Changes Occuring in Proteins in Alkaline Solution - ACS Symposium

7 Changes Occuring in Proteins in Alkaline Solution JOHN R. WHITAKER

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Department of Food Science and Technology, University of California, Davis, CA 95616

Proteins may be exposed to alkaline conditions during puri fication procedures, i n the characterization of proteins, as a step in a processing methodology or during storage. The proce dure developed by Leone (1) for purification of uricase recom mends the treatment of the crude extract with butanol at 35° and pH 10 for up to 18 hours. Carbohydrate chemists use a l k a l i treatment to distinguish between O-glycosyl linkages of carbohy drates to serine and threonine residues in proteins and amide linkages of carbohydrates to asparagine residues in proteins (2). A l k a l i treatment has also proved useful in studying structure -function relationships of glycoproteins such as the active a n t i freeze glycoproteins from fishes of the A r c t i c and Antarctic regions (3). In food processing, sodium hydroxide i s used for peeling of potatoes and peaches, s o l u b i l i z i n g plant proteins, neutralizing casein preparations (sodium caseinate), removal of toxic constituents such as aflatoxin and protease inhibitors in production of texturized foods and vegetable protein whipping agents and in preparation of some special Scandinavian fish products. Calcium hydroxide i s used i n processing o f dough from corn f o r t o r t i l l a s . The most common method f o r o b t a i n i n g p r o t e i n i s o l a t e w i t h low n u c l e i c a c i d content from m i c r o b i a l c e l l s con s i s t s o f e x t r a c t i n g the p r o t e i n s from mechanically d i s r u p t e d c e l l s w i t h concentrated a l k a l i followed by p r e c i p i t a t i o n o f the e x t r a c t e d p r o t e i n s a t pH 4.5 ( 4 - 7 J . During storage o f the egg, the pH increases from approximately 6.5 to above 9.5 due to l o s s of carbon d i o x i d e ( 8 ) . Adverse e f f e c t s o f exposing p r o t e i n s to a l k a l i n e c o n d i t i o n s are known. As e a r l y as 1913, i t was shown t h a t s e v e r e l y a l k a l i t r e a t e d c a s e i n fed to dogs was e l i m i n a t e d unchanged i n the f e c e s , t h a t i t was not attacked by p u t r e f a c t i v e b a c t e r i a and t h a t t r y p s i n o r pepsin was unable to hydrolyze i t ( 9 ) . Ten Broeck reported t h a t egg albumin t r e a t e d w i t h 0.5 JN NaOH f o r 3 weeks at 37° had no immunological p r o p e r t i e s (1_0). The n i t r o g e n d i g e s t i b i l i t y values o f 0.2 M and 0.5 M NaOH-treated c a s e i n (80°C, 1 h r ) , as determined i n r a t s , was 71 and 47%, r e s p e c t i v e l y , as 0-8412-0543-4/80/47-123-145$05.00/0 © 1980 American C h e m i c a l Society In Chemical Deterioration of Proteins; Whitaker, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.


146

C H E M I C A L DETERIORATION O F PROTEINS

compared to 90% f o r untreated c a s e i n (11 ) . The NPU value o f NaOH-treated soybean p r o t e i n s i n r a t s was decreased (12). Severely a l k a l i - t r e a t e d h e r r i n g meals d i d not support normal growth i n c h i c k s ; i n f a c t some t o x i c e f f e c t s were observed (13). D i s p e r s i n g soybean p r o t e i n concentrates w i t h sodium hydroxide r e s u l t e d i n decreased growth i n lambs ( V 3 ) . Cytomegalic renal l e s i o n s and n u c l e a r enlargement o f renal t u b u l a r c e l l s were observed i n r a t s fed a d i e t o f s e v e r e l y a l k a l i - t r e a t e d soybean p r o t e i n (14_) o r a d i e t c o n t a i n i n g up to 3% l y s i n o a l a n i n e ( l j [ ) . Other workers d i d not f i n d such r e s u l t s when l e s s s e v e r e l y t r e a t e d p r o t e i n was fed as 20% o f the t o t a l p r o t e i n and w i t h adequate c a l c i u m supplementation (12^,16) although a c i d hydrolysates o f the a l k a l i - t r e a t e d p r o t e i n or d i e t s c o n t a i n i n g l y s i n o a l a n i n e d i d show e f f e c t s i n r a t s (V7) s i m i l a r to those observed by Woodard and Short (14). Such e f f e c t s under s i m i l a r c o n d i t i o n s have not been observed i n mice, hamsters, q u a i l s , dogs or monkeys ( 2 8 ) . Gould and MacGregor (19) have r e c e n t l y d i s cussed some o f the f a c t o r s which may account f o r v a r i a b i l i t y i n observations o f the e f f e c t o f feeding a l k a l i - t r e a t e d p r o t e i n s . As w i l l be discussed l a t e r , the presence o f l y s i n o a l a n i n e i n foods cannot be used as an i n d i c a t o r o f a l k a l i treatment s i n c e l y s i n o a l a n i n e has been found i n foods prepared without use o f a l k a l i (20). Reactions i n A l k a l i n e S o l u t i o n In a l k a l i n e s o l u t i o n , p r o t e i n s are known to undergo the f o l l o w i n g types o f r e a c t i o n s : (a) d e n a t u r a t i o n , (b) h y d r o l y s i s of some peptide bonds, (c) h y d r o l y s i s o f amides (asparagine and g l u t a m i n e ) , (d) h y d r o l y s i s o f a r g i n i n e , (e) some d e s t r u c t i o n o f amino a c i d s , (f) 3 e l i m i n a t i o n and r a c e m i z a t i o n , (g) formation o f double bonds and (h) formation o f new amino a c i d s . Denaturation. P r o t e i n s are q u i t e s u s c e p t i b l e to denatura t i o n i n a l k a l i n e s o l u t i o n because o f decreased s t a b i l i z a t i o n o f the t e r t i a r y s t r u c t u r e by e l i m i n a t i o n o f e l e c t r o s t a t i c i n t e r a c t i o n s between c a r b o x y l a t e and protonated amino and guanidinium groupings (Equations 1 and 2) and hydrogen bonding between the hydroxyl group o f t y r o s i n e and c a r b o x y l a t e groups (Equation 3 ) .

