Chemical Reactions in Proteins Irradiated at Subfreezing

6 Chemical Reactions in Proteins Irradiated at

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Subfreezing Temperatures IRWIN A. TAUB and JOHN W. HALLIDAY Food Engineering Laboratory, U. S. Army Natick Research and Development Command, Natick, MA 01760 MICHAEL D. SEVILLA Department of Chemistry, Oakland University, Rochester, MI 48063

The course of chemical reactions in irradiated proteins is determined by factors that influence the reactivity of the primary free radicals, the kind of protein radicals formed, and the decay of these protein radicals to stable products. To understand these reactions, basic radiation chemical concepts are considered, chemical changes in several repre sentative proteins irradiated under different conditions are compared, and results from optical and electron spin reso nance studies on model systems are presented. Among the reactions described are those involving cation, anion, and α-carbon radicals of amino acids and peptides. Analogous reactions common to proteins are then summarized. These mechanistic considerations have important implications for the irradiation of hydrated muscle proteins at —40°C and for radiation sterilization of foods.

r

T he n

c h e m i c a l reactions

occurring i n a protein system exposed

i o n z i n g r a d i a t i o n a r e affected b y s e v e r a l factors.

to

T h e nature of the

p r o t e i n , i t s state o f h y d r a t i o n , t h e p h a s e a n d t e m p e r a t u r e o f t h e s y s t e m , a n d t h e presence

of r e a c t i v e c o m p o u n d s

are particularly important

factors. P r o t e i n s c o n t a i n i n g d i s u l f i d e g r o u p s , m e t a l ions, o r l a r g e p r o p o r tions of aromatic or heterocyclic amino acids w i l l undergo

reactions

d i f f e r i n g f r o m those w i t h o u t these constituents. P r o t e i n s t h a t a r e h y d r a t e d 0-8412-0484-5J79J33-180-109$8.00J0 © 1979 American Chemical Society Fennema; Proteins at Low Temperatures Advances in Chemistry; American Chemical Society: Washington, DC, 1979.

110

PROTEINS A T L O W T E M P E R A T U R E S

u n d e r g o different c h e m i c a l a n d p h y s i c a l processes t h a n p r o t e i n s t h a t are d e s i c c a t e d . M o s t i m p o r t a n t , h o w e v e r , is t h e p h y s i c a l state: fluid solutions a r e m u c h m o r e s u s c e p t i b l e to r a d i a t i o n i n d u c e d changes

than frozen

aqueous systems. T e m p e r a t u r e , p a r t i c u l a r l y as i t affects t h e v i s c o s i t y of t h e m e d i u m , c a n c h a n g e the c h e m i c a l consequences m a r k e d l y . Solutes i n these systems, s u c h as o x y g e n o r m e t a l ions, c a n alter t h e course of t h e reactions as w e l l .


I r r a d i a t i o n i n i t i a t e s a series of reactions as a c o n s e q u e n c e of electrons b e i n g ejected a n d b o n d s i n t h e p r o t e i n b e i n g r u p t u r e d . V a r i o u s i o n i c and

free r a d i c a l i n t e r m e d i a t e s are g e n e r a t e d

that ultimately become

s t a b i l i z e d b y the f o r m a t i o n of c o v a l e n t l y b o n d e d c o m p o u n d s .

Reactions

of t h e p r i m a r y r a d i c a l s c a n generate other r a d i c a l s a n d these i n t u r n c o m b i n e to f o r m the final p r o d u c t s . A n y factor t h a t affects t h e rates a n d routes of these i n t e r m e d i a t e s w i l l i n f l u e n c e w h i c h p r o d u c t s a r e f o r m e d . F o r t h e p r o t e i n s of interest h e r e , t h e final effects m i g h t b e o b s e r v a b l e as v a l e n c e c h a n g e , d e a m i n a t i o n , d e c a r b o x y l a t i o n , d i s u l f i d e loss or f o r m a t i o n , c h a i n d e g r a d a t i o n or a g g r e g a t i o n , or m o d i f i c a t i o n of i n d i v i d u a l a m i n o a c i d moieties. An

u n d e r s t a n d i n g of these reactions a n d h o w

t h e y are

affected,

p a r t i c u l a r l y b y i r r a d i a t i o n at s u b f r e e z i n g t e m p e r a t u r e s , c a n b e

achieved

b y considering certain basic concepts a n d major experimental observations. C o n s e q u e n t l y , t h e basic r a d i a t i o n c h e m i c a l concepts, t h e t e c h n i q u e s u s e d to d i s c e r n i n t e r m e d i a t e species a n d t h e i r e v e n t u a l p r o d u c t s ,

the

major

the

findings

o n s u c h p r o t e i n s as m y o g l o b i n

and myosin, and

g e n e r a l i z e d reactions of p r i m a r y a n d s e c o n d a r y r a d i c a l s as g l e a n e d f r o m studies o n a m i n o a c i d a n d p e p t i d e s i m p l i c a t i o n s of these f i n d i n g s , t h o u g h

w i l l be

considered

herein.

The

g e n e r a l l y r e l e v a n t to r a d i a t i o n

b i o l o g y , r e d o x processes i n b i o c h e m i s t r y , a n d p r o t e i n d y n a m i c s , w i l l b e d i s c u s s e d i n r e l a t i o n to l o w t e m p e r a t u r e r a d i a t i o n s t e r i l i z a t i o n of h i g h p r o t e i n foods. Basic

Radiation

Chemical

Concepts

Energy Deposition and Free Radical Distribution.

T h e interaction

of p e n e t r a t i n g g a m m a r a y s o r h i g h e n e r g y electrons w i t h the

valence

shells of the atoms c o m p r i s i n g t h e m o l e c u l e s of t h e c o n d e n s e d

medium

results i n e n e r g y b e i n g d e p o s i t e d i n the m e d i u m .

T h e s e electrons

can

b e p r o d u c e d d i r e c t l y i n m a c h i n e sources s u c h as a l i n e a r accelerator or a V a n d e G r a a f f accelerator, a n d w o u l d h a v e energies t y p i c a l l y i n t h e r a n g e of 2 - 1 0 M e V . G a m m a r a y s , s u c h as those f r o m c o b a l t - 6 0 o r c e s i u m 137 sources, as a c o n s e q u e n c e of i n t e r a c t i n g v i a the C o m p t o n process produce

energetic

electrons

as w e l l .

Because

of its c h a r g e , i t is t h e

e l e c t r o n t h a t is p a r t i c u l a r l y e xective i n e x c i t a t i o n a n d i o n i z a t i o n processes.

Fennema; Proteins at Low Temperatures Advances in Chemistry; American Chemical Society: Washington, DC, 1979.

Irradiated

111

Proteins

6.

TAUB E T A L .

