Isopeptides: The Occurrence and Significance of ... - ACS Publications

Jul 23, 2009 - MICHAEL S. OTTERBURN. Department of Chemical Engineering, The Queen's University of Belfast, 21 Chlorine Gardens, Belfast BT9 5DL, ...
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13 Isopeptides: The Occurrence and Significance of Natural and Xenobiotic Crosslinks in Proteins

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MICHAEL S. OTTERBURN Department of Chemical Engineering, The Queen's University of Belfast, 21 Chlorine Gardens, Belfast BT9 5DL, Northern Ireland

Proteins are biopolymers having a molecular weight arbi­ trarily greater than 5,000 daltons. They are essential for func­ tions in cellular structure, catalysis, metabolic regulation and contractile processes. They also play an important part in the defensive and protective mechanisms in animals. Protein chains are built up of monomeric units, the α-amino acids linked together by amido groups (peptide links). This polymerisation occurs through the α-amino and α-carboxyl groups of adjacent residues. The result is a complex random copolymer with many different functional groups and consequently complex chemical and physical properties. Whilst it is self-evident that the protein reactivity and many of its physical properties depend on the primary sequence, the secondary and tertiary structures are also important in defining the molecules functions. Many inter- and intrachenic forces and crosslinks are responsible for maintaining these structures and it is the purpose of this review to emphasize the importance of such crosslinks in protein struc­ ture, function and reactivity. Bonding and C r o s s l i n k i n g i n P r o t e i n s Bonding f o r c e s which occur i n p r o t e i n s f a l l into two groups, p h y s i c a l and c h e m i c a l , the l a t t e r being o f p a r t i c u l a r i n t e r e s t in t h i s review. P h y s i c a l f o r c e s include i o n i c i n t e r a c t i o n s b e ­ tween o p p o s i t e l y charged r e s i d u e s {Y) hydrogen bonds which a r e v i t a l i n s t a b i l i z i n g t h e α - h e l i c a l conformations and i n t e r c h e n i c a t t r a c t i o n s in the secondary and t e r t i a r y s t r u c t u r e s ( 2 , 3 ) . The r o l e o f hydrophobic i n t e r a c t i o n i s a l s o p a r t i c u l a r l y Impor­ tant i n the s t a b i l i z a t i o n process ( 4 - 6 ) . The common feature o f such p h y s i c a l s t a b i l i z a t i o n i s t h a t t h e a t t r a c t i o n s can be broken and reformed by v a r i o u s chemical and p h y s i c a l changes i n t h e p r o ­ t e i n s ' environment. An a l t e r n a t i v e method o f s t a b i l i z a t i o n i n p r o t e i n s i s t h a t 9

0097-6156/83/0234-0221 $06.00/0 © 1983 American Chemical Society

Finley and Schwass; Xenobiotics in Foods and Feeds ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