(1) H

C-C-R / NH

+

H0 9 2

2

In Chemical Deterioration of Proteins; Whitaker, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

(2)

7.

WHITAKER

0

Proteins in Mkaline Solution

0

-C-0®-H-0-(ll-

^

147

0

^ — * -C-OP +

+ H,0

(3)


H® T h e r e f o r e , adding a l k a l i t o p r o t e i n s may accomplish an increased s o l u b i l i z a t i o n o f the p r o t e i n w h i l e i n the a l k a l i n e s o l u t i o n . However, upon adjustment t o n e u t r a l o r a c i d pH, the p r o t e i n may be l e s s s o l u b l e than o r i g i n a l l y because o f d e n a t u r a t i o n . In t e x t u r i z a t i o n o f p r o t e i n s , t h i s denaturation may be an advantage. However, i n other cases such as a l k a l i treatment to destroy a f l a t o x i n o r protease i n h i b i t o r s i t may be a disadvantage. H y d r o l y s i s . Two types o f h y d r o l y t i c r e a c t i o n s occur i n p r o t e i n s a t a l k a l i n e pH. These a r e the h y d r o l y s i s o f peptide and amide bonds and the h y d r o l y s i s o f a r g i n i n e to o r n i t h i n e . Amide bonds a r e hydrolyzed r a p i d l y i n a l k a l i n e s o l u t i o n probably as shown i n Equation 4 ( 2 1 ) .

+ R-C-0®+ R'-NH

2

+ Orl® ( 4 )

Hi In t h i s r e a c t i o n , an hydroxide i o n a t t a c k s the carbonyl group o f the amide t o form an a n i o n i c t e t r a h e d r a l intermediate followed by e x p u l s i o n o f the -NHR moiety. Deamidation o f glutamine and asparagine residues leads t o a more a c i d i c p r o t e i n d e r i v a t i v e t h a t may have changed s o l u b i l i t y and f u n c t i o n a l p r o p e r t i e s . A l k a l i treatment a l s o leads t o l o s s o f some o f the amino a c i d s by the processes t o be d e s c r i b e d below. In a l k a l i n e s o l u t i o n a r g i n i n e s l o w l y decreases and o r n i t h i n e and/or c i t r u l l i n e i s formed probably by the r e a c t i o n shown i n Equation 5 . Z i e g l e r e t a l . ( 2 2 } reported t h a t treatment o f s e r i c i n e w i t h 0 . 1 M ^COQ a t 100°C f o r 6 0 min. l e d t o a decrease o f a r g i n i n e from 2 5 5 t o 2 2 0 vg per gram o f p r o t e i n (14% change) w h i l e d u r i n g the same time there was a decrease i n s e r i n e from 3 4 9 t o 2 0 6 yg per gram p r o t e i n ( 4 1 % change). Therefore, i t appears t h a t s e r i n e i s l e s s s t a b l e t o a l k a l i treatment than arginine, at least i n sericine. 1

β E l i m i n a t i o n and Racemization. There i s some l o s s o f the amino a c i d s c y s t i n e , c y s t e i n e , s e r i n e , t h r e o n i n e , l y s i n e and a r g i n i n e d u r i n g the a l k a l i n e treatment o f p r o t e i n s ( 1 2 , 2 2 - 3 0 ) . U n l i k e a r g i n i n e as shown above, l o s s o f the other amino a c i d s i s not due t o a h y d r o l y t i c r e a c t i o n but r a t h e r t o a 3 - e l i m i n a t i o n r e a c t i o n (Equation 6 ) . There i s a l s o some r a c e m i z a t i o n o f amino

American Chemical Society Library 1155 16th St. N.Whitaker, W. J., et al.; In Chemical Deterioration of Proteins; ACS Symposium Series; American Chemical Society: Washington, DC, 1980. Washington, D. C. 20030

148

C H E M I C A L DETERIORATION

O F PROTEINS

a c i d s which can be explained by the same 3 - e l i m i n a t i o n r e a c t i o n . X

ι

X

CHR

CHR

I

* X

ι

^

I

0

OTJ

CHR - ,

I

CHR II 0

--HN-C-C — ^ — H N—- CL —j CL— HN-C U--^=--HIN — ^^~ —HIN — = C— -> - H N - C - C -

H)(I) Downloaded by UNIV OF PITTSBURGH on January 21, 2015 | http://pubs.acs.org Publication Date: May 28, 1980 | doi: 10.1021/bk-1980-0123.ch007

Θ OH L-Amino a c i d residue

+ ) T (6)

( I I ) ( I V ) (V)

.Η 1[Η

Φ

φ

Η 0 ' }! -HIM-L-LCHR U

M

I

X

(in)