By

successive i o n i z a t i o n acts, the p r i m a r y e l e c t r o n g r a d u a l l y b e c o m e s

degraded

i n energy a n d c o n c o m i t t a n t l y p r o d u c e s

also c a p a b l e of i o n i z i n g the m o l e c u l e s .

secondary

electrons,

U l t i m a t e l y , these p r i m a r y , sec-

o n d a r y , a n d t e r t i a r y , etc. electrons c a n n o l o n g e r cause i o n i z a t i o n s , a n d lose t h e i r r e m a i n i n g e n e r g y

v i a electronic, vibrational, a n d rotational

e x c i t a t i o n . A n e n e r g e t i c a l l y d e g r a d e d e l e c t r o n m i g h t b e d r a w n b a c k to the positive i o n formed u p o n ionization, m i g h t be t r a p p e d if the meDownloaded by UNIV OF MASSACHUSETTS AMHERST on January 18, 2018 | http://pubs.acs.org Publication Date: September 1, 1979 | doi: 10.1021/ba-1979-0180.ch006

d i u m is p o l a r a n d b e c o m e s o l v a t e d , o r m i g h t react w i t h a n i m p u r i t y of h i g h e l e c t r o n affinity. S o m e of the e x c i t e d m o l e c u l e s f o r m e d d i r e c t l y o r produced

u p o n e l e c t r o n - p o s i t i v e i o n r e a c t i o n w i l l dissociate

into

free

radicals.

T h e o v e r a l l effect is the i n i t i a l f o r m a t i o n of ions a n d

free

r a d i c a l s n o n u n i f o r m l y d i s t r i b u t e d i n regions c a l l e d spurs a l o n g the t r a c k of the i o n i z i n g p a r t i c l e . T h e d i s t r i b u t i o n of these p r i m a r y species a n d t h e i r e v e n t u a l fate is d e t e r m i n e d b y the n a t u r e a n d state of the m e d i u m .

I f t h e v i s c o s i t y is

e x t r e m e l y h i g h as i n solids or glasses, t h e d i s t r i b u t i o n r e m a i n s n o n u n i f o r m a n d the reactions that o c c u r i n v o l v e species w i t h i n the same, or closely r e l a t e d , spur.

I f the v i s c o s i t y is l o w , s u c h as i n a

fluid

system, these

species t e n d to diffuse a p a r t a n d l e a d to a u n i f o r m d i s t r i b u t i o n t h r o u g h out t h e m e d i u m . I n this case, the reactions c o n f o r m to k i n e t i c l a w s f o r a h o m o g e n e o u s system. T h e y i e l d of a n y species f o r m e d as a d i r e c t c o n s e q u e n c e of

the

e n e r g y b e i n g a b s o r b e d b y t h e c o m p o n e n t m o l e c u l e s is g i v e n i n t e r m s of a G - v a l u e , w h i c h is defined as t h e n u m b e r of ions, free r a d i c a l s , or e x c i t e d m o l e c u l e s f o r m e d for every 100 e V of energy a b s o r b e d .

G-values

m a y also b e g i v e n for the stable p r o d u c t s t h a t are e v e n t u a l l y f o r m e d . F o r w a t e r , r a d i o l y s i s leads to t h e f o r m a -

Direct Effect on Water.

t i o n of species w i t h r a t h e r s p e c i a l features t h a t h a v e b e e n the subject of considerable

i n v e s t i g a t i o n (1,2,3).

E q u a t i o n 1 describes

the

overall

effect:

H

2

0 — ~ — >

(H 0 ) e 2

+

;

a q

-,OH-,H 0 ,H-,H ,H 0 +

3

2

2

2

(1)

T h e m o l e c u l a r i o n of w a t e r is s h o w n i n parenthesis b e c a u s e it r a p i d l y converts to O H - a n d H 0 . T h e s o l v a t e d e l e c t r o n (2), 3

+

c o r r e s p o n d i n g to

a n e l e c t r o n b o u n d to several w a t e r m o l e c u l e s i n a fluid system, is d e s i g n a t e d here as e ", b u t w i l l also be d e n o t e d as e ~ f o r a n e l e c t r o n b o u n d i n aq

other p o l a r m e d i a .

s

I t is h i g h l y m o b i l e , has a b r o a d , intense a b s o r p t i o n

s p e c t r u m w i t h a m a x i m u m at 720 n m , a n d is a p o w e r f u l r e d u c t a n t .

The

h y d r o x y l r a d i c a l , O H - , is also v e r y m o b i l e a n d is a s t r o n g o x i d a n t ; i t exhibits a w e a k a b s o r p t i o n i n the 240 n m r e g i o n .

T h e hydrogen atom,

H ·, is a r e d u c t a n t a n d exhibits o n l y a w e a k a b s o r p t i o n i n t h e u l t r a v i o l e t


112

PROTEINS A T L O W

TEMPERATURES

r e g i o n . A l l three r a d i c a l s h a v e b e e n d e t e c t e d a n d c h a r a c t e r i z e d b y v i r t u e of t h e i r e l e c t r o n s p i n resonance

( E S R ) spectra. E v i d e n c e f o r a n u n s o l -

v a t e d e l e c t r o n is extensive a n d is b a s e d o n c h e m i c a l a n d c o n d u c t o m e t r i c measurements, w h i c h w i l l be mentioned below.

G-values for the

e ", aQ

O H - , a n d H - u n i f o r m l y d i s t r i b u t e d i n w a t e r at r o o m t e m p e r a t u r e are 2.8, 2.7, a n d 0.55, r e s p e c t i v e l y ; G - v a l u e s f o r these same species i n i c e at — 5 ° C are a p p r o x i m a t e l y 0.3, 1.0, a n d 0.7, r e s p e c t i v e l y . Downloaded by UNIV OF MASSACHUSETTS AMHERST on January 18, 2018 | http://pubs.acs.org Publication Date: September 1, 1979 | doi: 10.1021/ba-1979-0180.ch006

For

p r o t e i n s o r o t h e r o r g a n i c constituents, r a d i o l y s i s p r e s u m a b l y

leads to analogous species, a l t h o u g h these h a v e not b e e n

unequivocably

e s t a b l i s h e d . E q u a t i o n 2 describes the o v e r a l l effect:

PH

( P H ) , e", Ρ ·, P H +

2

+

, Η •, H , P 2

(2)

2

T h e g e n e r a l i z e d p r o t e i n m o l e c u l e is s h o w n as P H . T h e e" is left u n s p e c i fied; its fate w o u l d d e p e n d o n the m e d i u m a n d other c o n d i t i o n s . A s w i l l b e d e s c r i b e d l a t e r , there are s e v e r a l free r a d i c a l s t h a t h a v e b e e n d e t e c t e d , b u t t h e g e n e r a l d e s i g n a t i o n P - is a l l t h a t is n e e d e d here. EfFect o f P h a s e , T e m p e r a t u r e , S o l u t e s , a n d D o s e o n t h e R a d i o l y s i s . S i n c e t h e p r i m a r y r a d i c a l species m u s t diffuse to other r a d i c a l species or m o l e c u l e s i n the system to transfer o r share electrons a n d b e c o m e stable, t h e phase a n d t e m p e r a t u r e , because t h e y affect v i s c o s i t y , d e t e r m i n e w h i c h reactions w i l l o c c u r .

