl ( 5 | .
Some p h y s i c a l a d s o r p t i o n may ta k e p l a c e a t t h e r e s i n
surface*. Among th e amino a c i d s , tr y p to p h a n seems t o he a d
so rb e d p r e f e r e n t i a l l y ; t h i s may be a g e n e r a l e f f e c t o f
a r o m a tic compounds ( 5 ) . P y r i d i n e h a s b e e n found e s p e c i a l l y
d i f f i c u l t t o remove from p h y s i c a l c o m b in a tio n with, th e
r e s i n (5 )^
S e v e r a l s t u d i e s on th e k i n e t i c s o f t h e exchange
r e a c t i o n s have been p e rfo rm e d i n r e c e n t y e a rs* Nachod and
Wood (8 ) r e p o r t t h a t th e r a t e o f i o n u p ta k e f i t s a sim p le
second o r d e r b i m o l e e u l a r r e a c t i o n e q u a t i o n , and c o n c lu d e
t h a t th e exchange r e s i n may be t r e a t e d a s th o u g h i t were a
d i s s o l v e d r e a c t a n t . They a l s o found a r e l a t i v e l y s m a ll
t e m p e r a tu re e f f e c t on th e r e a c t i o n r a t e , i n d i c a t i n g t h a t
t h e d i f f u s i o n o f io n s i n t o and o u t o f t h e e x c h a n g er i s
n o t th e r a t e d e te r m i n e r .
Boyd, Adamson and Myers ( 9 ) , h o w ev er, r e p o r t t h a t i n
s o l u t i o n s i n w hich th e t o t a l e l e c t r o l y t e c o n c e n t r a t i o n i s
0 . 1 M o r g r e a t e r th e v e l o c i t y o f th e exchange r e a c t i o n i s
c o n t r o l l e d by t h e r a t e o f d i f f u s i o n i n t o and th ro u g h th e
r e s i n p a r t i c l e . In v e r y d i l u t e s o l u t i o n s th e r a t e o f e x
change i s g o v ern ed by th e r a t e o f d i f f u s i o n th r o u g h a
l i q u i d f i l m a t th e p e r i p h e r y o f th e r e s i n p a r t i c l e .
7
Nachod and Wood r e p o r t in a n o t h e r p a p e r (10) t h a t
, th e exchange e q u i l i b r i u m o f a c a t i o n e x c h a n g e r o p e r a t i n g i n .
th e h y drogen c y c l e depends on th e s t r e n g t h o f th e a c i d form
e d ; th e w eaker th e a c i d th e more th e e q u i l i b r i u m i s s h i f t e d
t o th e s a l t form o f th e e x c h a n g e r . The r e a c t i o n v e l o c i t i e s ,
a r e th e same w h eth er weak or s tr o n g a c i d s a r e form ed. Mono
v a l e n t c a t i o n s exchange more r a p i d l y t h a n d i v a l e h t c a t i o n s ,
\ b u t th e exchange e q u i l i b r i u m a v e r a g e s 2 0 -2 5$ g r e a t e r f o r the
: d i v a l e n t c a t i o n s , p o s s i b l y due to t h e i r g r e a t e r e l e e t r o -
] s t a t i c a t t r a c t i o n . W ith in grou ps o f e le m e n ts , io n s w ith
lo w er p o s i t i o n s in th e p e r i o d i c sy stem have more r a p i d e x - ,
■ change f a t e s .
By p l o t t i n g th e h y d r a te d io n ic r a d i i v s . e q u i l i b r i u m
exchange f o r io n s o f t h e f i r s t and second g ro u p s , Nachod
and Wood (10) o b t a i n e d s t r a i g h t l i n e s o f th e same s l o p e .
; They have d e v elo p ed an i n t e r p r e t i v e e q u a t i o n which c o r r e -
' l a t e s exchange c a p a c i t y w ith th e h y d r a te d r a d i u s and v a le n c e
; I
o f t h e io n i n - q u e s t i o n . The l a r g e r th e h y d r a t e d io n ic
r a d i u s , th e s m a ll e r i s th e exchange c a p a c ity *
O th e r w ork ers have a l s o r e p o r t e d t h a t io n s o f h i g h e r '
v a le n c e a r e more f i r m l y bound t o th e e x c h a n g e r ( 4 ) . Among i
io n s o f th e same v a le n c e , th o s e o f h i g h e r atom ic w e ig h t
and s m a l l e r amounts o f h y d r a t i o n a r e more f i r m l y b ou nd.
W atson (3) a l s o r e p o r t s t h a t up t o a p o i n t , th e l a r g e r
th e u n s o l v a t e d ion., th e more f i r m l y i t i s h e ld by th e e x
ch an ger*
T here a r e few r e p o r t s i n th e l i t e r a t u r e o f th e u se
o f io n exchange r e s i n s in c o n h e c t i o n w ith p r o t e i n s t u d i e s *
R e id and Jo n e s (11) u sed exchange r e s i n s t o a l t e r th e s a l t
c o n c e n t r a t i o n o f serum , h u t do n o t r e p o r t any e f f e c t on th e
p r o t e i n l e v e l . C a r p e n t e r and S m ith (12) r e p o r t e d an i n
t e n t i o n t o a d s o rb a tom ato g l o b u l i n on exchange r e s i n s , b u t
g iv e no d a t a .