D-Ami no a c i d residue In Equation 6 , X = H , OH, 0 - g l y c o s y l , O-phosphoryl, - S H , -SCH2-R, a l i p h a t i c o r aromatic r e s i d u e ; R = Η o r CH3. In the case o f racemization a hydrogen can add back t o the carbanion intermediate ( I I ) t o g i v e e i t h e r the D o r L amino a c i d residue. Racemization o f amino a c i d s residues i n p r o t e i n s by a l k a l i treatment has been known f o r a long t i m e . Dakin i n 1912 (31) observed t h a t p r o t e i n s d i s s o l v e d i n d i l u t e a l k a l i underwent a p r o g r e s s i v e increase i n s p e c i f i c o p t i c a l r o t a t i o n from about -80 t o - 2 0 ° . He and coworkers a l s o observed t h a t the amino a c i d residues d i d not a l l undergo racemization a t the same r a t e which has been v e r i f i e d more r e c e n t l y (23,32-34). Table I shows the r e l a t i v e r a t e s o f racemization o f several amino a c i d residues i n lysozyme, p h o s v i t i n , and a n t i f r e e z e g l y c o p r o t e i n f r a c t i o n 8 as determined by g a s - l i q u i d chromatography. I t i s c l e a r the rates o f racemization vary markedly among the amino a c i d s w i t h i n a s i n g l e p r o t e i n ranging from 30% f o r s e r i n e t o 0.09% f o r l e u c i n e i n lysozyme and t h a t the r a t e s a r e s u b s t a n t i a l l y d i f f e r e n t among p r o t e i n s as shown by comparing the r a t e s o f racemization o f s e r i n e , threonine and a s p a r t i c a c i d i n lysozyme and p h o s v i t i n . F a c i l i t a t e d 3 e l i m i n a t i o n by having good l e a v i n g groups on the hydroxyl o f s e r i n e (phosphoserine i n p h o s v i t i n ) o r threonine (phosphothreonine i n p h o s v i t i n and g l y c o t h r e o n i n e i n the a n t i freeze g l y c o p r o t e i n ) appears t o lead t o a s m a l l e r percentage o f racemization because o f competition from the r e a c t i o n s l e a d i n g t o compound V (Equation 6 ) . The r e s u l t s shown i n Table I a r e i n agreement w i t h those o f others i n t h a t the a l i p h a t i c amino a c i d s g e n e r a l l y show the lowest r a t e s o f r a c e m i z a t i o n . These are followed by the b a s i c amino a c i d s , the aromatic amino a c i d s , the a c i d i c amino acids and



7.

WHiTAKER

Table I .

Proteins in Alkaline

Solution

0

Racemization o f Amino A c i d Residues i n P r o t e i n s '

Amino a c i d

c

Lysozyme

d

Phosvitin

e

Antifreeze glycoprotein

f

(% D-amino a c i d ) Alanine Valine Leucine Allo-Isoleucine Phenylalanine Tyrosine Proline Serine Allo-Threonine Aspartic acid Glutamic a c i d Lysine a

Ref.

1.02 0.42 0.09 0.50 2.98 2.62 0.68 30.1 12.0 16.2 2.82 0.96

0.89 0.00 1.20 0.45 3.91

-

1.05 2.47 5.52 6.82 1.33 2.61

3.56

0.06 4.87

34.

d e t e r m i n e d by gas chromatography as described i n Reference 35. d e t e r m i n e d a f t e r h y d r o l y s i s o f the a l k a l i t r e a t e d sample i n 6 Ν HC1 f o r 22 hours at 110°C. d

3 . 3 mg/ml lysozyme i n 0.5 Ν NaOH f o r 2.5 hours at 22°C.

e

3 . 8 mg/ml p h o s v i t i n i n 0.123 Ν NaOH f o r 30 min. at 60°C.

f

Antifreeze glycoprotein f o r 21 hrs a t 22°C.

f r a c t i o n 8 a t 5 mg/ml i n 0.5

NaOH



150

CHEMICAL

DETERIORATION

O F

PROTEINS

the a l i p h a t i c hydroxy amino a c i d s having the f a s t e s t r a t e o f 3 e l i m i n a t i o n . Amino a c i d residues undergo more r a p i d racemization i n p r o t e i n s than when f r e e . T h i s i s because the e l e c t r o n d e n s i t y o f the amino and c a r b o x y l a t e groups o f the free amino a c i d i n the v i c i n i t y o f the α-carbon decreases the attack by the hydroxide ion. Not a l l e l i m i n a t i o n o f hydrogen from the α-carbon o f an amino a c i d r e s i d u e leads to r a c e m i z a t i o n . In the case o f s e r i n e , t h r e o n i n e , c y s t i n e and c y s t e i n e intermediate I I can continue v i a the pathway o f intermediate IV to g i v e the dehydroamino a c i d ( V ) . The requirement f o r t h i s pathway i s t h a t X be a good l e a v i n g group such as -OH, -SH and -S-CH2-R. Attachment o f phosphoryl and g l y c o s y l groups lead to even f a s t e r r a t e s . In a l k a l i s o l u t i o n one might expect t h a t phosphate groups would be removed from O-phosphoserine-containing p r o t e i n s by h y d r o l y s i s and by 3 e l i m i n a t i o n . With p h o s v i t i n i t was found t h a t g r e a t e r than 85% o f the phosphate group was removed by 3 e l i m i n a t i o n as measured by increase i n absorbance a t 241 nm due to formation o f dehydroa l a n i n e ( 3 6 ) . Anderson and K e l l e y (37) had p o s t u l a t e d as e a r l y as 1959 t h a t the mechanism o f 3 e l i m i n a t i o n o f the phosphate group i n a l k a l i n e s o l u t i o n would f o l l o w the general mechanism o u t l i n e d i n Equation 6. The e f f e c t o f a l k a l i on the degradation o f c y s t i n e has been s t u d i e d e x t e n s i v e l y i n both model systems as w e l l as i n p r o t e i n s . Three models have been proposed to e x p l a i n the degradation. These a r e : (a) 3 e l i m i n a t i o n , (b) α e l i m i n a t i o n and (c) hy d r o l y s i s . In 3 e l i m i n a t i o n , the proposed r e a c t i o n s are shown i n Equation 7. Therefore, the s t o i c h i o m e t r y o f the r e a c t i o n i s two moles o f dehydroalanine, one mole o f elemental s u l f u r and one mole o f d i s u l f i d e as shown by the o v e r a l l r e a c t i o n (Equation 8 ) . HC-CH -S-S-CH -CH + 2 0 H ^ 2 HC=CH + S® + S + 2 H 0 2

2

(8)

2

2

Friedman (38) has proposed t h a t the two hydrogens on the α - c a r bons may be e l i m i n a t e d simultaneously thus l e a d i n g d i r e c t l y to the same f i n a l products. In the α - e l i m i n a t i o n mechanism (39) as shown i n Equation 9, hydrogen e x t r a c t i o n by the base alpha to the s u l f u r r e s u l t s i n formation o f a carbanion which may rearrange v i a route à or b c to g i v e c y s t e i n e and a thioaldehyde which decomposes i n a l k a l i n e s o l u t i o n to an aldehyde and hydrogen s u l f i d e . Thus, the s t o i c h i ometry o f t h i s r e a c t i o n i s (Equation 10): 9

Θ HC-CH -S-S-CH -C{j + 2 OH + HC-CHg-S + ÎÏC-CH0 + HsP + HgO 0

2

2

(10)

Therefore, the α - e l i m i n a t i o n mechanism alone could not lead to formation o f dehydroalanine.