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222

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b r o u g h t a b o u t by c o v a l e n t i n t e r a c t i o n s between r e s i d u e s o f a d ­ j a c e n t p o l y p e p t i d e c h a i n s . T h e most w i d e l y d i s t r i b u t e d o f a l l known c r o s s l i n k s i s c y s t i n e . T h i s m o l e c u l e a l l o w s two c h a i n s ( o r a s i n g l e f o l d e d c h a i n ) t o be c o v a l e n t l y l i n k e d t o g e t h e r by a d i s u l p h i d e b r i d g e . T h e i m p o r t a n c e o f t h i s m o i e t y and i t s i n h e r ­ e n t r e a c t i v i t y has l e d t o a c o n s i d e r a b l e amount o f work b e i n g r e ­ p o r t e d i n t h e s c i e n t i f i c l i t e r a t u r e (7_, 8 ) . O t h e r c r o s s l i n k s o f a more s p e c i a l i z e d o r i g i n have a l s o been n o t e d i n t h e l i t e r a t u r e . Such c r o s s l i n k s t e n d t o be s p e c i f i c t o a p a r t i c u l a r p r o t e i n rather than being ubiquitous l i k e c y s t i n e . Thus i n c o l l a g e n and e l a s t i n t h e a l d o l and a l d o l i m i d e c r o s s l i n k s o c c u r . Precursors f o r c r o s s l i n k i n g in these p r o t e i n s are the l y s i n e and h y d r o x y l y s i n e d e r i v e d a l d e h y d e s w h i c h a r e f o r m e d enzymically. The c r o s s l i n k s o f c o l l a g e n a r e f o r m e d by a s e r i e s o f s p o n t a n e o u s a l d o l and a l d i m i d e c o n d e n s a t i o n s . I n e l a s t i n t h e c r o s s l i n k s a r e f o r m e d by s i m i l a r r e a c t i o n s l e a d i n g i n t h i s c a s e t o t h e h i g h l y s t a b l e p y r i d i n i u m compounds d e m o s i n e and i s o d e m o s i n e . In the case o f c o l l a g e n , the f u n c t i o n o f the c r o s s l i n k s seems t o be t o c o n f e r o r d e r o n t h e a g g r e g a t e d p r o t e i n c h a i n s w i t h a r e s u l t i n g i n c r e a s e i n s t r e n g t h and d i m e n s i o n a l s t a b i l i t y . I n t h e c a s e o f e l a s t i n , the c r o s s l i n k s f u n c t i o n a p p e a r s t o be t h a t of r e s t r i c t i n g the e x t e n s i b i l i t y o f the polypeptide chain (9-11). Another group o f c r o s s l i n k s i n v o l v i n g t y r o s y l r e s i d u e s h a s been d e m o n s t r a t e d t o e x i s t i n t h e p r o t e i n r e s i l i n (12). T h i s c r o s s l i n k has been shown t o be f o r m e d f r o m d i m e r i c and t r i m e r i c r e s i d u e s o f t y r o s i n e ( 1 3 ) . The mechanism o f f o r m a t i o n o f t h e s e compounds i s b e l i e v e d t o be i n i t i a t e d by a p e r o x i d a s e . The e n ­ zyme c a t a l y z e s t h e o x i d a t i v e c o u p l i n g o f t h e t y r o s i n e r e s i d u e s through the three p o s i t i o n s o f the aromatic r i n g s . T a b l e 1 i l l u s t r a t e s some o f t h e c r o s s l i n k s known t o o c c u r i n p r o t e i n s . Isopeptide

Links

I s o p e p t i d e c r o s s l i n k s a r e f o r m e d by t h e c o v a l e n t r e a c t i o n o f t h e ε-amino g r o u p o f l y s i n e r e a c t i n g w i t h e i t h e r t h e 3 - c a r b o x y l group o f a s p a r t i c a c i d o r the α-carboxyl group o f glutamic a c i d . T h u s t h e g e n e r a l n o t a t i o n o f t h e s e l i n k s i s t o r e f e r t o them a s ω-ε i s o p e p t i d e c r o s s l i n k s t o d i f f e r e n t i a t e them f r o m t h e normal peptide 1 ink. The i d e a t h a t such c r o s s l i n k s m i g h t o c c u r i n p r o t e i n s was r a i s e d by F i s c h e r (14) and more f o r c e f u l l y by A s t b u r y ( 1 5 ) . P a u l i n g and Nieman a l s o a l l u d e d t o s u c h amide c r o s s l i n k s ( 1 6 ) . However, l i t t l e o r no work was c a r r i e d o u t o n t h e s e e a r l y s u g g e s ­ t i o n s , p o s s i b l y because o f the p r o t e i n chemistry obsession with c y s t i n e and t h e i n t r a c t a b l e c r o s s l i n k s o f c o l l a g e n . In s p i t e o f t h i s h i a t u s , much i n d i r e c t e v i d e n c e was amassed f o r t h e i n v o l v e ­ ment o f t h e ε-amino g r o u p o f l y s i n e i n b i n d i n g r e a c t i o n s . This c i r c u m s t a n t i a l e v i d e n c e has been a d e q u a t e l y r e v i e w e d by Loewy (J_7), P i s a n o e t a l . ( J 8 ) , F i n l a y s o n Q 9 ) and A s q u i t h e t a l . (20). As a c o n s e q u e n c e o f t h i s , t h e c r o s s l i n k s (and o t h e r s ) were i n -

Finley and Schwass; Xenobiotics in Foods and Feeds ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

Finley and Schwass; Xenobiotics in Foods and Feeds ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