B. MUCGPROTEIHS
E a r l y i n v e s t i g a t i o n s on th e s e serum p r o t e i n s were
c o n d u c te d b y B r d ie k a , who r e p o r t e d ( 1 3 ,1 4 ,1 5 , 1 6 ,1 7 ,1 8 , 1 9 )
t h a t a h i g h m o le c u la r w e i g h t, c y s t i n e c o n t a i n i n g "alb u m o se -.
l i k e ” s u b s t a n c e , g i v i n g a s p e c i f i c p o l a r o g r a p h i c c u r r e n t
wave, i s p r e s e n t in i n c r e a s e d am ounts in th e b lo o d o f c a n
c e ro u s and in fla m m a to ry d i s e a s e d p a t i e n t s . B rd ie k a and
o t h e r x?orkers ( 1 6 , 1 7 , 1 8 , 1 9 , 2 0 , 2 1 , 2 2 , 2 3 , 2 4 ) a tt e m p t e d t o
d e v e lo p a d i a g n o s t i c t e s t f o r c a n c e r u t i l i z i n g th e p o l a r o
g r a p h ic t e c h n i q u e , b u t th e i n t e r f e r e n c e o f in fla m m a to r y •
c o n d i t i o n s p r e v e n te d a c c u r a t e d i a g n o s is * A p o t e n t i a l
a p p l i c a t i o n o f t h e m u e o p ro te in l e v e l i s th e e l i m i n a t i o n o f
c a n c e r from d i a g n o s t i c c o n s i d e r a t i o n ; s e v e r a l w o rk e rs (22,24)
have r e p o r t e d t h a t n o rm a l s e r a i n v a r i a b l y have low mueo
p r o t e i n l e v e l s .
C hem ical c h a r a c t e r i z a t i o n o f m u c o p r o te in s was p e r -
9
formed by W in z le r and B urk (25) and b y W in z le r ejt a l (26) *
I t was th e l a t t e r g ro up w hich i d e n t i f i e d th e M a lb u m o s e - lik e
s u b s ta n c e w ith th e p r e v i o u s l y known se ro m u e o id , and term ed
i t m ueoprotein*.
G. PROTEIN ISOELECTRIC POINTS
M ehl, Humphrey and W in z le r (27) d e m o n s tra te d t h r e e
e l e c t r o p h o r e t i c com ponents o f human p lasm a m u e o p ro te in ,
h a v in g i s o e l e c t r i c p o i n t s o f 2 . 3 , 3 . 4 and 4 .3 r e s p e c t i v e l y .
The m a jo r component had th e lowest, i s o e l e c t r i c p o i n t , and
was a l s o th e component e x h i b i t i n g th e m ost i n c r e a s e i n th e
plasm a o f a g a s t r i c c a n c e r p a t i e n t *
M i c h a e li s and Rona r e p o r t e d in 1910 (28) t h a t t h e
i s o e l e c t r i c p o i n t o f serum g l o b u l i n i s 4.94 * T h e i r r e s u l t s
were o b ta in e d from p r e c i p i t a t i o n e x p e r im e n ts . S v e d b e rg and
P e d e r s o n (29) have t a b u l a t e d th e e l e c t r o p h o r e t i c a l l y ob
t a i n e d d a ta o f o t h e r w o r k e r s . T h e ir f i g u r e s , and th o se o f
Cohn (30) have been i n c lu d e d in T ab le I .
10
TABLE I
PROTEIN ISOELECTRIC POINTS
I s o e l e c t r i c P o i n t
P r o t e i n S v e d b e rg Cohn
Serum a lb u m in
y
4 .8 0
4 ,2 R 3 ^ -
4 .8 0 N— '
G lo b u lin s o c
y
5 .0 0
y
5 .0 0
P
y
5 .1 2
K
y
6 .0
y
6 .4
y '
6.3
y
h o r s e
K
human
y
h o v in e
METHODS AND MATERIALS
A. IOM EXCHANGE RESINS
1. The r e s i n s u se d i n t h i s i n v e s t i g a t i o n w ere:
A nion e x c h a n g e rs C a ti o n e x c h a n g e rs
A m b e rlite IRA 400 A m b e rlite HI 120
A m b e rlite IR 4B A m b e rlite IRC 5Q
These r e s i n s w ere p ro v id e d by th e R e sin o u s P r o d u c t s D iv i
s i o n , Rohm and H aas Company.