7.

wmTAKER

Proteins

in Alkaline

151

Solution

The proposed h y d r o l y s i s mechanism (40) shown i n Equation 11 leads to a f i n a l o v e r a l l s t o i c h i o m e t r y o f (Equation 12): 2[JC-CH -S-S-CH -CH + 4®0H + 3HC-CH -S® + HC-CH -S0® + 2H 0


2

2

2

2

2

(12)

The second molecule o f c y s t e i n e and a s u l f i n i e a c i d r e s u l t from the d i s p r o p o r t i o n a t e o f two molecules o f a s u l f e n i c a c i d (Equa t i o n 11). Nashef e t a l . (41) have c a r e f u l l y s t u d i e d the s t o i c h i o m e t r y of products formed from a l k a l i treatment o f lysozyme and a - l a c t albumin. The s t o i c h i o m e t r y was c o n s i s t e n t w i t h the 3 - e l i m i n a t i o n mechanism and could not be e x p l a i n e d by e i t h e r the α - e l i m i n a t i o n or the h y d r o l y s i s mechanisms. N e i t h e r the α - e l i m i n a t i o n nor h y d r o l y s i s mechanism alone w i l l e x p l a i n the formation o f dehydro a l a n i n e and the subsequent a d d i t i o n products t h a t are observed (see below). The 3 - e l i m i n a t i o n mechanism i s a l s o c o n s i s t e n t w i t h the products found when k e r a t i n i s t r e a t e d i n an a l k a l i n e s o l u t i o n c o n t a i n i n g S - s u l f i d e ( 4 2 ) . The observation t h a t c y s t i n e as the free amino a c i d cannot undergo l a n t h i o n i n e formation a l s o favors the 3 - e l i m i n a t i o n mechanism ( 4 3 ) . In the free amino a c i d , the amino group and the c a r b o x y l a t e anion attached to the α-carbon atom (see Equations 7, 9 , 11) generate a high e l e c t r o n d e n s i t y and prevent the a b s t r a c t i o n o f the α-hydrogen atom by base. In the presence o f c a l c i u m or strontium hydroxide, l a n t h i o n i n e i s formed from the free c y s t e i n e i n d i c a t i n g t h a t the c a l c i u m or s t r o n t i u m i o n , by complexation, can reduce the e l e c t r o n charge about the α-carbon atom (42). The 3 - e l i m i n a t i o n r e a c t i o n (Equation 6) i s s e n s i t i v e to pH, temperature and presence o f other i o n s . Table I I shows the e f f e c t o f hydroxide i o n c o n c e n t r a t i o n on the i n i t i a l r a t e o f 3 e l i m i n a t i o n o f phosphate from phosphoserine i n p h o s v i t i n (36) and g l y c o s y l groups from threonine i n a n t i f r e e z e g l y c o p r o t e i n s ~ T 4 4 ) . The i n i t i a l r a t e i s d i r e c t l y p r o p o r t i o n a l to hydroxide ion con c e n t r a t i o n over the range i n v e s t i g a t e d . The 3 e l i m i n a t i o n o f g l y c o s y l groups from threonine i n a n t i f r e e z e g l y c o p r o t e i n - 8 i s some 12 times f a s t e r at 60°C than the r a t e o f 3 e l i m i n a t i o n o f phosphate groups from phosphoserine i n p h o s v i t i n (Table I I ) . Nashef e t a l . (41) a l s o reported t h a t the r a t e o f 3 e l i m i n a t i o n from c y s t i n e was d i r e c t l y dependent on hydroxide ion concen t r a t i o n although the r e l a t i o n s h i p was not l i n e a r perhaps because o f the complexity o f the r e a c t i o n (Equation 7 ) . Sternberg and Kim (20) found the r a t e o f l y s i n o a l a n i n e formation i n c a s e i n to be dependent on hydroxide i o n c o n c e n t r a t i o n . Touloupais and V a s s i l i a d i s (45) a l s o found the r a t e o f l y s i n o a l a n i n e formation i n wool to be pH dependent. These workers d i d not measure the r a t e o f 3 e l i m i n a t i o n , t h e r e f o r e the r a t e determining step i s not known. These r e s u l t s on p r o t e i n s appear to be i n c o n t r a d i c t i o n to those o f Samuel and S i l v e r (46) who reported t h a t hydroxide ion c o n c e n t r a t i o n had no e f f e c t on the r a t e o f 3 e l i m i n a t i o n from free phosphoserine between pH 7 and 1 3 . 5 . Because o f the e f f e c t 3 5



152

C H E M I C A L DETERIORATION OF PROTEINS

Table I I . E f f e c t o f Hydroxide Ion Concentration and Calcium Ion on I n i t i a l Rate o f 3 E l i m i n a t i o n of P h o s v i t i n and A n t i f r e e z e G l y c o p r o t e i n - 8 (AFGP-8) Protein

Initial

-

[OH ]

(M

2

(M Χ 1 0 ) AFGP-8

3

rate/[0H"] - 1

1

min" )

b

4.13 0.614 0.570 0.226 0.408

0.001 0.01 0.1 1.0 50

ave = 0.454 (0.647 at 6 0 ° C ) Phosvitin

d

0.0370 0.0514

1.74 5.41 12.3 18.7

0.0622 (0.934Γ 0.0647 ave = 0.0538

R e f e r e n c e 44. Performed a t 50.0°C i n 0.2 M phosphate-NaOH buffers and a t 3.70 X 10-5 M AFGP-8. u

L e f t out o f average.

