Amino Acids and Serum Proteins - ACS Publications

1 The Nature and Origin of the Serum Proteins

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JULIUS SCHULTZ Institute for Biochemical Studies in Cancer, Department of Biochemistry, Hahnemann Medical College, Philadelphia, Pa.

The contributions of Richard Block to the serum protein problem originated from the hypothesis of Kossel. From recent data on the amino acid composition of the proteins found in animal sera, a formulation is derived which reflects the properties of a continuous system of molecular species originating from a common biosynthetic pathway, as if from mixed polymers of monomeric peptides of lower molecular weight. Indirect evidence of this is found in the amino acid interrelationship, and direct evidence is limited to the isolation of peptides of common composition, whose primary structures are still under investigation. These findings suggest that undifferentiated proteins may be continuous systems rather than discrete molecular species.

The a m i n o a c i d c o m p o s i t i o n of p r o t e i n s w a s one of the m a j o r i n t e r e s t s of Block's i n t e l l e c t u a l endeavor. H e carried w i t h h i m a g r e a t d e a l of the b i o c h e m i c a l heritage of the M e n d e l , O s b o r n e , and Vickery s c h o o l , i n w h i c h he w a s w e l l t u t o r e d . T h i s s c h o o l v i e w e d p r o t e i n s i n the light of t h e i r a m i n o a c i d c o m p o s i t i o n . Physiological

Meaning

It w a s B l o c k ' s tendency to r e a d as m u c h p h y s i o l o g i c a l m e a n i n g i n t o an a m i n o a c i d a n a l y s i s as h i s t r a i n i n g and e x p e r i e n c e a l l o w e d that l e d h i m i n t o the development of a hypothesis c o n c e r n e d w i t h a m i n o a c i d i n t e r r e l a t i o n s h i p of s e r u m p r o t e i n s . A l t h o u g h h i s concepts r e c e i v e d s o m e attention i n the 30's (graduate students at the U n i v e r s i t y of M i c h i g a n w e r e r e q u i r e d to r e a d h i s p a p e r s ) , t h e r e a r e few c i t a t i o n s to his w o r k in c o m p r e h e n s i v e r e v i e w s on the p l a s m a p r o t e i n s , p u b l i s h e d only a few

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Stekol; Amino Acids and Serum Proteins Advances in Chemistry; American Chemical Society: Washington, DC, 1964.

ADVANCES IN CHEMISTRY SERIES

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y e a r s ago (31). A s the knowledge of the p h y s i c a l c h e m i c a l p r o p e r t i e s of p r o t e i n s g r e w , the influence of the a m i n o a c i d s on the functional c h a r a c t e r of the p r o t e i n w a s given a m i n o r r o l e . Thus i n c o m m e n t i n g on H . B . V i c k e r y ' s statement, " i n r e c e n t y e a r s t h e r e has a r i s e n the c o n v i c t i o n that, not only the c h e m i c a l but a l s o the p h y s i c a l p r o p e r t i e s , o r at least many of t h e m , c a n be a s s i g n e d a r a t i o n a l explanation i n t e r m s of the a m i n o a c i d c o m p o s i t i o n " (45), B a i l e y a r g u e d that "one of the most d i s h e a r t e n i n g features of the a m i n o a c i d a n a l y s i s of p r o t e i n s i s that the r e s u l t s have l i t t l e m e a n i n g " (44). In view of the sudden i n v a s i o n of the o r g a n i c c h e m i s t ' s a p p r o a c h to p r o t e i n s t r u c t u r e , the e l u c i d a t i o n w i t h e x p l o s i v e f o r c e of the p r i m a r y s t r u c t u r e of a n u m b e r of p r o t e i n s and a c t i v e s i t e sequences, and a most recent statement that s t r u c t u r e and function m a y b e d e t e r m i n e d by p r i m a r y s t r u c t u r e (1), one must c o n s i d e r s e r i o u s l y that a g i v e n a m i n o a c i d m a y p l a y a p a r t i c u l a r l y i m p o r t a n t r o l e i n d e t e r m i n i n g function. T h u s w i t h the r e - e m e r g e n c e of the dominant r o l e of the a m i n o a c i d s , e n v i s i o n e d by V i c k e r y , B l o c k r e t u r n e d to the theater and began to r e p l a y h i s p a r t i n the i n v e s t i g a t i o n of s e r u m p r o t e i n s of a q u a r t e r of a century ago, only to be cut off as a r e s u l t of a n u n t i m e l y a c c i d e n t . Anlage