A m b e rlite IRA 4QQ1 i s a s t r o n g l y b a s i c s y n t h e t i c
r e s i n which b e h a v e s a s a s o l i d b a s e w ith o n ly i t s h y d r o x y l
io n s i n s o l u t i o n * I t a b s o r b s a n io n s from a c i d , n e u t r a l
and m i l d l y a l k a l i n e s o l u t i o n s . T h is r e s i n r e a c h e s e q u i l i
b riu m f o r co m p lete exchange i n l e s s th a n a m in u te , t h i s
v e r y r a p i d exchange r a t e b e in g made p o s s i b l e b y i t s s t r o n g
b a s i c i t y . The exchange gro up o f IRA 400 i s a m o d ifie d
amine w h ich may be r e p r e s e n t e d as R=N-GH* I t p r e f e r e n
t i a l l y a d s o r b s io n s o f h i g h e r v a le n c e , th e r e l a t i v e
a f f i n i t i e s f o r v a r i o u s a n io n s b e in g :
PQJ3 > SQ~2 > C l” > QH~
I •
The f o l lo w in g i n f o r m a t i o n on A m b e rlite r e s i n s i s t a k e n
from t h e Rohm & Haas t e c h n i c a l b u l l e t i n *
The a d s o rb e d io n s a r e n o t s u b j e c t t o h y d r o l y s i s fro m t h e
r e s i n s in c e th e e l e c t r o v a l e n t a s s o c i a t i o n i s s i m i l a r to t h a t
o f a n e u t r a l s a l t * T h is p r o p e r t y , p l u s t h e r e l a t i v e l y
s l i g h t a f f i n i t y o f IRA 400 f o r h y d r o x y l i o n , g r e a t l y i n
c r e a s e s th e amount o f a n io n e x c h a n g e . The r e s i n i s s t a b l e
i n s t r o n g a c i d s and b a s e s and i n o r g a n ic s o l v e n t s .
A m b e rlite IR 4B a c t s as a w e ak ly i o n i z e d b a s e , th e
exchange g ro u p s a g a i n b e i n g m o d if ie d a m in e s . I t s exchange !
c a p a c i t y i s g r e a t e r t h a n t h a t o f IRA 400 i n column p r o c e
d u r e , b u t th e g r e a t e r ex change r a t e o f th e l a t t e r r e s i n
makes i t s u p e r i o r f o r b a t c h o p e r a t i o n s . IR 4B i s e f f e c t i v e
o v e r a pH ra n g e o f 1 - 7 . I t s r e l a t i v e a f f i n i t y f o r v a r i o u s
a n io n s i s a s f o l lo w s :
OH- > POX3 > SO42 > G i
l t i s s t a b l e t o s t r o n g a c i d s o v e r l i m i t e d p e r i o d s o f tim e
and t o many t e s t e d o r g a n ic s o l v e n t s .
A m b e rlite IR 120 i s a s u l f o n i c a c i d ty p e c a t i o n e x
c h a n g e r c h a r a c t e r i z e d by a v e r y h ig h exchange c a p a c i t y .
I t i s an e x tr e m e l y s t a b l e r e s i n , b o th p h y s i c a l l y and chem i
c a l l y . I t i s an e f f e c t i v e e x c h a n g e r o v e r a pH ra n g e o f
1 - 1 4 .
A m b e rlite IRC 50 i s a c a r b o x y l ic a c i d c a t i o n e x ch an
g e r w ith o n ly c a r b o x y l g ro u p s a s a c t i v e ex change g r o u p s .
IRC 50 is_ s t a b l e _i_n s t r o n g l y a c i d and a l k a l i n e s o lu tio n ^ a n d
1 3
in. common s o lv e n ts * At i n c r e a s e d t e m p e r a tu r e s i t shows i n
c r e a s e d exchange r a t e and a f f i n i t y f o r h y d ro g e n i o n s .
2 . The p r o t e i n s o l u t i o n s w ere b r o u g h t i n t o c o n t a c t
w ith th e r e s i n s b y two m e th o d s, b a t c h and column p r o c e d u r e s
I n th e fo rm er te c h n iq u e an amount o f r e s i n i n a b e a k e r , o r
o t h e r s u i t a b l e v e s s e l , i s s l u r r i e d w i t h a s o l u t i o n u n t i l
exchange e q u i l i b r i u m i s r e a c h e d .
In colum n p r o c e d u r e , a tu b e o r colum n i s s e t up
c o n t a i n i n g th e exchange r e s i n . The i o n - c o n t a i n i n g s o l u t i o n
i s a llo w e d t o flow th ro u g h th e column a t a r a t e d e te rm in e d
by th e r e s i n i n u s e . As th e s o l u t i o n p a s s e s down th e tu b e
a s e r i e s o f exchange e q u i l i b r i a i s s e t up w ith d e c r e a s i n g
am ounts o f e x c h a n g e a b le io n from to p t o b o tto m . I f th e
colum n i s lo n g enough, co m p lete rem o v a l may be a c h i e v e d .
I t i s e s s e n t i a l f o r e f f i c i e n t colum n o p e r a t i o n t h a t th e
column be backw ashed b e f o r e u se to remove a i r b u b b le s and
e x c e s s i v e l y f i n e p a r t i c l e s w hich m ig h t o th e r w is e c lo g th e
f lo w . B ackw ashing sh o u ld be done a t a r a t e s u f f i c i e n t t o
c a u se 5 0 -70$ e x p a n s io n o f th e column b e d .