C a l c u l a t e d using E = 9.60 k c a l / m o l . a

d

Reference 36. Performed a t 60.0°C i n KCl-NaOH buffers w i t h 1.0 X 1 0 " to 1.1 Χ Ι Ο " M p h o s v i t i n . Rates c o r r e c t e d to i o n i c s t r e n g t h o f 0.170. 6

e

5

A s i n d except i n presence o f 1.12 mM C a C l . ?


C


At

60.0°C.

13.5

20.3

20.2

3

-20.4

8.71

f

4

9

-5

added.

Reactions were i n 0.1 H NaOH a t 1 X 10

As i n c except 7.47 X T O " M C a C l

Reference 41_. Reactions were i n 0.1 N_ NaOH a t 1 X 10 mucoid (GAX ovomucoid).

Reference 4 1 ·

6

of

M golden pheasant ovo

M lysozyme.

R e f e r e n c e 36. A t 0.170 i o n i c s t r e n g t h w i t h p h o s v i t i n c o n c e n t r a t i o n s 1 . 0 - 1 1 X"T0" M phosvitin.

R e f e r e n c e 36.

c

R e f e r e n c e 44. A n t i f r e e z e g l y c o p r o t e i n - 8 i n 0.5 Ν NaOH a t 2.22 Χ 1 0 " M p r o t e i n concentration.

a

14.2

23.1

-7.21

22.5

20.1

20.8

GAX ovomucoid^ (cystine)

?

23.8

CaCl

-13.8

-39.9

(cal/mol/deg)

24.1

22.4

(kcal/mol)

19.5

8.94

(kcal/mol)

a

20.2

9.60

Lysozyme (cystine)

with

d

Phosvitin (O-phosphoserine)

0

(kcal/mol)

AS+

E f f e c t o f Temperature on the 3 E l i m i n a t i o n o f O-Phosphoserine, O-Glycothreonine and C y s t i n e Groups i n P r o t e i n s

AFGP-8 (O-glycothreonine)

b

Protein

Table I I I .



154


o f the e l e c t r o n d e n s i t y o f the amino and carboxylate groups on 3 e l i m i n a t i o n (see above) i n free phosphoserine the r e s u l t s are not d i r e c t l y comparable. 3 E l i m i n a t i o n from s e r i n e , threonine and c y s t i n e i s tempera t u r e dependent as shown by the data o f Table I I I . The e f f e c t o f temperature (ΔΗΤ) on the i n i t i a l r a t e s o f 3 e l i m i n a t i o n i s i n the i n c r e a s i n g order o f removal o f g l y c o s y l groups from g l y c o t h r e onine i n a n t i f r e e z e g l y c o p r o t e i n , s u l f u r from c y s t i n e i n GAX ovomucoid, phosphoryl groups from phosphoserine i n p h o s v i t i n and s u l f u r from c y s t i n e i n lysozyme. Therefore, the environment i n the p r o t e i n s may have more e f f e c t on the i n f l u e n c e o f temperature on the r a t e than the type o f group undergoing 3 e l i m i n a t i o n (compare ΔΗΤ o f 13.5 and 23.1 kcal/mol f o r GAX ovomucoid and lysozyme, r e s p e c t i v e l y i n Table I I I ) . The r a t e o f 3 e l i m i n a t i o n i s a l s o i n f l u e n c e d by the type o f ions present i n the s o l u t i o n . Sen et a l . (36) showed t h a t the r a t e o f 3 e l i m i n a t i o n o f phosphoserine i n p h o s v i t i n was markedly enhanced by the a d d i t i o n o f c a l c i u m c h l o r i d e (Table I V ) . Touloupis and V a s s i l i a d i s (45) reported t h a t sodium phosphate enhanced the r a t e o f formation o f l y s i n o a l a n i n e i n wool several f o l d over t h a t found i n sodium carbonate s o l u t i o n s o f equal pH. I t i s l i k e l y t h a t the observed e f f e c t was on the r a t e o f 3 e l i m i n a t i o n r a t h e r than the a d d i t i o n r e a c t i o n . As reported Table I V . E f f e c t o f Calcium C h l o r i d e Concentration on I n i t i a l Rates o f 3 E l i m i n a t i o n o f P h o s v i t i n and A d d i t i o n o f Lysine to the Dehydroalanine Formed CaC1

I n i t i a l rate of addition

I n i t i a l rate of β-elimination

2

4

(M Χ 1 0 ) 0 3.36 5.60 8.96 11.2

(M

- 1

1

(M"

2

min" Χ 10 )

A t 60°C, 0.123 Ν NaOH, 5.54 X 1 0 '

2

min Χ 1 0 ) 2.5 1.4 1.9 4.3 6.4

4.74 24.8 43.2 53.8 93.4

R e f e r e n c e 36.

1

6

M phosvitin.

above, F e a i r h e l l e r et a l . (42) found t h a t calcium and s t r o n tium ions permitted the formation o f l a n t h i o n i n e from c y s t i n e i n free form presumably by r e d u c t i o n o f the high e l e c t r o n d e n s i t y i n the neighborhood o f the α - c a r b o n . We suspect the same general e f f e c t i s o p e r a t i v e i n the case o f c a l c i u m i o n on the r a t e o f 3 e l i m i n a t i o n o f phosphoserine i n p h o s v i t i n . The s p e c i f i c e f f e c t i s to mask the negative charges on the phosphate group thus p e r m i t t i n g the hydroxide i o n to a b s t r a c t more e a s i l y the hydrogen


7.