ι

0

ι

H C-CH -S-S-CH,-CH ι 2 2 ,

Naturally Occurring

C r o s s l i n k s in P r o t e i n s

Table 1

— C H

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— C H — I

ω-ε ISOPEPTIDE

I

224

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f e r r e d from binding experiments. This u n s a t i s f a c t o r y s i t u a t i o n was r e s o l v e d when an a d e q u a t e e n z y m i c d i g e s t i o n p r o c e d u r e was d e v e l o p e d w h i c h e n a b l e d c - ( - y - G L U T A M I C ) L Y S I N E (G-L) t o be i d e n t i f i e d f o r t h e f i r s t t i m e . T h i s b r e a k t h r o u g h o c c u r r e d i n 1968 w i t h t h e i d e n t i f i c a t i o n o f G-L i n b o v i n e and human f i b r i n . T h i s work showed t h a t a c r o s s l i n k o f G-L was f o r m e d a s t h e f i r s t s t e p i n t h e f i b r i n o g e n ^ f i b r i n t r a n s f o r m a t i o n i n b l o o d c l o t t i n g . The s t e p s l e a d i n g up t o t h e c r o s s l i n k f o r m a t i o n were i d e n t i f i e d and c h a r a c t e r i z e d {2]_ 2 2 ) . T h e p r e s e n c e o f G-L i n t h i s s y s t e m o p e n e d a new e r a i n c r o s s l i n k i n v e s t i g a t i o n and f r o m t h a t t i m e many w o r k e r s have f o u n d t h e m o i e t y i n a wide v a r i e t y o f o r g a n i s m s . T h i s l e d Loewy e t a l . t o c o n c l u d e t h a t t h e i s o p e p t i d e o c c u r s u n i v e r s a l l y in a l l l i v i n g systems (23). Subsequent r e s e a r c h y i e l d e d t h e c r o s s l i n k i n an i m p r e s s i v e a r r a y o f c e l l s , o r g a n i s m s and t i s s u e s . T h e o c c u r r e n c e o f G-L and i t s s i g n i f i c a n c e i n b i o l o g i c a l m a t e r i a l s was t h e s u b j e c t o f an e x c e l l e n t r e v i e w by F o l k and F i n i a y s o n ( 2 4 ) . F o l l o w i n g t h e work o n f i b r i n , t h e n e x t p r o t e i n t o be e x t e n s i v e l y s t u d i e d a s f a r a s t h e i s o p e p t i d e was c o n c e r n e d was t h e h e t e r o g e n e o u s s t r u c t u r a l p r o t e i n k e r a t i n . In t h e i r work, A s q u i t h e t a l . (25) i d e n t i f i e d and i s o l a t e d G-L f r o m n a t i v e wool k e r a t i n and s u b s e q u e n t l y i s o l a t e d t h e a s p a r t y l a n a l o g u e c-(-3-ASPARTIC) LYSINE (A-L) f r o m t h e same s o u r c e ( 2 6 ) . O t h e r w o r k e r s went o n t o i d e n t i f y t h e i s o p e p t i d e s i n human h a i r , g u i n e a p i g h a i r , p o r c u p i n e q u i l l s and p r o t e i n f r o m h a i r f o l l i c l e s (27, 2 8 ) . O t h e r t i s s u e s where i s o p e p t i d e s have been l o c a t e d i n c l u d e a v i a n and mouse m u s c l e ( 2 9 ) , human s t r a t u m c o r n e u m (_30), e r t h y r o c y t e membranes (31 ) , c e l l membranes {32) and b o v i n e c o l o s t r u m ( 3 3 ) . The mechanisms o f f o r m a t i o n o f G-L i n a l l t h e s e d i v e r s e t i s s u e s , w h e t h e r s l i m e m o u l d s o r human t i s s u e , i s b e l i e v e d t o be s i m i l a r . The c r o s s l i n k i n g b e i n g b r o u g h t a b o u t e n z y m i c a l l y v i a a t r a n s g l u t a m i n a s e , i n some c a s e s t h e enzyme s y s t e m , has been i d e n t i f i e d (24). The f u n c t i o n o f t h e i s o p e p t i d e s i n t h e v a r i o u s t i s s u e s i s n o t c l e a r , a l t h o u g h t h e y may c o n t r i b u t e d i r e c t l y t o t h e s t r u c t u r e o f the p a r t i c u l a r p r o t e i n . For example, p r i o r t o k e r a t i n i z a t i o n o r by s i m p l y i n c r e a s i n g t h e m o l e c u l a r w e i g h t o f a p r o t e i n s y s t e m c o n f e r s e n h a n c e d s t a b i l i t y , d e c r e a s e d s o l u b i l i t y and i n c r e a s e d r e s i s t a n c e t o enzymic h y d r o l y s i s .