T h o s e reactions i n v o l v i n g the p r i m a r y species

the m o l e c u l e s not d i r e c t l y affected species—secondary

and

b y t h e r a d i a t i o n g i v e rise to

new

r a d i c a l s — w h i c h are c o n s i d e r e d as b e i n g a n i n d i r e c t

c o n s e q u e n c e of t h e r a d i o l y s i s . I n fluid aqueous systems, e ", O H - , a n d aq

Η · r e a d i l y react w i t h e a c h o t h e r a n d w i t h solutes, e v e n those present at l o w concentrations impeded

by

(~

10"

4

the r i g i d i t y of

M).

I n f r o z e n systems, s u c h reactions are

the m e d i u m .

I n d i r e c t consequences

l i m i t e d to systems w i t h v e r y h i g h solute concentrations

1M)

are

a n d at

t e m p e r a t u r e s n e a r 0 ° C , b o t h factors c o n t r i b u t i n g to f o r m a t i o n of a m o r p h o u s or

fluid-like

regions.

Studies of the r e a c t i o n of e " w i t h v a r i o u s s

solutes i n p o l y c r y stalline systems at — 40 ° C h a v e s h o w n t h a t G - v a l u e s f o r p r o d u c t s are o n l y a b o u t 1 %

to 1 0 %

of t h e c o r r e s p o n d i n g

values

f o u n d f o r fluid systems ( 4 , 5 , 6 ) . W h e n c h l o r o a c e t i c a c i d w a s u s e d as a p r o b e f o r the electrons, the G - v a l u e f o r C I " f o r m a t i o n i n c r e a s e d a p p r o x i m a t e l y 0.03 to 0.8 as t h e c h l o r o a c e t i c

acid concentration

from was

i n c r e a s e d f r o m 10" M to 1 M . S i m i l a r l y , studies of reactions of O H · i n 2

p o l y c r y s t a l l i n e ice at — 4 0 ° C h a v e s h o w n t h a t t h i s species is e v e n m o r e r e s t r i c t e d t h a n e f i n its a b i l i t y to m i g r a t e a n d r e a c t ( 4 ) . m e n t s i n v o l v e d u s i n g f e r r o c y a n i d e as a p r o b e f o r Ο Η · . e s t i m a t e d that o n l y 4 %

of the a v a i l a b l e Ο Η ·

These experi I t has

can be scavenged


been using

6.

Irradiated

TAUB E T A L .

113

Proteins

0.5 M f e r r o c y a n i d e , t h e h i g h e s t p r a c t i c a l c o n c e n t r a t i o n o b t a i n a b l e . s e q u e n t l y , the o v e r a l l c h e m i c a l c h a n g e

Con

i n a f r o z e n or s o l i d system is

s i g n i f i c a n t l y r e d u c e d b y l i m i t i n g t h e i n d i r e c t f o r m a t i o n of o t h e r species. T h e course of p r i m a r y species reactions i n a fluid s y s t e m , w h i c h is d e p e n d e n t u p o n t h e rate constants f o r r e a c t i o n (see T a b l e I )

a n d the

c o n c e n t r a t i o n s of r e a c t i v e solutes, d e t e r m i n e s t h e n a t u r e of t h e i n d i r e c t effects.

T h e solutes c o u l d b e i n t r o d u c e d d i r e c t l y or c o u l d b e f o r m e d as a


c o n s e q u e n c e of t h e r a d i o l y s i s . F o r systems i n w h i c h h o m o g e n e o u s k i n e t i c laws apply, the predominant

r e a c t i o n is d e t e r m i n e d

by

competition

p r i n c i p l e s : t h e r e a c t i o n i n v o l v i n g t h e h i g h e s t p r o d u c t of r a t e constant k times concentration predominates.

I f a r e a c t i v e p r o d u c t is f o r m e d i n t h e

r a d i o l y s i s w i t h a r e a s o n a b l y h i g h G - v a l u e , i t w i l l t e n d to c o m p e t e as t h e dose is i n c r e a s e d . Table I. Rate Constants for Reaction of Primary Water Radicals w i t h Some Amino Acids and Peptides in F l u i d Aqueous Solutions' k , M'

1

Amino

AddJPeptide

Glycine Glycylglycine Alanine Lysine Arginine Aspartic acid Histidine Phenylalanine Tryptophan Methionine Cysteine Cystine e

e' χ Χ Χ χ Χ Χ Χ Χ Χ χ Χ Χ

(pH)

1

OH-

aq

8.2 3.7 5.9 2 1.8 1 6 1.5 4.0 3.5 8.7 1.3

s

10 10 10 10 10 10 10 10 10* 10 10 10 6

8

6

7

8

7

7

8

7

9

1 0

(6.4) (6.4) (6.4) (7) (6) (7.3) (7) (6.8) (6.8) (6.0) (6.3) (6.1)

1.6 4.4 4.7 6.0 3.5 2.1 5.0 6.6 1.4 — — —

Χ χ Χ Χ Χ χ Χ Χ Χ

10 10 10 10 10 10 10 10 10

H(5.2) (5.2) (6) (2) (6.5-7.5) (6.8) (6-7)

7 8 7 8 9 7 9 9 1 0

(6.1)

9 2.6 2.9 1.6 4.9 2.9 2.5 8.0 2.3 — 4 8

Χ Χ Χ Χ Χ Χ Χ Χ Χ

10 10 10 10 10 10 10 10 10

(7)

4 6 5 6 6

(7) (7)

6

8

Χ 10 Χ 10

8 9

9 9

FromRefs.2,S,£2,50.

Major Pbysicochemical

and Analytical

Techniques

Techniques for Examining and Characterizing Irradiated Proteins. S e v e r a l t e c h n i q u e s h a v e b e e n e m p l o y e d to detect t h e presence of i n t e r m e d i a t e species i n t h e i r r a d i a t e d p r o t e i n o r t o c h a r a c t e r i z e t h e b r o u g h t a b o u t i n its s t r u c t u r e o r c o m p o s i t i o n .

changes

O p t i c a l techniques

have

b e e n u s e d b o t h f o r d e t e c t i n g i n t e r m e d i a t e s as w e l l as f o r d e t e r m i n i n g final

changes.