WHITAKER


Solution

155


from the α-carbon (Equation 6 ) . A d d i t i o n R e a c t i o n . The double bond o f dehydroalanine and 3-methyldehydroalanine formed by the 3 - e l i m i n a t i o n r e a c t i o n (Equation 6) i s very r e a c t i v e w i t h n u c l e o p h i l e s i n the s o l u t i o n . These may be added n u c l e o p h i l e s such as s u l f i t e ( 4 4 ) , s u l f i d e ( 4 2 ) , c y s t e i n e and other s u l f h y d r y l compounds ( 2 0 , 4 7 ) , amines such as α - Ν - a c e t y l l y s i n e (47) o r ammonia ( 4 8 ) . Or the nucleo p h i l e s may be c o n t r i b u t e d by the s i d e chains o f amino a c i d r e s i dues, such as l y s i n e , c y s t e i n e , h i s t i d i n e or tryptophan, i n the p r o t e i n undergoing r e a c t i o n i n a l k a l i n e s o l u t i o n . Some o f these r e a c t i o n s are shown i n F i g u r e 1. Friedman (38) has p o s t u l a t e d a number o f a d d i t i o n a l compounds, i n c l u d i n g stereo-isomers f o r those shown i n F i g u r e 1, as w e l l as those compounds formed from the r e a c t i o n o f 3-methyldehydroalanine (from 3 e l i m i n a t i o n o f threonine). He has a l s o suggested a systematic nomenclature f o r these new amino a c i d d e r i v a t i v e s (38). As pointed out by Friedman the stereochemistry can be complicated because o f the number o f asymmetric carbon atoms (two to three depending on d e r i v a t i v e ) possible. A d d i t i o n to the double bond o f dehydroalanine (or 3-methyldehydroalanine) i n v o l v e s n u c l e o p h i l i c attack by compounds con t a i n i n g S, 0 o r NH as shown by F i g u r e 1. The o v e r a l l r e a c t i o n may be w r i t t e n as shown i n Equation 13 --HN-C-C0-II CHR

+

R'-X-H +

H -HN-C-C0-I XCHR

(13)

where R i s H or CH3 and R' may represent the p r o t e i n , the r e mainder o f the s i d e chain o f an amino a c i d d e r i v a t i v e or H as i n H S. The r a t e o f a d d i t i o n o f protein-bound l y s i n e to dehydro a l a n i n e has been shown to be pH dependent and temperature de pendent but r e l a t i v e l y independent o f i o n i c strength and c a l c i u m c h l o r i d e c o n c e n t r a t i o n (36). Because o f the n u c l e o p h i l i c nature o f the a d d i t i o n r e a c t i o n , i t i s not s u r p r i s i n g t h a t the i n i t i a l r a t e o f a d d i t i o n should be pH dependent u n t i l a l l the n u c l e o p h i l e i s i n the c o r r e c t form (unprotonated ε-amino group i n the case o f l y s i n e , pK ^ 1 0 . 5 ) . The e f f e c t o f temperature on the r a t e o f the a d d i t i o n r e a c t i o n to dehydroalanine i n p h o s v i t i n i s shown i n Table V . I t i s i n t e r e s t i n g t h a t , although CaCl2 appears not to a f f e c t the r a t e o f the a d d i t i o n r e a c t i o n at 60°C (Table IV), i t has a r a t h e r marked e f f e c t on AST. F e a i r h e l l e r e t a l . (42) found t h a t added c a l c i u m and strontium hydroxides permitted l a n t h i o n i n e formation from free c y s t i n e . Touloupis and V a s s i l i a d i s (45) reported t h a t the r a t e o f formation o f l y s i n o a l a n i n e i n wool was f a s t e r i n sodium phosphate than sodium carbonate at the same pH and temperature. While the r a t e o f 3 e l i m i n a t i o n o f phospho2



NrL

NrL

_ I^H'°-H HO^C^

ι

NH

NH

ι

ι 2

y u

Λ

(CH )

3

2

,

or

Arginine

NH,

(CH ),

—>

9

9

/

(CH ),

2

+ C=0

9

I

—NH-CH-CO—

\

—NH-CH-CO—

NH

Ornithine

Urea

Λ

\*

NH

I

\ I

-H%?

3

— N H - C H - C O — î = i —NH-CH-CO—


J H NH

H

ι

Η

(CH )

0-C-NrL

I HO-C-NH,

£

(5)

Q

/ \ H H*NH

0

Ί ^C-NH,

0=C-NH

NH

NH

(CH ) 2

9

—

3

(ÇH ) 2

--NH-CH-C0--

3

NH

+

3

«HN-CH-CO-Citrul 1 irre

--HN-C-C0--

II

CH —HN

C0-

CH

and

S (sulfur)

5

S

I CH

I

2

(Dehydroalanine)

I

CH

9

I

2

-HN-C-COH

(Cystine)

9

and

S® (sulfide) and

CH

-HN-C-COH

(7)

CH

2

II

2

0

--HN-C-CO— -HN—C^-CO--

(A persulfide)

(Dehydroalanine) (Cysteine)

H -HN-C-CO-

Η -HN-C-CO-

1

HCP^HCH

:CH S

S

—• S S ι I or CH I 2 --HN-C-CO-—HN-C-CO-H H

(9)

9

(Cyttine)

Ο -C-CO-(Thioaldehyde)

II CH —HN-C-C0-H (Aldehyde)

Figure 1. New amino acids which may be formed through reaction of a dehydro alanine residue with internal or external nucleophiles in alkali treated proteins.


7.

WHITAKER

—HN-C-CO--

--HN-C-CO--

CH


--HN-C-CO--

CH,

0

OH

4OH

—

--HN—Ç—C0--

CH,

0

*

.