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9

I s o l a t i o n and A n a l y s i s o f I s o p e p t i d e s The m a j o r p r o b l e m i n a l l work c a r r i e d o u t o n i s o p e p t i d e s was r e l a t e d t o t h e f a c t t h a t t h e i s o p e p t i d e bond i s c h e m i c a l l y a n amide bond and a s a c o n s e q u e n c e o f t h i s i s s u s c e p t i b l e t o a t t a c k by a c i d s o r a l k a l i s , t h u s d e s t r o y i n g t h e i s o p e p t i d e . The o n l y p o s s i b l e methods were m i c r o b i o l o g i c a l o r e n z y m i c , b o t h o f w h i c h o b v i a t e t h e p r o b l e m o f random h y d r o l y s i s . Methods o f e n z y m i c d i g e s t i o n had p r e v i o u s l y been known and a d e q u a t e l y u s e d ; however, such methods, a l t h o u g h s u i t a b l e f o r g l o b u l a r p r o t e i n s , p r o v e d t o

Finley and Schwass; Xenobiotics in Foods and Feeds ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

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13.

OTTERBURN

Natural

and Xenobiotic

Crosslinks

in

Proteins

225

be i n s u f f i c i e n t f o r p r o t e i n s w i t h a h i g h c r o s s l i n k d e n s i t y due t o h i g h c o n c e n t r a t i o n s o f c y s t i n e . In t h e l a t e 1960's and e a r l y 1970's, new methods i n v o l v i n g r e d u c i n g and b l o c k i n g o f c y s t i n e f o l l o w e d by e n z y m i c d i g e s t i o n were d e v e l o p e d . T h e s e p r o v e d t o be s u c c e s s f u l enough t o a l l o w i d e n t i f i c a t i o n and i s o l a t i o n o f t h e i s o p e p t i d e s (22, 3 4 ) . S i n c e t h e n , o t h e r methods have been s u g g e s t e d which m o d i f y t h e r e d u c t i o n and b l o c k i n g s t a g e s and a l s o i n t r o d u c e p r e - d i g e s t i o n s t a g e s i n t o t h e schemes ( 3 5 ) . In a l l t h e s e methods, p a r t i c u l a r l y w i t h t h e I c e r a t i n s , t h e e f f i c i e n c y o f r e d u c t i o n , b l o c k i n g and d i g e s t i o n i s i n q u e s t i o n and c o n s e q u e n t l y t h e t r u e c o n c e n t r a t i o n o f i s o p e p t i d e . I f t h e r e d u c t i o n s t a t e i s not 100% e f f i c i e n t , a r e a s o f t h e p r o t e i n w i l l be u n a b l e t o be d i g e s t e d and t h u s any i s o p e p t i d e s p r e s e n t i n such r e g i o n s w i l l n o t be m o n i t o r e d . T h u s t h e r e a r e f r u i t f u l areas o f r e s e a r c h a v a i l a b l e i n t o enzymic d i g e s t i o n o f c r o s s l i n k e d p r o t e i n s and, u n t i l a t o t a l l y e f f i c i e n t s y s t e m i s d e v e l o p e d , t h e t r u e s i g n i f i c a n c e o f i s o p e p t i d e s i n p r o t e i n s w i l l n o t be r e v e a l e d . The o r i g i n a l method o f a n a l y s e s o f t h e i s o p e p t i d e s i n p r o t e i n d i g e s t s was t o use i o n e x c h a n g e c h r o m a t o g r a p h y u s i n g s o d i u m b u f f e r s (22-25). However, a l t e r n a t i v e l y l i t h i u m b u f f e r s were u s e d in o r d e r t o o b t a i n b e t t e r s e p a r a t i o n and r e s o l u t i o n ( 3 6 ) . More r e c e n t l y G r i f f i n e t a l . (37) have d e v e l o p e d HPLC t o q u a n t i f y G-L, w h i l s t o t h e r w o r k e r s have d e v e l o p e d r a p i d i o n e x c h a n g e methods (38., 21). Formation o f Isopeptides