Hypothesis

It often t a k e s as m u c h courage to d r a w a p h y s i o l o g i c a l l y o r i e n t e d i n f e r e n c e f r o m c h e m i c a l data b a s e d on c r u d e m a t e r i a l obtained f r o m e x p e r i m e n t a l a n i m a l s o r patients as to d e r i v e these i n f e r e n c e s f r o m the data on dead, h i g h l y p u r i f i e d c h e m i c a l entities obtained under r i g i d p h y s i c a l c h e m i c a l c o n t r o l s . Thus when B l o c k d e t e r m i n e d the b a s i c a m i n o a c i d s a r g i n i n e , l y s i n e , and h i s t i d i n e i n the a l b u m i n and g l o b u l i n f r a c t i o n s of patients whose a l b u m i n - g l o b u l i n ( A / G ) r a t i o s v a r i e d , he found that, i n s p i t e of the m a r k e d v a r i a t i o n of the p r o p o r t i o n of the two p r o t e i n f r a c t i o n s , the m o l a r r a t i o s of the b a s e s r e m a i n e d constant (4, 8). T h i s was l i k e a n a l y z i n g a s e r i e s of m i x t u r e s of a l k a l i phosphates; r e g a r d l e s s of the p r o p o r t i o n s of N a to Κ to H , the r a t i o of Ρ to Ο would r e m a i n 1 to 4. In t h i s r e s p e c t the P 0 d e t e r m i n e s the p r i n c i p a l p r o p e r t i e s of the m i x t u r e s ; l i k e w i s e , w i t h g r e a t e n t h u s i a s m B l o c k a s s u m e d that the d e t e r m i n e r s h e r e w e r e the b a s i c a m i n o a c i d s , the " a n l a g e , " which" K o s s e l i n 1905 (21) c a l l e d p r o t a m i n , a b a s i c p r o t e i n . So w i t h a s t r o n g tendency t o w a r d " r e a d i n g i n t o " the a m i n o a c i d data, he p r o p o s e d that the s e r u m p r o t e i n s w e r e one great m o l e c u l e f r o m w h i c h a l l i s o lated p r o d u c t s w e r e obtained as a r e s u l t of c h e m i c a l t r e a t m e n t . In the w o r k of Sorensen (43) and H a r d y (15) B l o c k found p h y s i c a l c h e m i c a l support of t h i s notion. B l o c k i g n o r e d the b r i l l i a n t i n v e s t i g a t i o n s t a k i n g p l a c e i n C o h n ' s l a b o r a t o r y at H a r v a r d , w h e r e a multitude of p u r i f i e d p l a s m a p r o t e i n f r a c t i o n s w e r e being i s o l a t e d , because c h e m i c a l reagents w e r e u s e d i n the p r o c e s s . But when T i s e l i u s p r o v i d e d a means of d e m o n s t r a t i n g the e x i s t e n c e of a number of s e r u m e l e c t r o p h o r e t i c components w i t h a m i n i m a l exposure of the s e r u m to c h e m i c a l r e a g e n t s , B l o c k gave up. F o r about 20 y e a r s he l o s t i n t e r e s t i n the subject and p u b l i s h e d nothing. B r a n d ' s a n a l y s i s of the Conn f r a c t i o n s offered no support f o r h i s h y 4


f.