Because

o n l y species

w i t h u n p a i r e d electrons

can

be

d e t e c t e d b y E S R , this t e c h n i q u e has b e e n a p p l i e d f o r d e t e c t i n g i n t e r mediates

d i r e c t l y or, most r e c e n t l y , f o r d i s c e r n i n g t h e i r n a t u r e after

t r a p p i n g w i t h stable free r a d i c a l s ( 7 ) .

A m o n g the more standard bio-


114

PROTEINS A T L O W

TEMPERATURES

c h e m i c a l approaches for c h a r a c t e r i z i n g the p e r m a n e n t m o d i f i c a t i o n s i n t h e p r o t e i n are t h e e l e c t r o p h o r e t i c m e t h o d s , c h e m i c a l analyses of p r o d ucts or constituent moieties, a n d several s t r u c t u r e - r e l a t e d m e t h o d s . trophoresis, isoelectric focusing,

and chromatographic

Elec

separations

are

u s e f u l f o r d i s c e r n i n g changes d u e to loss or m o d i f i c a t i o n of moieties t h a t i n f l u e n c e size, shape, a n d c h a r g e o n t h e p r o t e i n . A m i n o a c i d analyses, S H g r o u p analysis, a n d d e t e r m i n a t i o n of a m i d e a n d f a t t y a c i d p r o d u c t s Downloaded by UNIV OF MASSACHUSETTS AMHERST on January 18, 2018 | http://pubs.acs.org Publication Date: September 1, 1979 | doi: 10.1021/ba-1979-0180.ch006

p e r t a i n to s i m i l a r m o d i f i c a t i o n s .

Experiments involving enzymatic activ

i t y , s e d i m e n t a t i o n rates, a n d b i n d i n g of r a d i o a c t i v e labels also p e r t a i n to s t r u c t u r a l alterations or specific m o i e t y m o d i f i c a t i o n s . analysis of p e r m a n e n t changes

I n g e n e r a l , the

is r e l a t i v e l y i n s e n s i t i v e , a n d v e r y h i g h

doses, o f t e n i n t h e r a n g e of 3000 k G y ( 1 M r a d =

10 k G y ) , m u s t b e u s e d .

C o n s i d e r a b l y greater s e n s i t i v i t y is a v a i l a b l e a n d a m o r e d i r e c t u n d e r s t a n d i n g of the c h e m i s t r y is p o s s i b l e i f one uses the t e c h n i q u e s r e c e n t l y d e v e l o p e d for s t u d y i n g the i n t e r m e d i a t e s . Detection i r r a d i a t i n g (8) lived

of

Transient

intermediates

properties.

(Short-Lived)

By

Intermediates.

pulse

a system, i t is possible to detect t h e presence of short

The

and

to

technique

characterize involves

their structural and kinetic

the use

of

electron

accelerators

c a p a b l e of d e l i v e r i n g a h i g h dose to a system i n a t i m e t h a t is short c o m p a r e d to the l i f e t i m e of the species b e i n g s t u d i e d . T y p i c a l l y , s u c h m a c h i n e s d e l i v e r doses of a b o u t 200 G y ( 1 k r a d =

10 G y ) i n a square

w a v e p u l s e l a s t i n g a b o u t 1 Jxs. F o r systems i n w h i c h i n t e r m e d i a t e is f o r m e d w i t h a G - v a l u e of 3, t h e instantaneous c o n c e n t r a t i o n after t h e p u l s e is 6 χ

10'

5

M.

If the species

has a r e l a t i v e l y h i g h e x t i n c t i o n

coefficient ( ~ 1 0 M " c m ) , i t c a n b e m o n i t o r e d w i t h fast 3

metric techniques.

1

- 1

spectrophoto-

T h i s o p t i c a l a p p r o a c h has b e e n u s e d p r i m a r i l y f o r

aqueous solutions; b u t i t has also b e e n a p p l i e d to aqueous glasses

(9).

W h e r e the m a g n e t i c resonance p r o p e r t i e s of t h e r a d i c a l s d o not l e a d to b r o a d fines, E S R c a n also b e u s e d (10,11,12).

Provided a particular

species has a r e a s o n a b l y h i g h o r sufficiently different c o n d u c t i v i t y , electro c h e m i c a l d e t e c t i o n is e s p e c i a l l y effective f o r c h a r g e d i n t e r m e d i a t e s

(13).

A r e c e n t v a r i a t i o n o n these t e c h n i q u e s has b e e n d e s c r i b e d b y W a r m a n (14) , i n w h i c h microwave

devices

u n s o l v a t e d electrons i n ice.

A l t h o u g h not c o n s i d e r e d a p u l s e d o r fast

are u s e d to d e t e c t h i g h - m o b i l i t y

reaction approach, the technique developed (15) , involving continuous e s p e c i a l l y effective.

by Eiben and

Fessenden

irradiation a n d i n situ E S R detection,

is

W i t h this t e c h n i q u e , r a d i c a l s t h a t h a v e a t t a i n e d a

r e q u i s i t e steady-state c o n c e n t r a t i o n c a n b e e x a m i n e d , a n d a s u b s t a n t i a l catalogue of spectra f o r free r a d i c a l s i n fluid aqueous solutions has b e e n compiled. I n systems

of

h i g h v i s c o s i t y , t h e free r a d i c a l s of interest i n p r o t e i n r a d i o l y s i s c a n

be

Detection of Trapped or Stabilized Intermediates.


6.

TAUB

Irradiated

E T AL.

115

Proteins

i m m o b i l i z e d a n d e x a m i n e d w i t h s t a n d a r d o p t i c a l or E S R t e c h n i q u e s . Transparent media must be used for examining the radicals optically a n d for g e n e r a t i n g t h e m p h o t o l y t i c a l l y ( 1 6 ) .