in Heated P r o t e i n s

The f a c t t h a t t h e i s o p e p t i d e s c o u l d be f o r m e d a s a c o n s e quence o f t h e r m a l t r e a t m e n t o f p r o t e i n s was f i r s t d e m o n s t r a t e d by A s q u i t h and O t t e r b u r n who a l s o showed t h a t t h e i s o p e p t i d e s h a d c r o s s l i n k i n g f u n c t i o n s and t h a t t h e i r c o n c e n t r a t i o n s i n c r e a s e d w i t h i n c r e a s i n g s e v e r i t y o f h e a t i n g ( 4 0 ) . T h e mechanism o f f o r m a t i o n c o u l d e i t h e r be v i a a c o n d e n s a t i o n o r t r a n s a m i n a t i o n r e a c t i o n d e p e n d i n g on w h e t h e r t h e f r e e c a r b o x y l i c a c i d o r t h e amide i s i n v o l v e d ( F i g u r e 1) ( 2 0 ) . L a t e r , o t h e r w o r k e r s c o n f i r m e d t h e s e f i n d i n g s which a r e summ a r i z e d i n T a b l e 2. T h u s i s o p e p t i d e s t o date have been f o u n d t o be f o r m e d d u r i n g h e a t i n g i n k e r a t i n ( 4 0 ) , m i l k p r o t e i n s ( 4 1 ) , m u s c l e p r o t e i n s ( 4 2 ) , r i b o n u c l e a s e ( 4 3 j and o t h e r p r o t e i n s o f n u t r i t i o n a l i n t e r e s t (44). The ease o f formation o f the isopept i d e i n a n y s p e c i f i c p r o t e i n s h o u l d be d e p e n d e n t on two f a c t o r s : f i r s t l y , t h e c o n c e n t r a t i o n o f g l u t a m i c and a s p a r t i c a c i d s i n r e l a t i o n t o t h e c o n c e n t r a t i o n o f l y s i n e and, s e c o n d l y , t h e p r o x i m i t y o f t h e l y s y l and c a r b o x y l i c r e s i d u e s w h i c h w i l l be d e t e r mined by t h e c o n f o r m a t i o n o f t h e p r o t e i n c h a i n . I t has been shown by O t t e r b u r n e t a l . (44) t h a t t h e r e i s no o b v i o u s c o r r e l a t i o n between t h e q u a n t i t y o f i s o p e p t i d e s f o r m e d and t h e c o n c e n t r a t i o n o f t h e i n v o l v e d r e s i d u e s i n t h e p r o t e i n . T h e s e a u t h o r s a l s o showed t h a t i n o n e s p e c i f i c c a s e , v i z . l y s o zyme, t h e s t e r e o s t r u c t u r e o f t h e p r o t e i n c o u l d be u s e d a s a g u i d e

Finley and Schwass; Xenobiotics in Foods and Feeds ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

XENOBIOTICS IN F O O D S A N D F E E D S

226

COOH HC-(CH )2-COOH or

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2

NH

NH

2

\ ^

HC-CH -COOH

COOH

NH

2

COOH 4HC-(CH2)4-NH NH

N H^2 5

COOH

NH

COOH

2

2

2

COOH

2

ε - (-Y-GLUTAMIC) LYSINE reaction

J

NH

2

H C - C H 2 - C O - N H - (CH2)4-CH

HC- (CH2 )2 - C O - N H - (CH >4-CH

1. Proposed

/ 2

Lysine

Lysine

Figure

2

Asparagine

Aspartic acid

j

+HC-(CH2)4-NH2

NH

or HC-CH2-CONH2

2

NH

2

Glutamine

Glutamic acid

COOH

COOH

COOH HC-CC^-CONHz

NH

COOH

2

É - ( - β - A S P A R T I C ) LYSINE scheme for the formation heated proteins.

of ω - e isopeptides

Finley and Schwass; Xenobiotics in Foods and Feeds ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

in

Finley and Schwass; Xenobiotics in Foods and Feeds ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

0.80

9.70 14.70

10.50

121

27

muscle

Chicken

0.40

9.70 14.70

10.50

121

8

muscle

Chicken

0.30

9.70 14.70

10.50

121

4

muscle

Chicken

0.14

7.88 19.99

6.80

115

27

Casein

0

0.43 5.75

4.92

16.80

121

27

Lysozyme

0.74

1.04

10.37 15.46

10.07

115

57

Lactalbumin

0.06

0.04 10.37

15.46

10.07

85

27

Lactalbumin

0

0.11

0.05

22.16

5.06

115

57

Zein

0.27

6.82

9.24

115

albumen

57

Egg

0.90

0.40

0.20

0.48

0.12

0.44

13.05

0.25

7.56

9.16

9.62

high temperature

0.32

B l o o d meal

0.33

8.92

9.20

9.50

0.19

0.75

9.85

G-L

A-L

7.14

Lysine

low temperature

9.73

121

27

Bovine haemoglobin

Glutamic

gN)