SCHULTZ

The Nature and Origin of the Serum Proteins

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p o t h e s i s (9, 10). In the late 5 0 ' s , f o l l o w i n g the development of paper c h r o m a t o g r a p h i c p r o c e d u r e s for the a m i n o a c i d s , to w h i c h he c o n t r i b uted (6), and c h r o m a t o g r a p h y f o r s e p a r a t i o n of p r o t e i n s , he renewed h i s i n t e r e s t and p r o d u c e d a s e r i e s of p a p e r s w h i c h w e r e r e v i e w e d i n an a d d r e s s before the New Y o r k A c a d e m y of S c i e n c e s (3). W i t h these newer methods of p r o t e i n s e p a r a t i o n and a m i n o a c i d a n a l y s i s he p r e p a r e d s e r u m p r o t e i n f r a c t i o n s by s e r i a l s a l t i n g out w i t h a m m o n i u m sulfate and by the Sober and P e t e r s o n D E A E c e l l u l o s e c o l u m n s (42), u s i n g the s e r a of r e p t i l e , f o w l , and m a m m a l i a n b l o o d . Some of the a m i n o a c i d a n a l y s e s w e r e c a r r i e d out by the automatic a m i n o a c i d methods of H i r s , M o o r e , and Stein (18). F o r t i f i e d w i t h t h i s p l e t h o r a of data, B l o c k now had the opportunity t o r e - e x a m i n e not only the r a t i o of the b a s i c a m i n o a c i d s , but at least 12 a m i n o a c i d s i n a v a r i e t y of p r o t e i n f r a c t i o n s p r e p a r e d by at least two different p r o c e d u r e s . W i t h the a i d of a s t a t i s t i c i a n he d e t e r m i n e d the s i g n i f i c a n c e of the constancy of the m o l a r r a t i o s of p a i r s of a m i n o a c i d s and found that i n spite of the m a r k e d v a r i a t i o n of the absolute amounts of an a m i n o a c i d , the m o l a r r a t i o s of c e r t a i n p a i r s r e m a i n r e l a t i v e l y constant among the n u m e r o u s p r o t e i n components of a n i m a l s e r a . F o u r t e e n of the 18 a m i n o a c i d s a n a l y z e d w e r e found to have r e l a t i v e l y constant m o l a r r a t i o s , w i t h two other a m i n o a c i d s at the 1% l e v e l , and a l e s s than 10% d e v i a t i o n f r o m the mean m o l a r r a t i o s . B l o c k r e peated these findings f r o m e a c h s p e c i e s and f r o m p r o t e i n s obtained by the two methods of p r e p a r a t i o n . A s a check on the v a l i d i t y of the s t a t i s t i c s he p r e s e n t e d to the s t a t i s t i c i a n the d e t e r m i n a t i o n of a m i n o a c i d i n eight a p p a r e n t l y u n r e l a t e d p r o t e i n s : bovine s e r u m a l b u m i n , human h e m o g l o b i n , c r y s t a l l i n e egg a l b u m i n , i n s u l i n , β - l a c t o g l o b u l i n , a c t i n , g e l a t i n , and r i b o n u c l e a s e . The s t a t i s t i c i a n , l e d t o b e l i e v e that these w e r e eight p r o t e i n f r a c t i o n s of the s e r u m , r e p o r t e d 1 0 5 p a i r s ; only five p a i r s w e r e s i g n i f i c a n t l y r e l a t e d at the 1% l e v e l and t h r e e p a i r s w e r e a c t u a l l y negatively c o r r e l a t e d . T h i s i s expected by chance. In c o n t r a s t w e r e the r e s u l t s obtained f r o m a s e r i e s of p r o t e i n s of c h i c k e n s e r u m , for e x a m p l e , w h e r e t h e r e w e r e a l s o 105 p a i r s ; 26 w e r e c o r r e l a t e d at the 5% l e v e l of s i g n i f i c a n c e . T o s u m m a r i z e B l o c k ' s most recent c o n ception of the s e r u m p r o t e i n s i n h i s own w o r d s (3): T h e s e findings suggest that a l l s e r u m p r o t e i n s a r e c o m p o s e d of a m i n o a c i d anlagen— that i s , a l i m i t e d n u m b e r of different peptides, e a c h of w h i c h has a r e l a t i v e l y constant a m i n o a c i d content but d i f f e r s f r o m the o t h e r s i n a m i n o a c i d c o m p o s i t i o n . B l o c k ' s r e s u l t s appear to support the concept that the p r o t e i n s of the s e r u m a r e not a m i x t u r e of v a r i o u s u n r e l a t e d p r o t e i n s , but the p r o tein f r a c t i o n s , whether homogeneous o r not, a r e i n t e r r e l a t e d and may e x i s t as a c o m p l e x of e a s i l y d i s s o c i a b l e m o l e c u l e s that he had named o r o s i n i n 1934 (5). In e s s e n c e the development of the anlage hypothesis a r o s e f r o m K o s s e l ' s u s e of an e m b r y o l o g i c a l t e r m as a p p l i e d t o p r o t a m i n e as a b a s i c unit of p r o t e i n s t r u c t u r e , w h i c h B l o c k i n the 1930's t r a n s l a t e d into the constancy of the a r g - l y s - h i s r a t i o i n the s e r u m p r o t e i n s . He changed t h i s i n h i s m o s t recent suggestion that t h i s anlage w a s r e p r e s e n t e d by