In th e b a t c h p ro c e d u re work r e p o r t e d h e r e i n , pH
v a l u e s were d e te rm in e d d i r e c t l y w ith th e g l a s s e l e c t r o d e .
When column p r o c e d u r e was u s e d pH was c o n t r o l l e d th r o u g h
th e u s e o f 0 .0 1 M a c e t a t e s o l u t i o n o f th e d e s i r e d a c i d i t y .
B e fo re u se the colum ns w ere washed w ith t h e s e s o l u t i o n s
u n t i l c o n s t a n t t y r o s i n e v a lu e s were o b t a i n e d fo r t h e
e f f l u e n t . I n s e v e r a l c a s e s c o n s t a n t v a lu e s c o u ld n o t be
o b t a i n e d and th e colum ns were d i s c a r d e d . The p r o t e i n s , dis-t
s o lv e d i n e i t h e r p u re w a te r o r t h e a c e t a t e , were th e n
p o u red o n to th e colum ns, a llo w e d to e n t e r th e r e s i n b ed a n d '
washed t h r o u g h w ith a d d i t i o n a l a c e t a t e ; th e e f f l u e n t was
c o l l e c t e d d ro p w ise a t a r a t e o f a b o u t 1 m l. p e r m in u te .
The e f f l u e n t was d i v i d e d i n t o f r a c t i o n s and a l i q u o t s o f
t h e s e were a n a ly z e d f o r t y r o s i n e . I
The colum ns u s e d i n t h i s work c o n ta in e d 30-35 gram s ,
o f r e s i n (wet w e i g h t ) .
B. PROTEINS
Serum a lb u m in and m u e o p ro te in p r e p a r a t i o n s u s e d i n
t h i s i n v e s t i g a t i o n were p r o v id e d by D r. H enry Weimer, and
w ere p r e p a r e d by t h e m ethod d e s c r i b e d i n F ig u re 1 .
I
A c o m m ercial p r e p a r a t i o n o f c r y s t a l l i z e d b o v in e :
serum a lb u m in , d i s t r i b u t e d by Armour and C o ., was a l s o used..
C . TYROSINE DETERMINATION
The F o l i n (p h e n o l) r e a g e n t , a p h o s p h o t u n g s t i c -
phosphom olybdic compound ( 3 1 ) , was u se d t o d e te rm in e p r o
t e i n s by t h e i r t y r o s i n e c o n t e n t . As was f i r s t shown b y
A b d e rh ald en (32), t h i s r e a g e n t i s n o t s p e c i f i c f o r ty r o s in e ,
b u t a l s o form s a c o l o r w ith t r y p t o p h a n .
1 5
FIGURE 1
PROTEIN PREPARATION PROCEDURE
Plasm a
F i l t r a t e A
To one volume o f plasm a
w ere added o n e -te n th ,
volume o f m o la r sodium,
a c e t a t e and 0 .9 volume
o f d i s t i l l e d w a t e r .
Ammonium s u l f a t e was
added to a f i n a l c o n ce n
t r a t i o n o f 2 .7 3 m o la r .
P r e c i p i t a t e A
pH re d u c e d t o
4 .9
U l t r a 1 ;e B P r e c i p i t a t e B Albumin'
pH r e d u c e d t o
3 .7
F i l t r a t e C P r e c i p i t a t e C
Ammonium, s u l f a t e
added to s a t u r a t i o n
a t 4° C.
F i l t r a t e D P r e c i p i t a t e D
M u e o p ro te in
1 6
The f o llo w in g p ro c e d u re was u s e d :
1. V a ry in g amounts o f a t y r o s i n e o r p r o t e i n s t a n
d a rd o r unknown s o l u t i o n were p i p e t t e d i n t o t u b e s .
2. D i s t i l l e d w a te r was added to f i v e m l.
3 . 1 .5 m l. o f s a t u r a t e d NagCOg was a d d e d .
4 . 0 .5 m l. o f p h e n o l r e a g e n t was ad d ed ; t h e co n
t e n t s o f th e tu b e s were m ixed.
5 . The tu b e s w ere p la c e d i n th e d a rk f o r one h o u r .
6 . The c o l o r was r e a d on th e K l e t t p h o t o e l e c t r i c
■ c o lo rim e te r u s i n g a r e d f i l t e r .
When s o l u t i o n s a t low pH xxrere u s e d , i t was found
n e c e s s a r y t o s u b s t i t u t e 5 N NaOH f o r th e NagCOg.
S ta n d a rd c u r v e s have b een in c lu d e d f o r th e t y r o s i n e ,
serum alb u m in and m u e o p r o te in s t a n d a r d s , F ig u re s 2 , 3 , and
, 4*
Klett Readings
FIGURE 2
TYROSIKE STANDARD CURVE
900 _
800
700 .
600 -
600 ,
400 .
300 .
200
100
0.16
0*05
> Tyrosine
Klett Readings
FIGURE 3
SERUM ALBUMIN STANDARD CURVE
400.
300
200.
100.