A q u e o u s glasses m a d e f r o m

h y d r o x i d e , p e r c h l o r a t e , or e t h a n e d i o l solutions are u s e d at l o w t e m p e r a tures (
2


CH

L

2

0·

F o r t h e t r i p e p t i d e , P h e G l y G l y , i n w h i c h the c a t i o n is n o t

favorably

d i s p o s e d f o r c h a r g e transfer, o n l y t h e ττ-cation r a d i c a l of p h e n y l a l a n i n e is observed

( t h i s species w o u l d r e a d i l y h y d r o l y z e i n w a t e r to f o r m

the

O H - a d d u c t r a d i c a l of p h e n y l a l a n i n e ) . T h e s e results h a v e a d i r e c t b e a r i n g o n t h e reactions i n a n i r r a d i a t e d system. R a d i o l y t i c studies o n ices c o n t a i n i n g p e p t i d e s acids h a v e b e e n

conducted

recently

(51)

c a t i o n i c processes. ( E l e c t r o n s g e n e r a t e d

and N-acetylamino

that p r o v i d e

evidence

concurrently w i t h the

for

solute

cations p a r t i c i p a t e i n reactions d e s c r i b e d b e l o w , b u t those f o r m e d i n the i c e d o not c o n t r i b u t e t o t h e o b s e r v e d reactions. ) T h e a p p e a r a n c e of t h e d e c a r b o x y l a t e d species, t h e y i e l d of w h i c h d e p e n d s o n t h e n a t u r e of t h e c o m p o u n d , p r o v i d e s this e v i d e n c e . A t — 1 9 6 ° C , a p p r o x i m a t e l y 2 5 % jof t h e r a d i c a l s o b s e r v e d f o r N - a c e t y l a l a n i n e corresponds indicating that C 0

2

to

CH CONDCCH , 3

3

has b e e n lost. F o r the d i p e p t i d e G l y A l a , t h e d e c a r

b o x y l a t e d r a d i c a l is o b s e r v e d to b e e q u i v a l e n t i n y i e l d to t h e f r o m t h e e l e c t r o n r e a c t i o n , i n d i c a t i n g a n efficient m e c h a n i s m f o r

product decar

b o x y l a t i o n . T h e findings f o r other d i p e p t i d e s are v e r y s i m i l a r . C o n s i s t e n t w i t h the p h o t o l y t i c results, t h e extent of d e c a r b o x y l a t i o n is p H d e p e n d e n t , r e f l e c t i n g a n i n f l u e n c e of c h a r g e state o n the m e c h a n i s m . T h e s e c o m p e t i t i v e p a t h w a y s f o r r e a c t i o n of c a t i o n i c species

imply

t h a t s e v e r a l different r a d i c a l s c o u l d b e f o r m e d i n p r o t e i n s , d e p e n d i n g o n


6.

TAUB

E T AL.

Irradiated

127

Proteins

their structure a n d the conditions.

T h e e v o l u t i o n of C 0

2

t h a t has b e e n

o b s e r v e d c a n b e u n d e r s t o o d as a r i s i n g f r o m s u c h c a t i o n i c p r e c u r s o r s .

The

f o r m a t i o n of O H - a d d u c t s of p h e n y l a l a n i n e moieties a n d f o r m a t i o n o f t h e p h e n o x y l r a d i c a l s f r o m t y r o s i n e moieties m i g h t also r e s u l t f r o m

such

cations a n d n e e d to b e i n v e s t i g a t e d . O x i d a t i o n of t h e a m i n o a c i d moieties i n i r r a d i a t e d aqueous

systems

b y r e a c t i o n w i t h O H - is w e l l e s t a b l i s h e d f o r fluid systems, b u t i t is n o t Downloaded by UNIV OF MASSACHUSETTS AMHERST on January 18, 2018 | http://pubs.acs.org Publication Date: September 1, 1979 | doi: 10.1021/ba-1979-0180.ch006

l i k e l y to b e e n c o u n t e r e d i n f r o z e n systems. OH-

B e i n g a strong oxidant, the

reacts b y e l e c t r o n transfer. I t also a d d s r e a d i l y to d o u b l e b o n d s

a n d abstracts H f r o m C — Η , r e a c t i o n r a t e constants.

Ν—Η,

s o l u t i o n has b e e n p u b l i s h e d ( 5 2 )

of rate constants f o r

aqueous

a n d a f e w r e p r e s e n t a t i v e values

a m i n o a c i d s are s h o w n i n T a b l e I . p r e d o m i n a n t sites f o r

and S — H bonds, but w i t h lower

A compendium

reaction i n amino

acids a n d peptides

w h i l e abstraction from

the peptide

backbone

is less

the

can

i n f e r r e d f r o m these v a l u e s , w h i c h i n d i c a t e t h a t t h e r i n g groups favored,

for

A s discussed b y S i m i c (53),

be are

likely.

H y d r o x y l a t i o n of the p h e n y l a l a n i n e r i n g also occurs as w a s f o u n d f o r t h e prototype reaction w i t h benzene

F o r m a t i o n of p h e n o x y l r a d i c a l

(54).

f o l l o w i n g O H - a d d i t i o n to t y r o s i n e s h o u l d b e s i m i l a r t o t h e m e c h a n i s m e s t a b l i s h e d for p h e n o l (55)

i n w h i c h e l i m i n a t i o n of w a t e r occurs as is

shown i n reaction 12:

—CHRCONHCHCONH— CH

2

OH (12)

CH

2

+

H 0 2

H o w e v e r , these a d d u c t f o r m a t i o n reactions a r e n o t e x p e c t e d to o c c u r w i t h m u c h efficiency

i n f r o z e n , h y d r a t e d p r o t e i n s , b e c a u s e of

the

m o b i l i t y of O H · i n t h e r i g i d m a t r i x .


limited

PROTEINS A T L O W

128

TEMPERATURES

F o r m a t i o n o f α-carbon r a d i c a l s e i t h e r f r o m c a t i o n d e c o m p o s i t i o n

or

t h r o u g h a b s t r a c t i o n of H b y O H - is c o n s i d e r e d h e r e b e c a u s e i t c a n b e c o n s t r u e d f o r m a l l y as loss of a n e l e c t r o n f o l l o w e d b y p r o t o n t r a n s f e r . I f t h e c a t i o n deprotonates f r o m t h e p e p t i d e c h a i n b y transfer t o of

components

h i g h p r o t o n affinity t h e n t h e α-carbon r a d i c a l is f o r m e d

directly.

A b s t r a c t i o n b y O H · i n fluid systems gives t h e same result. ( A b s t r a c t i o n of H b y H ·

is also p o s s i b l e i n b o t h fluid a n d f r o z e n


systems. R a t e constants f o r H - w i t h a m i n o a c i d s are k n o w n (56,57)

and

the intermediates formed have been characterized ( 5 3 ) . ) Accept

Electron Acceptance: Reduction and A d d u c t Formation.

ance of electrons at specific sites o n a m i n o acids a n d p e p t i d e s d e p e n d s o n t h e i r r e a c t i v i t i e s a n d p r o d u c e s different c h e m i c a l consequences.

Among

t h e sites of p a r t i c u l a r i m p o r t a n c e a r e t h e t e r m i n a l a m i n o a n d c a r b o x y l g r o u p s , the r i n g g r o u p s , t h e p e p t i d e c a r b o n y l , a n d t h e s u l f u r

bonds.

R e a c t i v i t i e s of these are reflected i n the rate constants f o r r e a c t i o n of s o l v a t e d electrons w i t h i n d i v i d u a l a m i n o a c i d s i n aqueous s o l u t i o n s , as s h o w n i n T a b l e I a n d as d i s c u s s e d b y S i m i c ( 5 3 ) .