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yet to be found c o m m o n peptides. N o m a t t e r what one may think of the conceptual i m p l i c a t i o n s of s u c h data f r o m K o s s e l ' s t i m e to the p r e s e n t , the fact r e m a i n s that the s e r u m p r o t e i n s appear to be m o r e c l o s e l y i n t e r r e l a t e d than i s c u r r e n t l y taught o r thought i n b i o c h e m i s t r y today, w h e r e a l b u m i n , a l p h a - 1 - , a l p h a - 2 - , b e t a - , and g a m m a - g l o b u l i n s as seen i n T i s e l i u s ' p a t t e r n s , the 18 c o l u m n f r a c t i o n s of Sober and P e t e r son (42), and the multicomponent s y s t e m s of g e l e l e c t r o p h o r e t i c c o m ponents of S m i t h i e s (41) a r e u s u a l l y t r e a t e d as d i s c r e t e i n d i v i d u a l s o r m i x t u r e s of p r o t e i n s , and d i s c u s s i o n of any i n t e r r e l a t i o n s h i p has only v e r y r e c e n t l y been i n d i c a t e d i n the c a s e of the g a m m a - g l o b u l i n s (2,13). In s o m e r e v i e w s , s u c h as the two v o l u m e s of " P l a s m a P r o t e i n s , " s u c h i n t e r r e l a t i o n s have not been r e c o g n i z e d , except to speculate that the g a m m a - g l o b u l i n m a y be a f a m i l y of p r o t e i n s (32), without r e f e r e n c e to Block's work. It i s p o s s i b l e that i n the future we m a y r e c o g n i z e K o s s e l ' s i d e a of the anlage, a b a s i c p r o t e i n d e t e r m i n e r found i n a l l c e l l s , t o be the m o d e r n - d a y equivalent of the " c o a t " o r " m a s k i n g " p r o t e i n w h i c h a c t u a l l y d e t e r m i n e s the p a r t i c u l a r a r e a s of the D N A ( d e o x y r i b o n u c l e i c acid) m o l e c u l e w h i c h a r e to function i n R N A ( r i b o n u c l e i c a c i d ) f o r m a t i o n i n a g i v e n cell—that i s , K o s s e l ' s anlage may be the i n t e l l e c t u a l a n tecedent of the p r i n c i p l e of c e l l u l a r d i f f e r e n t i a t i o n a s v i e w e d by many today. On the other hand, i f s u c h k i n d n e s s to o u r p r e d e c e s s o r s i s to be extended to the i d e a s of R i c h a r d B l o c k , who t r a n s f o r m e d K o s s e l ' s a n lage f i r s t to the b a s i c a m i n o a c i d s (7) and then to " c o m m o n p e p t i d e s , " it c a n e a s i l y be s a i d that the concept of the c o m m o n a c t i v e s i t e s e quence of many e n z y m e s (20) i s what B l o c k meant when he i n f e r r e d that a " p e p t i d e " anlage d e t e r m i n e d the function of many p r o t e i n s . A s f a r as the s e r u m p r o t e i n s a r e c o n c e r n e d , no one has yet i s o lated peptides whose sequence of a m i n o a c i d s f r o m m o r e than one c o m ponent of the s e r u m i s i d e n t i c a l . T h e s i g n i f i c a n c e of t h i s s e a r c h i s d i s c u s s e d i n the r e m a i n d e r of t h i s p r e s e n t a t i o n . Biosynthesis

of Serum

Proteins

T h e p r e s e n t a u t h o r ' s l i n e of r e s e a r c h c r o s s e d that of B l o c k i n the a u t h o r ' s attempt t o d e t e r m i n e c r i t e r i a other than m o b i l i t y to d i s t i n g u i s h p r o t e i n s r e s p o n s i b l e f o r a l t e r a t i o n s of the g a u s s i a n envelopes of the c l a s s i c a l T i s e l i u s e l e c t r o p h o r e t i c p a t t e r n s of the s e r u m p r o t e i n s found i n t u m o r - b e a r i n g r a t s (38). In these e x p e r i m e n t s , when s i x a m i n o a c i d s w e r e d e t e r m i n e d on the e l e c t r o p h o r e t i c f r a c t i o n s obtained b y s t a r c h s l a b e l e c t r o p h o r e s i s of dog, rat, and human s e r a , c e r t a i n r e l a t i o n s b e c a m e evident, w h i c h gave r i s e to a s p e c u l a t i o n as to the mode of b i o s y n t h e s i s of the s e r u m p r o t e i n s (36, 37). T h i s hypothesis depended an the d e m o n s t r a t i o n of the p r e s e n c e of c o m m o n peptides i n the p r o t e i n s of the s e r u m . In v i e w of the fact that even a c e l l u l a r s y s t e m s of p r o t e i n b i o s y n t h e s i s s o r e m a r k a b l y i l l u m i n a t e d by recent i n v e s t i g a t o r s (28) do not y i e l d s i n g l e p r o t e i n s on the a d d i t i o n of g i v e n p o l y n u c l e o t i d e f r o m a n a t u r a l s o u r c e , i t i s evident that a s e r i e s o r a s y s t e m of p r o t e i n s may be p r o d u c e d f r o m a g i v e n s i t e , whether t h i s t a k e s p l a c e d i r e c t l y on the s i t e i t s e l f o r peptides f r o m the t e m p l a t e appear as m o n o m e r s w h i c h