Mg. Albumin
t— *
03
Klett Readings
FIGURE 4
MUCOPROTEIN STANDARD CURVE
20CL
ioa
Mg. Mueoprotein
to
EXPERIMENTAL
The e x p e r i m e n t a l r e s u l t s have "been d i v id e d i n t o fo u r
s e c t i o n s b a se d on th e p a r t i c u l a r exchan ge r e s i n u s e d . These
s e c t i o n s a r e i n t u r n d i v i d e d by t h e two p r o c e d u r e s ( b a tc h
and column) and by th e v a r i o u s p r o t e i n p r e p a r a t i o n s u s e d .
A l l d a t a o b t a i n e d have b e en c o l l e c t e d i n T able I I .
A. IRA 400
1* B a tc h p ro c e d u re - To 1 gnu o f th e h y d r o c h l o r i d e
form o f t h i s a n io n exchange r e s i n , was added 18 mg, o f
serum a lb u m in , d i s s o l v e d i n 9 m l. o f w a t e r . The r e a c t i o n o f
th e s o l u t i o n was a d j u s t e d to pH 6 .2 w ith d i l u t e NaOH and the
m ix tu r e was s t i r r e d v i g o r o u s l y f o r s e v e r a l m in u t e s . A 0 .5
m l, sam ple was w ith d ra w n . The pH was g r a d u a l l y lo w ered by
t h e a d d i t i o n o f d i l u t e HG1 u n t i l pH 3 .2 was r e a c h e d ; f r e
q u e n t 0 .5 m l. sam ples were w ithdraw n a f t e r s t i r r i n g . No
s i g n i f i c a n t change i n p r o t e i n c o n c e n t r a t i o n was fo u n d .
I n a n o th e r e x p e r im e n t, 10 mg, o f m u c o p ro te in d i s
s o lv e d i n 10 m l. o f a c e t a t e s o l u t i o n a t pH 2 .6 were added
t o 1 gm. o f t h e r e s i n and s t i r r e d f o r s e v e r a l m in u t e s .
S e v e r a l sam p les were w ithdraw n and a n a ly s e d f o r t y r o s i n e .
A b la n k was s i m u lt a n e o u s ly ru n by s t i r r i n g a gram o f th e
r e s i n i n 10 m l. o f th e a c e t a t e . No p r o t e i n u p ta k e c o u ld be
d e m o n s tr a te d . U s in g th e above p r o c e d u r e , 10 mg, o f th e
m u c o p ro te in were t r e a t e d w ith 10 gm, o f th e r e s i n ; 32% o f the
2 1
TABLE I I
EXPERIMENTAL RESULTS
R e s in T echnique P r o t e i n pH
P r o t e i n
U ptake
IRA 400 b a tc h
serum
a lb u m in 6*2 - 3*2 hone
IRA 400 b a tc h
muco
p r o t e i n 2*6 n o n e ; 32$
IRA 400 colum n
serum
a lb u m in 2*9 none
IRA 400 column
muco
p r o t e i n 2 . 6 none
IR 4B b a tc h
serum
alb u m in 2*7 - 9*4 19$ (?)
IR 4B b a t c h
serum
a lb u m in 5 .7 none
IRC 50 column
serum
a lb u m in 2 .5 none
IRC 50 column
serum
alb u m in 2 .5 40$
IRC 50 column
b e e f
a lb u m in 2 .6 46$; 58$
m i s o column
serum
a lb u m in 2 .9 none
IR 120 column
muco
p r o t e i n 1*5: non e
T U l l e r ’ s
e a r t h column
b e e f
albuniin 2*6 100$
P u l l e r ’ s
e a r t h column
muco
p r o t e i n 2 .6 100$
22
p r o t e i n was ta k e n up by th e r e s i n .
2* Column p r o c e d u r e - T h ir t y - tw o mg. o f serum a l b u
min d i s s o l v e d in. 2 m l. o f w a te r were added t o a column o f
IRA 400 w hich had b een washed w ith a c e t a t e s o l u t i o n a t
pH 2 . 9 . The p r o t e i n was th e n washed down the column w ith
a d d i t i o n a l a c e t a t e . F r a c t i o n s o f th e e f f l u e n t were c o l l e c t
ed and a l i q u o t s a n a l y s e d . As was t h e n t o be e x p e c te d , a l l
o f th e p r o t e i n added t o th e column was found in th e e f f l u e n t .
The r e c o v e r y c u rv e i s r e c o r d e d in F ig u re 5 .
A column o f IRA- 400 was washed w ith a c e t a t e s o l u t i o n
a t pH 2 . 6 . Ten mg. o f - m u c o p r o te in , d i s s o l v e d i n 1 m l. o f
th e a c e t a t e , were added t o th e column and washed th r o u g h
w ith th e same s o l u t i o n . E s s e n t i a l l y . a l l o f th e p r o t e i n
(93fo) was r e c o v e r e d . I n o t h e r ru n s u n d e r th e same c o n d i
t i o n s r e c o v e r i e s o f 81 $ and 115$ were r e c o r d e d . R eco v e ry
c u rv e s o f the 93$ and 115$ r e c o v e r i e s a re i n c lu d e d i n
F ig u re 6 .
