Isopeptides: The Occurrence and Significance of Natural and

13 Isopeptides: The Occurrence and Significance of Natural and Xenobiotic Crosslinks in Proteins

Downloaded by UNIV OF ARIZONA on April 25, 2017 | http://pubs.acs.org Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch013

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.


222

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

FEEDS

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 -



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


— C H — I

ω-ε ISOPEPTIDE

I

224


FEEDS

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 .


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



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


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

226

COOH HC-(CH )2-COOH or


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


in


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




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



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.


230


FEEDS


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 .

Literature Cited 1. Speakman, J . B.; Hirst, M. C. Nature 1931, 128, 1073. 2. Mirsky, A. E.; Pauling, L. Proc. Nat. Acad. Sci. (U.S.A.) 1936, 22, 439. 3. Pimentel, G. C . ; McClellan, A. L. "The Hydrogen Bond"; W. H. Freeman: San Francisco, 1960. 4. Klotz, I. M. Brookhaven Symp. Biol. 1960, 13, 25. 5. Zahn, H. Palette 1965, 21, 19. 6. Crewther, W. G.; Fraser, R. D. B.; Lennox, F. G.; Lindley, H. Adv. Protein Chem. 1965, 20, 191. 7. Friedmann, M. "The Chemistry and Biochemistry of the Sulfhydral Group in Amino Acids, Peptides and Proteins"; Pergamon Press, 1973. 8. Asquith, R. S.; Leon, Ν. H. "Chemistry of Natural Protein Fibres"; Asquith, R.S., Ed.; Plenum Press, New York, 1977, Chap. 5. 9. Piez, K. A. Annu. Rev. Biochem. 1968, 37, 547. 10. Traub, W.; Piez, K. A. Adv. Protein Chem. 1971, 25, 243. 11. Feeney, R. E.; Blankenhorn, G.; Dixon, H. B. F. Adv. Protein Chem. 1975, 29, 135. 12. Weis-Fogh, T. J. Exp. Biol. 1960, 37, 889. 13. Andersen, S. O. Biochim. Biophys. Acta 1964, 93, 213. 14. Fischer, E. Ber. Desch. Chem. Ges. 1906, 39, 510. 15. Astbury, W. T.; Woods, H. J. Phil. Trans. Roy. Soc. (London) Series A 1934, 232, 333. 16. Pauling, L.; Niemann, C. J . Am. Chem. Soc. 1939, 61, 1860. 17. Loewy, A. G. "Fibrinogen"; Laki, E., Ed.; Dekker, New York, 1968, p. 185. 18. Pisano, J . J.; Finlayson, J . S.; Peyton, M. P. Thromb. Diath. Haemorrh. Suppl. 1970, 39, 113. 19. Finlayson, J . S. Sem. Thromb. Haemostasis 1974, 1, 33.