B l o o d meal

Aspartic

Temp. °C

Time o f Heating (h)

Protein

I s o p e p t i d e s i n H e a t e d P r o t e i n s (g/16

Table 2

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X E N O B I O T I C S IN F O O D S A N D

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228

FEEDS

t o t h e t y p e o f c r o s s l i n k most l i k e l y t o be f o r m e d . Weder e t a l . (43) have r e c e n t l y shown a s i m i l a r example w i t h r i b o n u c l e a s e where t h e y showed t h a t i n t e r m o l e c u l a r b o n d i n g o c c u r r e d between d i m e r s and o l i g o m e r s o f r i b o n u c l e a s e . T h e a b s o l u t e v i n d i c a t i o n o f t h i s argument a w a i t s more work b e i n g c a r r i e d o u t on p r o t e i n s w i t h known s e c o n d a r y s t r u c t u r e s . The p r e s e n c e o f r e d u c i n g s u g a r s and l i p i d s m i l i t a t e s a g a i n s t t h e f o r m a t i o n o f i s o p e p t i d e s : i n t h e f o r m e r c a s e , b e c a u s e o f com­ p e t i t i v e M a i l l a r d r e a c t i o n s , a n d i n t h e l a t t e r , by c o m p e t i t i o n t o f o r m e s t e r s o r by a c t i n g a s a h y d r o p h o b i c b a r r i e r between t h e l y s y l and g l u t a m y l o r a s p a r t y l r e s i d u e s ( 4 4 ) . T h u s t h e r e i s now c o n s i d e r a b l e e v i d e n c e t h a t i s o p e p t i d e s a r e f o r m e d d u r i n g t h e h e a t i n g o f p r o t e i n s , t h e amounts f o r m e d d e p e n d ­ i n g o n t h e p u r i t y o f t h e s t a r t i n g m a t e r i a l and t h e s e v e r i t y o f heating. N u t r i t i o n a l Consequences o f Isopeptide

Formation

I t has been known f o r some t i m e t h a t h e a t i n d u c e s c h a n g e s i n t h e p h y s i c a l p r o p e r t i e s o f p r o t e i n s and t h a t such c h a n g e s i n ­ f l u e n c e t h e p r o t e i n s d i g e s t i b i l i t y and hence i t s n u t r i t i o n a l v a l u e a s a f o o d (45, 4 6 ) . T h e n u t r i t i o n a l v a l u e o f h e a t s t e r i l i z e d p r o t e i n s was i n v e s t i g a t e d by C a r p e n t e r e t a l . (47) and o t h e r s (48, 4 9 ) . T h i s work e s t a b l i s h e d t h a t t h e d e c r e a s e i n n u t r i t i o n a l v a l u e was r e l a t e d t o t h e i n v o l v e m e n t o f r e a c t i v e l y s i n e g r o u p s . From t h i s work i t became a p p a r e n t t h a t t h e ε-amino g r o u p o f t h e l y s i n e r e s i d u e s were b e i n g b l o c k e d by p h y s i c a l i n h i b i t i o n o r chem­ i c a l c o m b i n a t i o n (40, 50) t o f o r m m o i e t i e s w h i c h were u n a t t a c k e d by enzymes i n t h e g u t , t h u s e f f e c t i v e l y r e d u c i n g t h e i n t a k e o f l y s i n e by t h e a n i m a l . B j a r n a s o n and C a r p e n t e r (51^, 52) showed t h a t s i m p l e a c y l a t i o n o f t h e ε-amino g r o u p was i n i t s e l f i n s u f f i ­ c i e n t t o c a u s e t h e e f f e c t . I n d e e d , a c y l a t e d l y s i n e was shown t o have g r o w t h p r o m o t i n g p r o p e r t i e s when f e d t o y o u n g r a t s on a l y ­ sine d e f i c i e n t d i e t . T h e s e o b s e r v a t i o n s l e d t o t h e s p e c u l a t i o n t h a t t h e ε-amino g r o u p o f l y s i n e was i n v o l v e d i n c r o s s l i n k f o r m a t i o n w h i c h e i t h e r prevented o r i n h i b i t e d the d i g e s t i v e p r o c e s s e s . The p o s s i b l e c r o s s l i n k s were t h e i s o p e p t i d e s and l y s i n o a l a n i n e . T h i s l a t t e r c r o s s l i n k can be f o r m e d v i a a M i c h a e l a d d i t i o n r e a c t i o n o f t h e ε-amino g r o u p o f l y s i n e a c r o s s t h e d o u b l e bonds o f d e h y d r o a l a n i n e , t h e l a t t e r b e i n g t h e d e g r a d a t i o n p r o d u c t o f a l k a l i o r heat d e ­ g r a d e d c y s t i n e ( 5 3 - 5 5 ) . L y s i n o a l a n i n e i s known t o be s t a b l e t o a c i d and e n z y m i c h y d r o l y s i s and was s u b s e q u e n t l y i d e n t i f i e d i n heated p r o t e i n s (40). The c o n c e n t r a t i o n o f l y s i n o a l a n i n e i n ­ c r e a s e s c o n s i d e r a b l y i n a l k a l i / h e a t c o m b i n a t i o n s (55). The b i o ­ l o g i c a l s t a b i l i t y o f t h e i s o p e p t i d e s has a l s o been i n v e s t i g a t e d . Thus O t t e r b u r n e t a l . (44) showed t h a t G-L was s t a b l e t o t h e r e d u c t i o n / b l o c k i n g and enzyme s y s t e m s u s e d t o i s o l a t e t h e com­ pound i n p r o t e i n s . C a r p e n t e r and W a i b l e (56) i n v e s t i g a t e d t h e s t a b i l i t y and u t i l i z a t i o n o f f r e e i s o p e p t i d e s a s a s o u r c e o f