B . IR 4B
T h is r e s i n was u se d w ith a lb u m in s o l u t i o n s i n b a tc h
t e c h n i q u e . Two gram s o f th e r e s i n were washed t h r e e tim e s
w ith d i l u t e HG1, t h e n w ith w a t e r . To th e r e s i n was added
20 mg. o f serum alb u m in d i s s o l v e d in 10 m l. o f w a t e r . A f t e r
s t i r r i n g , t h e pH o f t h e s u p e r n a t a n t was found to be 2 . 7 .
S u f f i c i e n t NaOH was added to b r i n g th e m ix tu re to pH 9 . 4 .
Sam ples were w ith d raw n a t th e two e x tre m e s and a n a l y s e d .
Percent Protdin Recovered
2 3
FIGURE 5
SERUM ALBUMIN ON IRA 4 0 0 COLUMN AT pH 2 . 9
100
80
60
40
20
10
M is . E f f l u e n t
20
Percent Protein Recovered
2 4
FIGURE 6
MUCOPROTEIN ON IRA 4 0 0 COLUMN AT pH 2 . 6
ioa
4Q,
2Q
40
M is . E f f l u e n t
25
U ptake o f 19$ o f the p r o t e i n was d e te r m in e d ; t h i s a p p a r e n t
•uptake may have been due to d e c r e a s e d s o l u b i l i t y due t o
d e n a t u r a t i o n a t t h e e l e v a t e d pH.
I n a s i m i l a r e x p e rim e n t an a lb u m in s o l u t i o n was added
to a s m a ll amount o f th e r e s i n w hich had b een washed w ith
w a t e r . The p r o t e i n s o l u t i o n was a t pH 5 . 7 ; th e pH o f t h e
r e s i n wash w a te r was 7* 0. Sam ples o f t h e alb u m in s o l u t i o n
were removed b e f o r e t r e a t m e n t w ith t h e r e s i n and a f t e r v a r y
in g i n t e r v a l s o f s t i r r i n g w ith th e r e s i n . T here was no u p
ta k e o f p r o t e i n .
C. IRC 50
T his r e s i n was u se d w ith v a r i o u s a lb u m in s o l u t i o n s
in column t e c h n i q u e . Columns o f IRC 50 were washed w i t h
a c e t a t e s o l u t i o n a t pH 2 . 5 . To one column was added 10 mg.
o f serum alb u m in d i s s o l v e d i n 5 m l. o f w a t e r . A c e ta te was
added t o th e column and f r a c t i o n s o f th e e f f l u e n t were
c o l l e c t e d and a n a l y s e d . A l l o f th e p r o t e i n was found i n
th e f i r s t 18 m l. o f e f f l u e n t . To th e secon d column was
added 10 mg. o f serum alb u m in d i s s o l v e d i n 2 m l. o f the
a c e t a t e . W ashing th e colum n w i t h a c e t a t e removed 60$ o f
th e p r o t e i n w ith t h e f i r s t 2 8 'm l. o f e f f l u e n t . B o th o f
th e s e r u n s a r e r e c o r d e d i n F ig u re 7 a s an a v e ra g e c u r v e .
A c r y s t a l l i z e d b e e f alb u m in p r e p a r a t i o n was n e x t u se d on
IRC 50 c o lu m n s. To one such colum n, p r e v i o u s l y washed w ith
a c e t a t e s o l u t i o n a t pH 2 . 6 , t h e r e was added 10 mg. o f b e e f
Percent Protein Recovered
2 6
FIGURE 7
SERUM ALBUMIN ON IRC 5 0 COLUMN AT pH 2 . 5
1 0 0.
8 0 -
6 0 -
2 0 -
0 10 40 20 30
M i s . E f f l u e n t
a lb u m in d i s s o l v e d i n 1 m l. o f w a t e r . W ashing w ith a c e t a t e
removed 54$ o f th e p r o t e i n from th e column w ith th e f i r s t
i
'28 m l, o f e f f l u e n t , a . c o n s t a n t b la n k v a lu e b e in g o b ta in e d
i
t h e r e a f t e r . T his r u n h a s b e en r e c o r d e d i n F ig u re 8 .
I
J To a second column o f IRC 50, was added 10 mg. o f
I
ibeef a lb u m in and 8 mg. o f g lu c o s e d i s s o l v e d i n 1 m l. o f
i
w a te r * T h is was done t o d e te rm in e w h ether th e c h a rg e d p r o - ,
j te in p a r t i c l e s were r e t a r d e d i n p a s s a g e th r o u g h the colum n, J
jit b e in g assum ed t h a t t h e u n c h a rg e d s u g a r would be washed '
th r o u g h w ith no im pedance. The column was washed w ith
a c e t a t e s o l u t i o n a t pH 2 .6 ; 42$ o f th e p r o t e i n was found i
iin t h e f i r s t 37 m l. o f e f f l u e n t . The p r e s e n c e o f g lu c o s e I
I
i i
; was t e s t e d f o r w ith B e n e d i c t ’ s r e a g e n t ; i t was found o n ly :
i i
;in th o s e c u t s w hich a l s o c o n ta in e d th e p r o t e i n . The column ■
i i
jWas th e n a llo w e d t o d r a i n d ry and was washed w ith 25 m l. o f ,
w a t e r . T h is t r e a t m e n t removed an a d d i t i o n a l 44$ o f th e 1
' !
p r o t e i n . The colum n o p e r a t i o n i s r e c o r d e d - i n F ig u re 9 . j
i A sam ple o f th e c r y s t a l l i z e d b e e f a lb u m in p r e p a r a - 1
i ' *
I X 1
t i o n was d i a l y z e d , l y o p h i l i z e d and t e s t e d f o r t y r o s i n e . !