CH

^

(a sulfenic acid) CH

157

Proteins in Alkaline Solution

9

n

{

$

(Cysteine)

n

)

(a sulfinic acid)

and

2

--HN-C-C0-

S

0

CH,

H

(Cystine)

.. N-C-C0H

H (Cysteine)

HOOC X

^COOH CH-CH -NH-(CH ) -CH; 2

2

4

m ,

2

Lysinoalanine OH

(Bohak, 1964; Ref. 50) Ν

r

CH -CH-C00H

Ν

9

d

••••

|ΓΝΗ

0

Lysine

CH -CH-C00H 2 ι

HN(CH ) CH-COOH

9

2 3

NH

2

3-T-Histidinoalanine

Arginine derivative

HOOC

(postulated by Finley and Friedman, 1977; R é f . 5JJ

(postulated by Finley and Friedman, 1977; R é f . 51J

C=CH

0

Tryptophan^ Unknown (postulated by Finley and Friedman, 1977; Ref. 51.)

' HOOC

Dehydroalanine

2

H00C

x

C00H CH-CH -S-CH -CH NH x

9

HN 2

9

2

(Horn et al_., 1941; Ref. 52)

Figure 1.

V

2

(Ziegler et a i . , 1967; Ref. 22)

V

ù

Lanthionine

B-Aminoalanine (Asquith and Skinner, 1970; Ref. 49)

L

9

2

Ornithinoalanine

Cysteine HOOC

^COOH CH-CH -NH-(CH )~-CH NH 9

HN

Continued


158

CHEMICAL

DETERIORATION

O F

PROTEINS

Table V . E f f e c t o f Temperature on the A d d i t i o n o f I n t e r n a l Lysine to 3 E l i m i n a t e d P h o s v i t i n a

Reaction

No C a C l


CaCl

AG+

(kcal/mol)

25.0

24.3

24.6

-0.90

27.6

26.9

24.2

+8.11

2

R e f e r e n c e 36.. at

7.47 Χ 10"

(cal/mol/deg)

(kcal/mol)

2

c

b

(kcal/mol)

c

b

AS+

b

Rate data c o r r e c t e d to i o n i c strength o f 0.170.

60.0°C. 4

M CaCl . 2

s e r i n e i n p h o s v i t i n was q u i t e dependent on i o n i c s t r e n g t h , the a d d i t i o n r e a c t i o n was completely independent o f i o n i c strength (36).

The r a t e o f n u c l e o p h i l e a d d i t i o n to the double bond i s dependent on the nature o f the n u c l e o p h i l e as would be expected. F i n l e y e t a l . (47) have measured the r e l a t i v e e f f e c t i v e n e s s o f the s u l f h y d r y l group o f L - c y s t e i n e and the ε-amino group o f α - Ν - a c e t y l - L - l y s i n e i n adding to the double bond o f N-acetyl dehydroalanine. A t equal concentrations o f the r e a c t i v e s p e c i e s , c y s t e i n e adds some 31 times more r a p i d l y to the double bond than does a - N - a c e t y l - L - l y s i n e ( 4 7 ) . However, when one compares these two compounds a t the same pH the r e l a t i v e rates i n favor o f c y s t e i n e (pK o f s u l f h y d r y l group = 8.15) versus a - N - a c e t y l - L l y s i n e (pK o f ε-amino group = 10.53) are most impressive at lower pH's (Table V I ) . Therefore, i t has been recommended t h a t c y s Table V I . R e l a t i v e Rates o f A d d i t i o n o f the S u l f h y d r y l Group o f C y s t e i n e and the ε-Amino Group o f α - Ν - A c e t y l - L - L y s i n e to the Double Bond o f N-Acetyldehydroalanine at D i f f e r e n t pH V a l u e s d

pH

Relative rate Cysteine SH/Lysine ε-ΝΗ,

7.0 8.0 9.0 10.0 11.0 12.0

5000 2300 410 133 43 34

Adapted from Reference 47.


7.

WHiTAKER


t e i n e be added to foods during a l k a l i processing i n order to minimize l y s i n o a l a n i n e formation (20,47) and prevent l o s s o f the e s s e n t i a l amino a c i d l y s i n e .