!.

SCHULTZ

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a r e r e f o r m e d into m i x e d p o l y m e r i c m a c r o m o l e c u l e s . N e v e r t h e l e s s p r o t e i n s of the s e r u m s y n t h e s i z e d i n the l i v e r (26) do have the c h a r a c t e r i s t i c s of a s y s t e m ; i n a d d i t i o n , they a r e without s p e c i f i c e n z y m a t i c function and a r e t h e r e f o r e undifferentiated. I n t h i s way the s e r u m p r o t e i n s m a y s e r v e a s a p h y s i o l o g i c a l m o d e l f o r the m a t r i x p r o d u c t s of the m e s s e n g e r R N A of the hepatic p a r e n c h y m a l c e l l . T h i s a p p r o a c h , i n

Table I. Chemical Relationships among 16 Isolated Electrophoretic Components of Sera of Dog, Rat, and Man (37)

Molar Ratio

No. of Proteins Included a

16 14 16

(Ala + gly)/asp Glu/ala Thr/asp a

b

Range 1.3-1.5 1.3-1.5 0.5-0.6

b

Gamma-globulins excluded. Alpha-2, rat = 1.1 Albumin, dog = 1.6.

Table II. Molar Ratios of Amino Acids Characteristic of Serum Proteins Compared to Proteinsof Other Sources

Proteins Serum Myokinase ATP creatine P0 kinase Cytochrome c LDH Hemoglobin α-chain β -chain Tryptophan-synthetase Rat albumin Pancreatic protein Trypsinogen Chymotrypsinogen Β Ribonuclease Carboxypeptidase Egg proteins Ovalbumin Lysozyme Conalbumin Ovomucoid Histones A Β C 4

Ala+Gly Asp

Glu Ala

Thr Asp

Ref.

(1.3-1.5) 3.0 1.3 2.2 1.4

(1.3-1.5) 3.0 3.0 2.0 1.5

(0.5-0.6) 1.2 0.6 1.2 0.4

(Table I) (23) (29) (24) (25)

2.3 2.1 3.0 1.5

0.2 0.7 0.8 1.3

0.8 0.5 0.4 0.6

(17) (17) (16) (30)

1.8 2.0 1.1 1.4

0.8 0.6 1.0 1.2

0.5 1.1 0.7 0.9

(27) (19) (18) (40)

1.7 0.7 1.25 0.8

1.5 0.5 1.6 1.7

0.6 0.3 0.5 0.5

(22) (22) (22) (22)

0.2 0.7 0.3

2.7 0.9 1.7

(12) (12) (12)

15.1 3.6 8.0


6


addition to the efforts of S o r m (44) i n s e a r c h i n g f o r a r e a s of c o m m o n sequences a m o n g the highly d i f f e r e n t i a t e d p r o t e i n s of s p e c i f i c function, m a y contribute to u n d e r s t a n d i n g s o m e of the steps between the r a w t e m p l a t e m a t e r i a l and the end p r o d u c t . W h i l e B l o c k ' s i n t e r e s t w a s c e n t e r e d on the constancy of the m o l a r r a t i o of c e r t a i n p a i r s of a m i n o a c i d s , we w e r e c o n c e r n e d not only w i t h that p a r a m e t e r (Tables I and Π) but a l s o w i t h the o b s e r v a t i o n that the m o l a r r a t i o s of other p a i r s v a r i e d s y s t e m a t i c a l l y w i t h the m o b i l i t y of the p a r t i c u l a r p r o t e i n f r a c t i o n ( F i g u r e 1). S i n c e one might expect t h i s

MM Protein cc-2 MM Protein MM Protein

ί

Ç

BJ /,

ProteiJ»