, i
The r e s u l t s i n d i c a t e d t h a t a low m o le c u la r w e ig h t m a t e r i a l j
was p r e s e n t in th e o r i g i n a l sam ple and had p a s s e d th ro u g h
t h e d i a l y s i s membrane, b u t i t was e i t h e r n o n - p r o t e i n o r a
s p l i t p r o d u c t c o n t a i n i n g n e g l i g i b l e am ounts o f t y r o s i n e and
t r y p t o p h a n . The d i a l y z e d p r o t e i n have r e a d i n g s w i t h th e
p h e n o l r e a g e n t ; w h i c h a v e r a g e d 18$ h i g h e r th a n e q u i v a l e n t
Percent Protein Recovered
2 8
FIGURE 8
BEEF ALBUMIN ON IRC 5 0 COLUMN AT pH 2 . 6
100
80
60
40
20
40
20 30
10
M is . E f f l u e n t
Percent Protein Recovered
2 9
FIGURE 9
BEEF ALBUMIN ON IRC 50 C O L U M N AT pH 2 . 6 j ■
C R U D E QUANTITATIVE GLUCOSE ANALYSIS
100
80
60
40
20
GLUCOSE
40
M is . E f f l u e n t
I
3 0
amounts o f th e o r i g i n a l p r e p a r a t i o n . The m a t e r i a l h e ld by
th e r e s i n w a s . e v i d e n t l y p r o t e i n .
D. IR 120
T h is r e s i n was u se d i n column p ro c e d u r e w i t h serum
alb u m in and m u c o p r o t e i n . p r e p a r a t i o n s .
A column' o f IR 120 was washed w ith 20% a c e t i c a c i d .
To i t was th e n added 32 mg. o f serum a lb u m in d i s s o l v e d i n
2 m l. o f a c e t a t e s o l u t i o n a t pH 2 . 9 . The colum n was th e n
washed w i t h a d d i t i o n a l a c e t a t e s o l u t i o n . A l l o f t h e p r o
t e i n was r e c o v e r e d i n th e f i r s t 27 ml*, o f e f f l u e n t . T h is
r u n i s r e c o r d e d i n F ig u r e 1 0 .
A second column o f t h i s r e s i n was washed w ith a c e
t a t e a t pH 1 .5 * To t h i s column was added 10 mg. o f muco
p r o t e i n d i s s o l v e d in 1 m l. o f th e a c e t a t e * E s s e n t i a l l y
a l l o f t h e p r o t e i n (.96$) was r e c o v e r e d i n the f i r s t 45 m l.
o f e f f l u e n t * T h is r u n i s re c o r d e d i n F ig u re 11*
Percent Protein Recovered
3 1
FIGURE 1 0
SERUM ALBUMIN ON IR 1 2 0 COLUMN AT pH 2 , 9
100 .
80 -
60
40
2 0 *
20 10
M is* E f f l u e n t
i
Percent Protein Recovered
32
FIGURE 11
MUCOPROTEIN ON IR 1 2 0 COLUMN AT pH 1 . 5
100
80
60.
40-
20 .
Mis. Effluent
1 _ _
DISCUSSION AND CONCLUSIONS
The i n a b i l i t y o f io n exchange r e s i n s t o q u a n t i t a t
i v e l y ta k e up p r o t e i n m o le c u le s o f p r o p e r ch arg e may p r o
b a b l y be a t t r i b u t e d t o the l a r g e s i z e o f t h e s e p r o t e i n ions*
Nachod and ffood ( 1 0 ) c o r r e l a t e d t h e exchange c a p a c i t y o f a
r e s i n f o r an io n d i r e c t l y w i t h the v a le n c e and i n v e r s e l y
w ith th e h y d r a t e d io n ic r a d i u s ; a lth o u g h th e c h a rg e d p r o
t e i n m o le c u l a r may be c o n s i d e r e d an io n o f h i g h v a l e n c e ,
i t s s i z e o r r a d i u s i s trem en d o u s r e l a t i v e t o i n o r g a n i c io n s
o f lo w e r v a l e n c e .