M o r e detailed informa

t i o n , h o w e v e r , r e g a r d i n g t h e stepwise p r o g r e s s i o n f r o m a t t a c h m e n t to specific r a d i c a l f o r m a t i o n has b e e n o b t a i n e d f r o m l o w t e m p e r a t u r e studies. ATTACHMENT

TO

AMINO

GROUP.

The

electron

reacts

with

the

p r o t o n a t e d t e r m i n a l a m i n o g r o u p i n a l i p h a t i c a m i n o a c i d s , l e a d i n g to d e a m i n a t i o n (53,58).

T h e rate constants i n s o l u t i o n d e p e n d o n p H , k

d e c r e a s i n g as p H is i n c r e a s e d .

E S R studies o n e l e c t r o n r e a c t i o n w i t h

a m i n o a c i d s i n n e u t r a l glasses at

indicate that dissociative at

—196°C

t a c h m e n t r e a d i l y occurs p r o d u c i n g a f a t t y a c i d r a d i c a l : e." +

+

N H C H R C O O " -> N H 3

3

+

-CHRCOO"

(13)

T h e f o r m a t i o n of a m m o n i a does n o t necessarily i m p l y d i r e c t r e a c t i o n w i t h the N H +

3

group.

ATTACHMENT TO T H E CARBOXYLATE

GROUP.

I n b a s i c systems w h e r e

t h e a m i n o g r o u p is n o t p r o t o n a t e d , a t t a c h m e n t occurs o n t h e c a r b o x y l a t e i o n , as e v i d e n c e d f r o m E S R studies o n h y d r o x i d e glasses (16).

At — 196°C

s p e c t r a are o b s e r v e d f o r t h e d i a n i o n r a d i c a l of s e v e r a l a m i n o a c i d s .

At

h i g h e r t e m p e r a t u r e s , t h e f a t t y a c i d r a d i c a l s are f o r m e d , i n d i c a t i n g t h a t d e a m i n a t i o n has t a k e n p l a c e : H 0 2

e.- + N H C H R C 0 - - » 2

N H

2

3

+

NH CHRC0 2

2

=

-

-CHRCOjf+ OH-

(14)

T h e t w o - s t e p s e q u e n c e d e m o n s t r a t e d f o r f r o z e n systems c o u l d also o c c u r i n fluid systems, b u t at rates too fast to observe.


6.

TAUB

E T AL.

Irradiated

τ Downloaded by UNIV OF MASSACHUSETTS AMHERST on January 18, 2018 | http://pubs.acs.org Publication Date: September 1, 1979 | doi: 10.1021/ba-1979-0180.ch006

129

Proteins

•

Τ \

r

Irradiated

ι ι

1—

__ι

250 300 WAVELENGTH, nm

Figure 5. Optical spectrum of hydrogen adduct of phenylalanine in an ethanediol-water glass at —196°C. Glass was formed from an equimolar mixture of ethanediol and water containing 4 X 10~ M phenylalanine, irradiated to ap proximately 4 kGy, and exposed at —196°C to visible light to bleach trapped electrons. Solid curve corresponds to phenylalanine in the unirradiated glass; dotted curve is displaced vertically for clarity and corresponds to the NH CH(CH (C H )-)CO ' radical 2

+

2

6

6

B


3

130

PROTEINS A T L O W

ATTACHMENT

TO

TEMPERATURES

Electrons

GROUPS.

AROMATIC AND HETEROCYCLIC

r e a c t m o r e r a p i d l y w i t h h i s t i d i n e , t r y p t o p h a n , p h e n y l a l a n i n e , a n d tyrosine than w i t h the aliphatic amino acids, f o r m i n g radicals i n v o l v i n g the r i n g groups.

S u c h e l e c t r o n - a d d u c t r a d i c a l s r e a d i l y p r o t o n a t e i n a q u e o u s sys-

tems ( 5 9 ) g i v i n g t h e e q u i v a l e n t of a n H - a d d u c t r a d i c a l . S i m i l a r reactions o c c u r i n f r o z e n systems f o r w h i c h extensive E S R e v i d e n c e has obtained ( I S ) .

w a t e r - e t h a n e d i o l glass (60) Downloaded by UNIV OF MASSACHUSETTS AMHERST on January 18, 2018 | http://pubs.acs.org Publication Date: September 1, 1979 | doi: 10.1021/ba-1979-0180.ch006

been

O p t i c a l e v i d e n c e for t h e H - a d d u c t of p h e n y l a l a n i n e i n a is s h o w n i n F i g u r e 5. R e a c t i o n 1 5 i n d i c a t e s

the s e q u e n c e :

e." +

+

NH CHC0 " 3

2

+

NH CHC0 3

+

2

NH CHC0 - + OH3

2

o. ÇH

(15)

2

A t t a c h m e n t to t h e r i n g is n o t t h e e x c l u s i v e fate of t h e e l e c t r o n , b u t i t is p a r t i c u l a r l y c o m p e t i t i v e w i t h other p a t h w a y s for r e a c t i o n . ATTACHMENT

TO

T H E PEPTIDE

CARBONYL.

AS

the peptide

increases, t h e rate constant f o r e l e c t r o n r e a c t i o n increases (61),

length

indicating

t h a t r e a c t i o n o c c u r s at a p e p t i d e c a r b o n y l . T h e r e s u l t i n g r a d i c a l c a n b e p r o t o n a t e d d e p e n d i n g o n the p K f o r t h e f o l l o w i n g e q u i l i b r i u m (62) : H *± +

—CHRCHNR— I 0"

—CHRCNHR— . I OH

(16)

E S R e v i d e n c e f o r this e l e c t r o n a d d u c t of t h e c a r b o n y l g r o u p i n f r o z e n solutions of a c e t y l a m i n o acids a n d d i - a n d t r i p e p t i d e s is extensive ( 5 0 ) . A t y p i c a l s p e c t r u m is s h o w n i n F i g u r e 6 f o r the a n i o n r a d i c a l of β-alanylg l y c i n e at — 1 5 3 ° C . D e p e n d i n g o n t h e p e p t i d e a n d p H , d e a m i n a t i o n c a n o c c u r b y a process i n v o l v i n g either i n t e r - or i n t r a m o l e c u l a r e l e c t r o n transfer. D i s s o c i a t i o n at other C — Ν b o n d s is also p o s s i b l e , l e a d i n g to a n a m i d e a n d a " f a t t y a c i d " r a d i c a l (20,63); as " d e a m i d a t i o n " o r " s e c o n d a r y

t h i s process w i l l b e r e f e r r e d to

deamination.

,,

A s w i l l be

mentioned

b e l o w , this r e a c t i o n a n d t h e c h e m i s t r y t h a t f o l l o w s are i m p o r t a n t f o r peptides a n d proteins. ATTACHMENT

TO SULFUR

GROUPS.