Finley and Schwass; Xenobiotics in Foods and Feeds ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

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13.

OTTERBURN

Natural

and Xenobiotic

Crosslinks

in

Proteins

229

l y s i n e . They were a b l e t o show w i t h c h i c k s t h a t t h e f r e e i s o p e p t i d e G-L was m e t a b o l i z e d and had some growth p r o m o t i n g e f f e c t . T h e s e w o r k e r s a l s o showed t h a t none o f t h e f r e e i s o p e p t i d e c o u l d be d e t e c t e d i n t h e u r i n e , a l t h o u g h t r a c e s were f o u n d i n t h e p l a s ma. T h i s o b s e r v a t i o n i n d i c a t e d t h a t some i s o p e p t i d e had been a b s o r b e d t h r o u g h t h e i n t e s t i n a l w a l l a l t h o u g h n o t a l l t h e G-L was a c c o u n t e d f o r . R a c z y n s k i e t a l . (57) o b s e r v e d t h a t t h e s u p e r n a t a n t f r a c t i o n o f k i d n e y homogenates c a u s e d g r e a t e s t e n z y m i c h y d r o l y s i s o f G-L. However, O t t e r b u r n and H e a l y (58, 59) f o u n d t h a t s u p e r n a t a n t f r a c t i o n s o f s m a l l i n t e s t i n a l homogenates e x h i b i t e d maximum e n z y m i c a c t i v i t y t o w a r d s t h e i s o p e p t i d e s . T h e s e r e s u l t s were s u b s t a n t i a t e d by r e s u l t s o b t a i n e d i n s t u d y i n g t h e t r a n s f e r e n c e o f G-L a c r o s s the wall o f the small i n t e s t i n e s o f r a t s using the e v e r t e d s a c - t e c h n i q u e ( 5 9 ) . T h e s e l a t t e r r e s u l t s showed t h a t G-L was h y d r o l y z e d t o a c o n s i d e r a b l e d e g r e e (71.03%) i n t h e j e j u n a l r e g i o n o f t h e gut and t o a l e s s e r e x t e n t (24.4%) i n t h e d u o d e n a l a n d i l e a l (8.72%) r e g i o n s . A g a i n , t h e s e r e s u l t s a r e a t v a r i a n c e w i t h t h o s e o f R a c z y n s k i e t a l . (57) whose i n v i t r o s t u d i e s w i t h e v e r t e d i n t e s t i n a l s a c s showed t h a t r a d i o l a b e l l e d G-L p a s s e d a c r o s s t h e gut w a l l u n c h a n g e d . The r e s u l t s o f O t t e r b u r n and H e a l y , however, a g r e e w i t h t h e s u g g e s t i o n s o f W a i b l e and C a r p e n t e r (56) t h a t h y d r o l y s e s o f i n g e s t e d G-L can o c c u r w i t h i n t h e i n t e s t i n a l w a l l a s a c o n s e q u e n c e o f which o n l y t r a c e s o f G-L a r e f o u n d i n t h e b l o o d p l a s m a . V e r y l i t t l e work has been c a r r i e d o u t on t h e n u t r i t i o n a l s i g n i f i c a n c e o f s e v e r e l y h e a t e d p r o t e i n s w h i c h a r e known t o c o n t a i n i s o p e p t i d e c r o s s l i n k s . One such s t u d y was c a r r i e d o u t b y H u r r e l l e t a l . (42) u s i n g s e v e r e l y h e a t e d c h i c k e n m u s c l e p r o t e i n fed a s p a r t o f the d i e t t o r a t s . These workers a n t i c i p a t e d t h a t t h e i n a c c e s s i b l e l y s i n e and l y s i n e r e s i d u e s i n v o l v e d w i t h i s o p e p t i d e s w o u l d have s i g n i f i c a n t l y r e d u c e d d i g e s t i b i l i t y v a l u e s comp a r e d w i t h the o v e r a l l p r o t e i n . Thus i t was e x p e c t e d t h a t such h i g h l y s i n e f r a c t i o n s w o u l d a c c u m u l a t e i n t h e i l e a l and f a e c a l c o n t e n t s . However, i t was f o u n d t h a t a l t h o u g h p r o t e i n d i g e s t i b i l i t y was g r e a t l y r e d u c e d a f t e r h e a t t r e a t m e n t , t h e i s o p e p t i d e s t h e m s e l v e s were a t l e a s t a s d i g e s t i b l e a s t h e t o t a l p r o t e i n comp o n e n t , t o t a l l y s i n e and FDNB r e a c t i v e l y s i n e . T h e r e d u c t i o n i n i l e a l p r o t e i n d i g e s t i b i l i t y o n l y p a r t l y a c c o u n t e d f o r t h e much l a r g e r r e d u c t i o n i n n u t r i t i v e v a l u e a s m e a s u r e d by t h e Net P r o t e i n R a t i o (NPR). One e x p l a n a t i o n f o r t h i s r e s u l t may be f o u n d i n t h e f a c t t h a t i f i s o p e p t i d e s are not absorbed through the ileum they w i l l p a s s t o t h e c o l o n where i n t e s t i n a l b a c t e r i a and m i c r o f l o r a a r e a b l e t o h y d r o l y z e them ( 4 4 ) . T h u s t h e r e i s e v i d e n c e t o show t h a t b o t h G-L and A-L a r e f o u n d i n p r o t e i n s w h i c h have been s u b j e c t e d t o h e a t t r e a t m e n t s the q u a n t i t y o f i s o p e p t i d e s formed being r e l a t e d t o the s e v e r i t y of treatment. F u r t h e r , i t seems t h a t t h e i s o p e p t i d e s a r e i m p l i c a t e d t o some e x t e n t i n t h e r e s u l t i n g f a l l i n n u t r i t i o n a l v a l u e a s m o n i t o r e d by NPR.