In a d d i t i o n t o t h e s i z e f a c t o r , th e n e c e s s i t y o f
a d j u s t i n g t h e pH o f t h e p r o t e i n s o l u t i o n i n t r o d u c e s a n
e l u t i o n f a c t o r * Any p r o t e i n m o le c u le t a k e n up b y th e r e s i n
would have t o compete w ith t h e s m a l l e r io n s o f th e b u f f e r i n g
s o l u t i o n , and e l u t i o n w ould ta k e p la c e t o an e x t e n t d e t e r
mined b y th e t o t a l num ber, s i z e and c h arg e o f th e b u f f e r
io n s *
That th e e l u t i o n f a c t o r i s n o t t h e m ajo r c a u s e o f
p o o r p r o t e i n u p ta k e b y i o n exchange r e s i n s i s e v i d e n t from
th e b a tc h p r o c e d u r e work i n which no b u f f e r was p r e s e n t *
I n t h i s w ork , u s i n g IRA 140 and IR 4B, no p r o t e i n u p ta k e
o c c u rre d *
C e r t a i n r e s i n s a r e a b l e t o a d s o r b p r o t e i n s t o some
e x t e n t . T hus, a l a r g e amount o f IRA 400 was a b l e t o ta k e
up 30fo o f added m u c o p r o te in i n b a t c h t e c h n i q u e . The c a t i o n .
| e x c h a n g e r , IRC 50, was found to he c a p a b le of. a d s o r b in g
i - |
I 4 0 -60$ o f added p r o t e i n . Such a d s o r p t i o n c o u ld c o n c e iv - j
| - I
1 a b ly be u t i l i z e d to s e p a r a t e a p r o t e i n o r p r o t e i n s from !
> o t h e r s w ith lo w e r i s o e l e c t r i c p o i n t s . About h a l f o f th e
1
j p o s i t i v e l y c h a rg e d p r o t e i n s , o r a t l e a s t th o s e o f s i z e and ;
I j
{ n e t c h a rg e s i m i l a r t o a lb u m in , m ig h t be ta k e n up by th e
r e s i n and c o u ld be e l u t e d by w a sh in g t h e colum n w ith a i
I
j s o l u t i o n a d j u s t e d t o a pH above th e i s o e l e c t r i c p o i n t s o f
th e a d s o rb e d p r o t e i n s .
S e p a r a t i o n o f m u c o p ro te in and serum a lb u m in w ould ;
I
a l s o a p p e a r p o s s i b l e from a c o m p a riso n o f F igures 10 and 1 1 . j
At pH 1*5 i t ? v ’o u ld a p p e a r t h a t th e a lb u m in would be a lm o st I
i
c o m p le te ly r e c o v e r e d b e f o r e the m u c o p r o te in would a p p e a r
i n th e e f f l u e n t .
| S
' The o t h e r r e s i n s s t u d i e d a p p ea r to be in c a p a b le o f f
I . i
; a d s o r b i n g p r o t e i n s t o an y e x t e n t . A b r i e f s tu d y o f an |
I |
i a d s o r p t i v e m a t e r i a l , F u l l e r f s e a r t h , showed t h a t t h i s m a t- [
i e r i a l i s c a p a b le o f t a k i n g up a ll . o f th e p r o t e i n ad d ed r e - 1
I
! g a r d l e s s o f c h a r g e . Columns o f F u l l e r r s e a r t h rem oved j
10 m gs. o f m u c o p ro te in and o f b e e f a lb u m in , b o t h a t pH 2 . 6 .
I A p o s s i b i l i t y e x i s t s t h a t d i f f e r e n t e l u t i n g a g e n ts m ig h t
j be a b le t o s e p a r a t e th e d i f f e r e n t l y c h a rg e d p r o t e i n s on
i
• su c h c o lu m n s. I t i s c o n c e iv a b le t h a t one p r o t e i n i s h e ld 1
j by b o th p h y s i c a l f o r c e s and i o n ex change ty p e f o r c e s w h ile
i ;
| th e o t h e r , o p p o s i t e l y c h a rg e d p r o t e i n , i s h e ld o n ly b y th e |
i ____________________ : ____________________________________________________ : ____I
5 I
j p h y s i c a l f o r c e s . ^
♦
, I t may be s t a t e d i n c o n c l u s i o n t h a t t h e e x p e r i m e n t a l '
i
' r e s u l t s do n o t i n d i c a t e t h a t an e f f i c i e n t p r o t e i n s e p a r a - 1
!
j t i o n can be a c h ie v e d by th e u s e o f t h e io n exchange r e s i n s ■
I ;
i studied*. Com plete p r o t e i n u p ta k e by a t r u e exchange r e s i n
i
jwas n e v e r a c h ie v e d * th e m a jo r f a c t o r a g a i n s t . s u c h u p ta k e
I
.a p p e a r in g t o be t h e l a r g e s i z e o f p r o t e i n m o le c u l e s .
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(1 9 1 3 ). 1
i
l
UMI Number: EP41316
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Asset Metadata

Creator Kallman, Burton Jay (author)

Core Title A study of protein separation using ion exchange resins

Contributor Digitized by ProQuest (provenance)

School Department of Biochemistry and Nutrition

Degree Master of Science

Degree Program Biochemistry

Degree Conferral Date 1951-02

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

Tag chemistry, analytical,OAI-PMH Harvest

Language English

Advisor [illegible] (committee chair), [illegible] (committee member), Visser, Donald (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c17-777092

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