O f a l l t i e amino acids, the t w o

m o s t r e a c t i v e i n s o l u t i o n t o w a r d t h e e l e c t r o n are c y s t i n e a n d cysteine. R e a c t i o n w i t h t h e f o r m e r leads to t h e d i s u l f i d e a n i o n r a d i c a l , +

N H C H (COO") C H [ S - ^ S ] " C H (COO") C H N H 3

2

2

3

+

,


TAUB

Irradiated

E TA L .


6.

J

131

Proteins

I

I

I

MAGNETIC FIELD, GAUSS

L

•

Figure 6. ESR spectrum of the peptide anion radical derived from electron attachment to β-alanylglycine at —196°C in a LiCl glass. Sample contained 5 X I 0 " M of β-alanylglycine and the electrons were generated photolytwally (64). Spectrum was recorded at —153°C and is attributed to the radicat+ND CH CH CONDCH C0 : 2

s

2

2

2

2


132

PROTEINS

AT LOW

TEMPERATURES

a n d w i t h the l a t t e r , to N H C H ( C O O ) C H - a n d H S ' . R e a c t i o n w i t h t h e +

3

2

—CH2SCH3 g r o u p i n m e t h i o n i n e also o c c u r s a n d appears to p r o d u c e t w o t y p e s of r a d i c a l s : e - + aQ

~SCH

3

+

+

+

NH CH(COO-)CH CH SCH 3

2

NH CH(COO-)CH CH 3

2

2

3

-»

(17) and NH CH(COO-)CH CH S"+ -CH +


2

3

2

2

3

E S R studies of the a m i n o acids i n glasses c o n f i r m t h e f o r m a t i o n of t h e d i s u l f i d e a n i o n ( 2 0 ) , w h i c h is e s p e c i a l l y stable, a n d t h e m e t h y l r a d i c a l (20).

S i m i l a r reactions m a y b e e x p e c t e d to o c c u r d i r e c t l y o r i n d i r e c t l y

o n peptides. COMPETITION

FOR

ELECTRONS

BY

REACTIVE

GROUPS

ON

PEPTIDES.

S i n c e there are m a n y sites for r e a c t i o n o n p e p t i d e s , t h e fate of t h e e l e c t r o n w i l l b e i n f l u e n c e d b y t h e specific moieties a n d t h e i r d i s p o s i t i o n . S e v e r a l illustrations c a n b e g i v e n . F o r p e p t i d e s w i t h a n a r o m a t i c g r o u p , d e a m i n a tion competes w i t h r i n g attachment, but peptide carbonyl p r e d o m i n a t e s as t h e n u m b e r of p e p t i d e groups increases. (a)

GlyPhe, (b)

for ( a ) ,

ring

PheGly, and (c)

attachment

I n t h e series

P h e G l y G l y : d e a m i n a t i o n is p r e f e r r e d

a t t a c h m e n t a n d d e a m i n a t i o n are e q u i v a l e n t f o r ( b ) ,

p e p t i d e a t t a c h m e n t begins to c o m p e t e w i t h these t w o processes i n F u r t h e r m o r e , r i n g a t t a c h m e n t is e x c l u s i v e i n H i s G l y , b u t a b o u t

and (c). 40%

of t h e electrons r e a c t w i t h G l y H i s t o d e a m i n a t e i t . I n a c e t y l p e p t i d e s , f o r w h i c h n o sites f o r d e a m i n a t i o n exist, e l e c t r o n a t t a c h m e n t to

the

peptide c a r b o n y l predominates, b u t competition b y methionine, cysteine, p h e n y l a l a n i n e , t y r o s i n e , t r y p t o p h a n , g l u t a m i c a c i d , a n d a s p a r t i c a c i d is extensive. elsewhere

D e t a i l e d comparisons

of

these processes w i l l b e

reported

(64).

Reaction of Peptide Radicals.

O n t h e basis of t h e k i n d of p h y s i c o -

c h e m i c a l e v i d e n c e p r e s e n t e d here a n d of the c h e m i c a l e v i d e n c e d e s c r i b e d elsewhere (46,47),

i t is a p p a r e n t t h a t the v a r i o u s r a d i c a l s f o r m e d i n i t i a l l y

i n the i r r a d i a t i o n of p e p t i d e s a n d p r o t e i n s c o n v e r t to other r a d i c a l s t h a t s u b s e q u e n t l y react to f o r m p r o d u c t s . already been mentioned.

C o n v e r s i o n of c a t i o n r a d i c a l s has

C o n v e r s i o n s of t h e p e p t i d e α-carbon r a d i c a l s

a r e e s p e c i a l l y i m p o r t a n t to u n d e r s t a n d i n g t h e r a d i o l y s i s of p r o t e i n s , so some i l l u s t r a t i v e examples w i l l b e g i v e n .

T h e e v e n t u a l r e a c t i o n of t h e

α-carbon r a d i c a l s is n o t w e l l u n d e r s t o o d , b u t c e r t a i n assumptions c a n b e m a d e r e l e v a n t t o t h e systems s t u d i e d . DECOMPOSITION O F T H E PEPTIDE C A R B O N Y L RADICALS.

Depending on

t h e n a t u r e of t h e c o n s t i t u e n t g r o u p s , these r a d i c a l s c a n d e c o m p o s e b y t r a n s f e r r i n g t h e e l e c t r o n t o the t e r m i n a l a m i n o g r o u p o r b y s p l i t t i n g off an amide.

B o t h processes, d e a m i n a t i o n a n d d e a m i d a t i o n , l e a d to

the

f o r m a t i o n of · C H R C O N H — r a d i c a l s , d e a m i n a t i o n c o r r e s p o n d i n g to c h a i n


6.

TAUB

Irradiated

ET AL.

Proteins

133

scission. A s a n i l l u s t r a t i o n , F i g u r e 7 s h o w s t h e s p e c t r a f o r D 0 ices of 2

N - a c e t y l a l a n i n e ( 1 9 0 m g J m l ) i r r a d i a t e d a t —196 ° C a n d t h e n a n n e a l e d (64).

T h e c a r b o n y l a n i o n p r e d o m i n a t e s i n t h e s p e c t r u m after a n n e a l i n g

a t — 1 5 3 ° C ( t o e l i m i n a t e O D - ) a n d a m o u n t s t o a b o u t 6 5 % of t h e r a d i cals; the next m o s t a b u n d a n t r a d i c a l is t h e d e c a r b o x y l a t e d species.