A

BJ

Λ

Protein·\

m A J r \ \

A

-GLOBULINS«-2 I t. L Biosynfhefic

Source

of

LIVER-

Normal

Serum

Proieins

ExtraHepatic Tissue

Figure 1. Systematic variation of molar ratio of ala to thrwith mobility of electrophoretic components of serum proteins to be t r u e of c e r t a i n a m i n o a c i d s s u c h a s l y s i n e , a r g i n i n e , o r t h e d i c a r b o x y l i c a c i d s , w h i c h , because of t h e i r e x c e s s c h a r g e , c o n t r i b u t e h e a v i l y to the m o r e b a s i c o r a c i d i c p r o t e i n s , we chose alanine and t h r e o n i n e , n e i t h e r of w h i c h w o u l d be c h a r a c t e r i s t i c of the l o w e r o r f a s t e r m o v i n g p r o t e i n s because of t h e i r e l e c t r i c a l p r o p e r t i e s , p e r s e . T h e o b s e r v a t i o n that a l a n i n e and t h r e o n i n e v a r i e d s y s t e m a t i c a l l y w a s f i r s t seen i n our s i n g l e - d i m e n s i o n a l a m i n o a c i d c h r o m a t o g r a m s of the f i v e e l e c t r o p h o r etic components, p u b l i s h e d i n 1955 (37). F i g u r e 1 shows d i a g r a m m a t i c a l l y the a r r a n g e m e n t of the p r o t e i n s of the s e r u m i n a c c o r d a n c e w i t h t h e i r m o b i l i t i e s , as would be found i n a c l a s s i c a l e l e c t r o p h o r e t i c a l p a t t e r n obtained b y e l e c t r o p h o r e s i s of n o r m a l s e r u m at p H 8.6 i n v e r o n a l buffer. T h e B e n c e - J o n e s and m y e l o m a g l o b u l i n s a r e a l s o p l a c e d i n a r e a s w h e r e they w e r e found i n r e spect to the s e r u m p r o t e i n s i n d i c a t e d (see T a b l e V f o r other data on these p r o t e i n s ) . In e a c h s y s t e m , i n both the hepatic and extrahepatic p r o t e i n s (26), the a l a - t h r r a t i o s change i n the s a m e d i r e c t i o n a s the


7.

SCHULTZ


7

m o b i l i t y , although these a m i n o a c i d s t h e m s e l v e s do not s i g n i f i c a n t l y c o n t r i b u t e to the e l e c t r i c a l p r o p e r t i e s of the p r o t e i n s , but i n t h i s m a y be a s s o c i a t e d w i t h an a c i d i c peptide c o m m o n to each p r o t e i n but i n c r e a s i n g i n p r o p o r t i o n t o other peptides w i t h i n c r e a s i n g m o b i l i t y of the component (see T a b l e II f o r the r e l a t i v e l y constant a l a - g l u r a t i o i n a l l p r o t e i n s of the s e r u m ) . The fact that c e r t a i n p a i r s of a m i n o a c i d s w e r e constant r e g a r d l e s s of m o b i l i t y , and others w e r e c h a r a c t e r i s t i c of p r o t e i n s of a g i v e n m o b i l i t y , gave r i s e t o the thought that t h e r e w e r e c o m m o n features s u g g e s t i v e of a s y s t e m of i n t e r w o v e n u n i t s . T h e r a t i o n a l e f o r t h i s was r e l atively simple. If a s e r i e s of peptides i s s y n t h e s i z e d f r o m the long t e m p l a t e s i t e , and these p r o d u c t s a r e o r g a n i z e d into p r o t e i n s c o n s i s t i n g of v a r i o u s p r o p o r t i o n s of these p r o d u c t s , the m i x t u r e of p r o t e i n s f o r m e d should have c e r t a i n i n h e r e n t features w h i c h w o u l d d i s t i n g u i s h t h e m f r o m a s i m i l a r m i x t u r e f o r m e d f r o m another t e m p l a t e s i t e . T h u s , a s e r i e s of t h r e e peptides, A , B , and C , f r o m a t e m p l a t e c o u l d be chosen s o that Β i s m o r e a c i d than A , and C i s m o r e b a s i c than A . T h e m o b i l i t y of a Hepati c t

AMINO ACID

POOL,

TEMPLATE GLOBULINS

Β

Β

«ι «• s « j

I1 1

ALBUMINS A

C ~l

Λ

+!