S i g n i f i c a n c e and a p p l i c a t i o n o f a l k a l i treatment o f p r o t e i n s The r e a c t i o n s o f p r o t e i n s i n a l k a l i n e s o l u t i o n are very im portant from a number o f s t a n d p o i n t s . We have already discussed s e v e r a l uses o f a l k a l i treatment i n food processing i n the i n t r o d u c t i o n . When contact between the food and a l k a l i i s kept to a minimum a t the lowest temperature p o s s i b l e w i t h adequate con t r o l o f m i x i n g , e t c . there i s p r e s e n t l y no apparent reason to d i s c o n t i n u e i t s use. Low l e v e l s o f l y s i n o a l a n i n e occur i n food which has been processed i n the absence o f added a l k a l i , even at pH 6 and i n the dry s t a t e ( 2 0 ) . For example, the egg white o f an egg b o i l e d three minutes contained 140 ppm o f l y s i n o a l a n i n e w h i l e d r i e d egg white powder contained from 160 to 1820 ppm o f l y s i n o a l a n i n e depending on the manufacturer ( 2 0 ) . No l y s i n o a l a n i n e was found i n fresh egg w h i t e , 3 E l i m i n a t i o n and a d d i t i o n o f l y s i n e to the double bond o f dehydroalanine reduce the l e v e l o f the e s s e n t i a l amino a c i d l y s i n e . T h i s can be prevented by adding other n u c l e o p h i l e s such as c y s t e i n e to the r e a c t i o n . Whether l y s i n o a l a n i n e (and other compounds formed by a d d i t i o n r e a c t i o n s ) i s t o x i c at low l e v e l s i n humans i s not known. The 3 - e l i m i n a t i o n and a d d i t i o n r e a c t i o n s may be important i n the t e x t u r i z i n g o f foods extruded from a l k a l i n e s o l u t i o n . Should t h i s prove to be r e q u i r e d i n t e x t u r i z a t i o n other means o f forming c r o s s l i n k a g e s between p r o t e i n molecules should be developed. In t h i s c o n n e c t i o n , 3 e l i m i n a t i o n i n the presence o f d i t h i o l com pounds may prove u s e f u l . F o l l o w i n g 3 e l i m i n a t i o n and a d d i t i o n o f the d i t h i o l compound to the double bond v i a one o f the s u l f h y d r y l groups, the modified p r o t e i n could then be allowed to o x i d i z e i n a i r to form d i s u l f i d e b r i d g e s . The p h y s i c a l and d i g e s t i b i l i t y p r o p e r t i e s o f such a p r o t e i n would be most i n t e r e s t i n g . Hydrogen s u l f i d e , which adds to the double bond ( 4 2 j , could a l s o be used f o r t h i s purpose. The 3 - e l i m i n a t i o n r e a c t i o n could a l s o be used to change the s o l u b i l i t y p r o p e r t i e s o f a p r o t e i n . For example, a l k a l i t r e a t ment i n the presence o f sodium s u l f i t e leads to i n c o r p o r a t i o n o f s u l f o n a t e groups i n t o the p r o t e i n (44,53) which would increase i t s water s o l u b i l i t y and probably change i t s f u n c t i o n a l proper ties. The 3 - e l i m i n a t i o n r e a c t i o n i s used r o u t i n e l y to d i s t i n g u i s h 0 - g l y c o s y l l i n k a g e s o f carbohydrate to s e r i n e and threonine i n p r o t e i n s from amide l i n k a g e s o f carbohydrates to asparagine residues i n p r o t e i n s ( 2 ) . In a l k a l i , the 0 - g l y c o s y l groups undergo 3 e l i m i n a t i o n to form dehydroalanine (from s e r i n e ) and 3-methyldehydroalanine (from threonine) w h i l e the a m i d e - l i n k e d carbohydrate i s not removed by such treatment. The 3 - e l i m i n a t i o n r e a c t i o n has been used to show the e s s e n t i a l i t y o f the carbohy-


159



160

d r a t e s i d e chains f o r the f r e e z i n g - p o i n t depressing a c t i v i t y and l e c t i n - i n h i b i t i n g p r o p e r t i e s o f the a n t i f r e e z e g l y c o p r o t e i n s ( 3 ) . 3 E l i m i n a t i o n has been used to show d i f f e r e n c e s among the d i s u l f i d e bonds i n various p r o t e i n s i n c l u d i n g the ovomucoids ( 5 4 ) . The 3 - e l i m i n a t i o n r e a c t i o n has a l s o been used to r e p l a c e the hydroxyl group o f the e s s e n t i a l s e r i n e residue o f s u b t i l i s i n w i t h a s u l f h y d r y l group ( 5 5 ) . The t h i o l s u b t i l i s i n had a small f r a c t i o n o f the a c t i v i t y o f s u b t i l i s i n but i t has been q u i t e useful i n mechanistic s t u d i e s o f the s e r i n e and s u l f h y d r y l proteases. As a consequence o f dehydroalanine and 3-methyldehydro a l a n i n e formation s p e c i f i c bond cleavage can o c c u r . Ebert e t a l . (56) have shown t h a t a d d i t i o n o f c y s t e i n e to the double bonds o f polydehydroalanine and copolymers o f dehydroalanine r e s u l t s i n increased s o l u b i l i t y and decrease i n molecular weight because o f peptide bond cleavage caused by formation o f a t h i a z o l i d i n e . T h i s r e a c t i o n can be used f o r s e l e c t i v e peptide chain cleavage o f c y s t e i n e - c o n t a i n i n g polypeptides and p r o t e i n s under r a t h e r m i l d c o n d i t i o n s . M i l d a c i d treatment o f d e h y d r o a l a n i n e - c o n t a i n i n g polypeptides and p r o t e i n s leads to s p e c i f i c peptide bond cleavage w i t h formation o f pyruvate and ammonia ( 5 7 - 5 9 ) ) . The d i f f e r e n c e i n s p e c t r a l p r o p e r t i e s o f t y r o s i n e i n n e u t r a l vs a l k a l i n e s o l u t i o n can be used to determine the t y r o s i n e con t e n t o f p r o t e i n s and by d i f f e r e n c e tryptophan ( 6 0 ) . Acknowledgments The author thanks V i c k y Crampton f o r checking the and C l a r a Robison f o r t y p i n g o f the manuscript.

references

Literature Cited 1. Leone, E. Biochem. J., 1953, 54, 393. 2. Downs, F.; Pigman, W. Meth. Carbohydr. Chem., 1976, 7, 200. 3. Ahmed, A. I.; Osuga, D. T.; Feeney, R. E. J. Biol. Chem., 1973, 248, 8524. 4. Cunningham, S. D.; Cater, C. M.; Matill, K. F. J. Food Sci., 1975, 40, 732. 5. Vananuvat, P.; Kinsella, J. E. J. Agric. Food Chem., 1975, 23, 216. 6. Lindblom, M. A. Lebensmittel.-Wiss U-Technol., 1974, 7, 295. 7. Lindblom, M. A. Biotechnol. Bioeng., 1974, 16, 1495. 8. Feeney, R. E.; Allison, R. G. "Evolutionary Biochemistry of Proteins. Homologous and Analogous Proteins from Avian Egg Whites, Blood Sera, Milk, and Other Substances"; John Wiley and Sons: New York, N.Y., 1969. 9. Dakin, H. D.; Dudley, H. W. J. Biol. Chem. 1913, 15, 271. 10. Ten Broeck, C. J. Biol. Chem. 1914, 17, 369. 11. Cheftel, C.; Cuq, J. L . ; Provansal, M.; Besancon, P. Rev.



7.

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161

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Changes Occuring in Proteins in Alkaline Solution - ACS Symposium

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