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OTTERBURN

Natural

and Xenobiotic

Crosslinks

in Proteins

231

Asquith, R. S.; Otterburn, M. S.; Sinclair, W. J. Angew. Chem. Internat. Edit. 1974, 13, 514. Matacic, S. S.; Loewy, A. G. Biochem. Biophys. Res. Commun. 1968, 30, 356. Pisano, J . J.; Finlayson, J . S.; Peyton, M. P. Science 1968, 160, 892. Loewy, A. G.; Matacic, S. S.; Showe, M. Fed. Proc., Fed. Am. Soc. Exp. Biol. 1971, 30, 1275. Folk, J. E.; Finlayson, J . S. Adv. Protein Chem. 1977, 31, 1. Asquith, R. S.; Otterburn, M. S.; Buchanan, J . H.: Cole, M.; Fletcher, J . C.; Gardner, K. L. Biochim. Biophys. Acta 1970, 221, 342. Asquith, R. S.; Otterburn, M. S.; Gardner, K. L. Experientia 1971, 27, 1388. Harding, H. W. J.; Rogers, G. E. Biochim. Biophys. Acta 1972, 257, 37. Harding, H. W. J.; Rogers, G. E. Biochemistry 1972, 11, 2858. Loewy, A. G.; Matacic, S. S. Biochim. Biophys. Acta 1981, 668, 167. Abernethy, J . L.; H i l l , R. L.; Goldsmith, L. A. J. Biol. Chem. 1977, 252, 1837. Lorand, L.; Siefring, G. E.; Lowe-Kilentz, L. J . Supramol. Struct. 1978, 9, 279. Haugland, R. B.; Lin, T. I.; Dowben, R. M.; Birckbichler, P. J . Biophys. J. 1982, 37, 191. Klostermeyer, H.; Rabbel, K.; Reimerdes, E.-H. HoppeSeyler's Z. Physiol. Chem. 1976, 357, 1197. Cole, M.; Fletcher, J . C.; Gardner, K. L.; Corfield, M. C. Appl. Polym. Symp. 1971, 18, 147. Schmitz, I.; Baumann, H.; Zahn, H. Proc. 5th Inter. Wolltextil-Forschungskonf., Aachen 1975, 2, 313. Otterburn, M. S.; Sinclair, W. J. J. Sci. Food Agric. 1976, 27, 1071. Griffin, M.; Wilson, J.; Lorand, L. Anal. Biochem. 1982, 124, 406. Weder, J . K. P.; Scharf, U. Z. Lebensm. Unters-Forsch. 1981, 172, 9. Sugawara, K.; Ouchi, T. Agric. Biol. Chem. 1982, 46, 1085. Asquith, R. S.; Otterburn, M. S. Appl. Polym. Symp. 1971, 18, 277. Schmitz, I.; Zahn, H.; Klostermeyer, H.; Rabbel, K.; Watanabe, Κ. Z. Lebensm. Unters-Forsch. 1976, 160, 377. Hurrell, R. F . ; Carpenter, K. J.; Sinclair, W. J.; Otterburn, M. S.; Asquith, R. S. Br. J. Nutr. 1976, 35, 383. Weder, J. K. P.; Scharf, U. Z. Lebensm. Unters-Forsch. 1981, 172, 104. Otterburn, M. S.; Healy, M. G.; Sinclair, W. J. Adv. Exp. Med. Biol. 1977, 86B, 239.



232

45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60.


FEEDS

Mecham, D. K.; Olcott, H. S. Ind. Eng. Chem. 1947, 39, 1023. Beuk, J . F.; Chornock, F. W.; Rice, E. E. J. Biol. Chem. 1948, 60, 291. Carpenter, K. J . Biochem. J. 1960, 77, 604. Lea, C. H.; Hannah, R. S. Biochim. Biophys. Acta 1950, 4, 1950. Asquith, R. S.; Chan, D. K.; Otterburn, M. S. J . Chromatogr. 1969, 43, 312. Asquith, R. S.; Speakman, J. B.; Tolgyesi, A. Nature 1957, 180, 502. Bjarnason, J.; Carpenter, K. J. Br. J. Nutr. 1969, 23, 859. Bjarnason, J.; Carpenter, K. J. Br. J. Nutr. 1970, 24, 313. Bohak, Z. J. Biol. Chem. 1964, 239, 2878. Ziegler, K. J . Biol. Chem. 1964, 239, P.C. 2713. Asquith, R. S. "Fibrous Proteins: Scientific, Industrial and Medical Aspects"; Parry, D. A. D.; Creamer, L. K., Eds.; Academic Press, 1979, I, p. 371. Waible, P. E.; Carpenter, K. J. Br. J. Nutr. 1972, 27, 509. Raczynski, G.; Snochowski, K.; Buraczewski, S. Br. J. Nutr. 1975, 34, 291. Otterburn, M. S.; Healy, M. G. Unpublished Results. Healy, M. G. M.Sc. Thesis, The Queen's University of Belfast, 1980. Gross, E. Adv. Exp. Med. Biol. 1977, 86B, 131.

RECEIVED

June 21, 1983


Isopeptides: The Occurrence and Significance of Natural and

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