ξ

+IBETA

4

-. 1, €

C

€

Figure 2. Proposed mechanism of biosynthesis of animal serum pro teins. A,B C are positions on tem plate available for certain groups of amino acids ; the arrows indicate the resultant proteins after assembly on the ribosomes. t

p r o t e i n made up of these peptides w o u l d be a function of A p l u s the r a t i o of B / C . On e l e c t r o p h o r e s i s , the T i s e l i u s p a t t e r n of the m i x t u r e of p r o t e i n s d e r i v e d f r o m t h i s p a r t i c u l a r template would p r o v i d e a s e r i e s of p r o t e i n s whose c o m p o s i t i o n w o u l d r e f l e c t components A , B , and C i n a m a n n e r that w o u l d d i s t i n g u i s h the f a m i l y f r o m a s i m i l a r s e r i e s a r i s i n g f r o m another t e m p l a t e o r other t e m p l a t e s . T o test t h i s h y p o t h e s i s , the only method a v a i l a b l e i n the e a r l y 50's w a s a c r u d e a p p r o x i m a t i o n a r r i v e d at by t a b u l a t i n g a l l the a m i n o a c i d v a l u e s i n t e r m s of m o l a r r a t i o s of a s p a r t i c a c i d , and f r o m the 16 p r o -


8


t e i n components of dog, r a t , and m a n a s s e m b l i n g the lowest r a t i o f o r e a c h a m i n o a c i d into a h y p o t h e t i c a l peptide r e p r e s e n t i n g A . Since the v a l u e s of a l a n i n e and g l u t a m i c a c i d i n c r e a s e d w i t h i n c r e a s i n g m o b i l i t y of the component, but r e m a i n e d i n constant m o l a r r a t i o to e a c h other, they w e r e s a i d to be c h a r a c t e r i s t i c of peptide B ; i n l i k e m a n n e r , s i n c e g l y c i n e and s e r i n e v a l u e s i n c r e a s e d w i t h d e c r e a s i n g m o b i l i t y of the components, they w e r e a s s i g n e d to the C peptide. A s we had seen w i t h the a l a - t h r r a t i o s , the r a t i o s of B / ( B + C ) , c a l l e d n / K , w e r e r e m a r k a b l y c h a r a c t e r i s t i c of the m o b i l i t y of e a c h of the e l e c t r o p h o r e t i c components of the s e r a of dog, r a t , and m a n [ T a b l e ΠΙ and (37)].

Table III. Formulation Interrelating Serum Proteins

Component

Β

C

n/K

Albumin Alpha-1 Alpha-2 Beta Gamma

117 37 28 2 20

0 36 56 72 138

1.0 0.5 0.33 0.03 0.13

It has been shown (37) that the m i n i m a l m o l a r r a t i o s p e r 100 m o l e s of a s p a r t i c a c i d found i n the 16 p r o t e i n s p r e p a r e d b y s t a r c h e l e c t r o p h o r e s i s of the s e r a of dog, r a t , and m a n w e r e : g l u 90, a l a 70, t h r 50, s e r 40, and g l y 30. T h i s r e p r e s e n t e d the h y p o t h e t i c a l A peptide. When these r a t i o s w e r e s u b t r a c t e d f r o m the v a l u e s of the m o l a r r a t i o of each a m i n o a c i d f r o m each p r o t e i n , the v a l u e s g i v e n i n T a b l e H I w e r e found. P e p t i d e Β i s r e p r e s e n t e d by the e x c e s s n u m b e r of m o l e s of a l a p l u s g l u , w h i c h equals η i n E q u a t i o n 1; peptide C i s r e p r e s e n t e d by the v a l ues f r o m e x c e s s g l y p l u s s e r . T h e s u m of these two v a l u e s f o r each p r o t e i n i s e q u a l to K , and μ = f [ A + n B + (K — n ) c ]

(1)

Since A has been s u b t r a c t e d f r o m e a c h p r o t e i n , the m o b i l i t y b e c o m e s a function of the n / K r a t i o , as seen i n the f i g u r e s of the last c o l u m n of T a b l e Ι Π . n / K v a l u e s w i l l not c o r r e s p o n d to the m o b i l i t y i n p r o t e i n s not made up f r o m peptide u n i t s a r i s i n g f r o m another s i t e of s y n t h e s i s , a s i n d i c a t e d h e r e by the g a m m a - g l o b u l i n s , w h i c h a p p a r e n t l y a r i s e f r o m a different s y s t e m ( F i g u r e 1). Together w i t h the c r i t e r i a set by the p a i r s of a m i n o a c i d s whose m o l a r r a t i o s w e r e constant, the n / K r a t i o s offered d i s t i n g u i s h i n g f e a t u r e s that c h a r a c t e r i z e d the s y s t e m found i n the s e r u m f r o m other p r o teins p i c k e d at r a n d o m . T h i s w o u l d be expected, because w h i l e the t o t a l a s p a r t i c and g l u t a m i c a c i d s m a y p r o v i d e the p r i n c i p a l s o u r c e of n e g a t i v i t y , other p r o t e i n s m a y c o n t a i n the two a m i n o a c i d s i n d i f f e r i n g p r o p o r t i o n s , and the alanine w o u l d not n e c e s s a r i l y be i n the s a m e r a t i o to g l u t a m i c a c i d a s i s found i n a l l of the s e r u m p r o t e i n s . F i g u r e 2 shows