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Conclusion The p r e s e n c e o f i s o p e p t i d e c r o s s l i n k s i n h e a t e d p r o t e i n s i s i n t e r e s t i n g f r o m a s c i e n t i f i c v i e w p o i n t a s i t i s a r a r e example o f t h e formation o f a x e n o b i o t i c substance which i s a l s o found i n n a t i v e p r o t e i n s . Other examples a r e l a n t h i o n i n e and l y s i n o a l a ­ n i n e w h i c h c a n be f o r m e d x e n o b i o t i c a l l y a n d have been shown by Gross t o occur n a t u r a l l y i n cinnamycin ( 6 0 ) . To date t h e r e i s no e v i d e n c e t h a t t h e i s o p e p t i d e s have any t o x i c o l o g i c a l manifestations i n heated foods, although l i t t l e o r no work h a s been c a r r i e d o u t i n t h i s a r e a . The o n l y e v i d e n c e o f any d e l e t e r i o u s e f f e c t s o f t h e x e n o b i o t i c s i s t h e r e d u c t i o n t h e y c a u s e i n n u t r i t i o n a l v a l u e a s measured by NPR. In o r d e r t o f u l l y understand the r o l e and importance o f t h e i s o p e p t i d e s i n p r o t e i n f o o d s t u f f s a n d n u t r i t i o n , more work s h o u l d be u n d e r t a k e n i n t o t h e i r n u t r i t i o n a l and t o x i c o l o g i c a l p r o p e r t i e s .

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RECEIVED

June 21, 1983

Finley and Schwass; Xenobiotics in Foods and Feeds ACS Symposium Series; American Chemical Society: Washington, DC, 1983.