î.

SCHULTZ


9

d i a g r a m m a t i c a l l y the template s i t e and the p o s s i b l e m a n n e r i n w h i c h a s y s t e m of p r o t e i n s m i g h t be a s s e m b l e d to satisfy the data. In m o d e r n t e r m i n o l o g y the m e s s e n g e r R N A would c o l l e c t the a m i n o a c i d of peptides r e p r e s e n t e d by A + Β + C , e t c . , and deposit them on a r i b o s o m e w h e r e they w o u l d be a s s e m b l e d to give r i s e to A + C , o r a l b u m i n ; A C , β-globulin; and v a r i o u s p r o p o r t i o n s of Β + C i n addition to A to give r i s e to a continuous s y s t e m . A , B , and C a r e m o d e l p e p t i d e s , but the s y s t e m i s not n e c e s s a r i l y l i m i t e d to these t h r e e , i n that these three a r e not d i s c r e t e peptides but r a t h e r a r e a s on the template; by this m e c h a n i s m " c o m m o n p e p t i d e s " should appear i n h i g h i n c i d e n c e among the s e r u m p r o t e i n s . N a t u r a l l y the i m p l i c a t i o n a r o s e that t h e r e should be a h i g h i n c i dence of c o m m o n peptides i n the p a r t i a l h y d r o l y z a t e s of e a c h of the s e r u m p r o t e i n s . T h i s , of c o u r s e , i s the s a m e i m p l i c a t i o n that a r o s e i n B l o c k ' s m i n d (3). Since 1956 t h i s l a b o r a t o r y has been busy t r y i n g to i s o l a t e c o m m o n peptides f r o m e a c h of the s e r u m p r o t e i n s as obtained f r o m e l e c t r o p h o r e s i s on s t a r c h s l a b s . In o r d e r to be able to p r e p a r e e a c h of the p r o t e i n h y d r o l y z a t e s s i m u l t a n e o u s l y we e x a m i n e d n o n e n z y m a t i c p r o c e d u r e s and d e v i s e d the d i l u t e a c i d p a r t i a l h y d r o l y z a t e method w h i c h has r e c e n t l y been d e s c r i b e d i n d e t a i l (33, 34). In t h i s method a s p a r t i c a c i d i s p r e f e r e n t i a l l y e l i m i n a t e d f r o m the peptide c h a i n , the r e -

100φ

90-

σ

1 "3 Ό "5
< ο α> ο» σ S ο

40

I

10

30 2

0

~0

8

16

24

32

40

48

Time in Hours

Figure 3. Liberation of aspartic acid during 48-hour partial hydrolysis of each serum protein in 0.03N HCl at 105°C. Ordinate indicates percentage of total aspartic acid found at time interval shown at abscissa, using aspartic acid value of a 24-hour hydrolysis in 6N HCl as equal to 100 (33, 34)


10


s u i t i n g m i x t u r e of peptides i s f i n g e r p r i n t e d , and the n i n h y d r i n - p o s i t i v e a r e a s a r e l o c a t e d and eluted. B y r e p e a t i n g the mapping p r o c e d u r e s many t i m e s , sufficient m a t e r i a l w a s a c c u m u l a t e d to obtain a n a m i n o acid analysis. Thus i n F i g u r e s 3 to 8 c a n be seen the a s p a r t i c a c i d r e l e a s e d f r o m e a c h of the p r o t e i n s under these c o n d i t i o n s , along w i t h the v a l u e s f o r other a m i n o a c i d s being s p l i t at the s a m e t i m e .

16

24

32

Time in Hours

Figure 4. Liberation of serine from um proteins at 105°C. in 0.03N

ser HCl

Φ Ο

τ* ο

Amino Acids and Serum Proteins - ACS Publications

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