Upon

a n n e a l i n g to — 8 0 ° C , t h e a n i o n c o n v e r t s b y d e a m i d a t i o n to t h e a m i d e a n d


fatty a c i d radical:

C H C (O") N D C H ( C H ) C < V -> 3

3

CH COND 3

+

2

-CH(CH )C0 3

(18)

2

T h e s p e c t r u m s h o w n c a n b e c o m p a r e d w i t h t h a t of the p r o p i o n i c a c i d r a d i c a l f o r m e d i n d e p e n d e n t l y o r b y t h e d e a m i n a t i o n of a l a n i n e .

This

s e q u e n c e is r e p r e s e n t a t i v e o f s e v e r a l a c e t y l a m i n o a c i d s s t u d i e d . ABSTRACTION

OF H

FROM

T H E PEPTIDE

BY

RADICALS.

CARBON

The

carbon radicals derived from deamination, deamidation, a n d decarboxyla tion react subsequently w i t h the peptides to abstract hydrogen, f o r m i n g t h e m o r e s t a b l e α-carbon p e p t i d e r a d i c a l s ( 6 3 ) .

This reaction can

be

d e m o n s t r a t e d i n i r r a d i a t e d ices c o n t a i n i n g a c e t y l a m i n o a c i d s . C o n t i n u i n g w i t h t h e N - a c e t y l a l a n i n e e x a m p l e , one c a n also see i n F i g u r e 7 t h a t u p o n f u r t h e r a n n e a l i n g t o — 50 ° C , a n o t h e r r a d i c a l a p p e a r s c o r r e s p o n d i n g

to

r e a c t i o n 19:

C H (CH ) C0 " + 3

2

C H C O N D C H ( C H ) C < V -> 3

CH (CH )CCV + 2

3

CH CONDCCH C0 -

3

3

3

(19)

2

T h e q u a r t e t s p e c t r u m s h o w n is a s s i g n e d t o t h e r a d i c a l f r o m a c e t y l a l a n i n e . T h e same t y p e of r e a c t i o n o c c u r s w i t h d i p e p t i d e s . C o n s e q u e n t l y , a series of a l i p h a t i c d i p e p t i d e s i n D 0 2

r a d i c a l s at a p p r o x i m a t e l y

ices w e r e i r r a d i a t e d a n d t h e r e s u l t a n t

— 50°C were

e x a m i n e d to

obtain

detailed

spectral data for comparison w i t h proteins. T a b l e I I shows the substituents o n t h e m o d e l p e p t i d e r a d i c a l , N D C H ( R 0 C O N D C R C O " , a n d t h e +

type of spectra observed.

3

Because peptides

2

with R

=

H

or

CH R"

p r e d o m i n a t e i n p r o t e i n s a n d b e c a u s e these w o u l d g i v e r i s e to

doublet

2

s p l i t t i n g s at l o w t e m p e r a t u r e s , i t is u n d e r s t a n d a b l e w h y the i r r a d i a t e d p r o t e i n s p e c t r a f o r a c t o m y o s i n , m y o s i n , a n d others a r e c h a r a c t e r i z e d b y a broad doublet

signal.

C o m p u t e r s i m u l a t i o n of t h e p r o t e i n s i g n a l b y

p r o p e r l y c o m b i n i n g t h e p e p t i d e s p e c t r a is u n d e r w a y . BIMOLECULAR

REACTION

OF T H E «-CARBON

RADICALS.

T h e eventual

f o r m a t i o n of stable c o v a l e n t b o n d s r e q u i r e s t h a t p r o t e i n α-carbon r a d i c a l s ( w h o s e d i s t r i b u t i o n is d e t e r m i n e d b y m o l e c u l a r c o n f o r m a t i o n a n d t h e conditions)

c o m b i n e or d i s p r o p o r t i o n a t e w i t h s i m i l a r o r o t h e r r a d i c a l s .


134


PROTEINS A T L O W T E M P E R A T U R E S

M A G N E T I C F I E L D , GAUSS

— •

Figure 7. ESR spectra of radicals derived from N-acetyl^-alanine in an irradiated D 0 ice plug. Sample contained 190 mgJml of N-acetyhlanine and was irradiated to 5 kGy at —196°C. Spectrum a was recorded at —196°C; spectra b and c at —135°C. All spectra have markers from Fremy's salt superimposed, (a) Composite spectrum of radicals present after annealing at — 1 5 3 C (approximately 65% corresponds to the anion), (b) Composite spectrum of the radicals formed upon annealing the ice plug to —80°C, of which 55% corresponds to the fatty acid radical, -CH(CH )CO ~. (c) Spectrum of the peptide radical, CH CONDC(CH )C0 ~, formed upon further annealing the ice plug to - 5 0 ° C . 2

e

s

3

3

z

2


6.

TAUB

Irradiated

E T AL.

Table II.

135

Proteins

E S R Spectral Features for Different

*ND CH(R')CONDC(R)C0 3


Parent

Radicals'

2

Dipeptide H CH H CH H

Glycylglycine Alanylglycine Glycylalanine Alanylalanine Glycylglutamic acid Glutlmylglutamic acid Glycylaspartic acid Glycylmethionine Glycylserine Lysyllysine

H H CH CH

3

3

CH2CH2CO2"

H H H (CH ) ND 2

Number of Lines"

R

R'

4

2 2 4 4 2 2 2 2 2 2

3 3

CH2CH2CO2" CH2CH2CO2" CH2CO2" (CH2) 2SCH CH 0H (CH )4ND

3

2

3

+

2

3

+

"Species stable after y -irradiating approximately 200 mgJml of the corresponding dipeptide in ice plugs at —196°C to a dose of about 5 k G y and annealing to -50°C. Hyperfme splittings are found to be 18-20 gauss for all the radicals observed. b

T h e i r l a r g e size a n d r e l a t i v e i m m o b i l i t y w o u l d r e n d e r s u c h

reactions

s l o w i n fluid m e d i a a n d i m p r o b a b l e i n viscous o r r i g i d m e d i a .

Reaction

i n d r y systems a n d i n f r o z e n systems w o u l d i n v o l v e either s m a l l m o b i l e r a d i c a l s d i f f u s i n g to t h e l a r g e r r a d i c a l s o r free r a d i c a l sites o n t h e l a r g e m o l e c u l e s b e i n g i n p r o x i m i t y to e a c h other. C H , C H R C 0 ~ , or S C H 3

2

globular polypeptides could combine

3

F r a g m e n t r a d i c a l s s u c h as

c o u l d a c c o u n t f o r s o m e of t h e d e c a y .

F o r large,

or proteins, the radicals o n folded-over

i f close e n o u g h .

F o r the long,

fibrous

chains

molecules, the

r a d i c a l s o n n e i g h b o r i n g chains m i g h t b e a b l e t o react i f sufficient b e n d i n g occurs to p l a c e these sites i n a n a p p r o p r i a t e d i s p o s i t i o n . S i n c e m y o s i n has t w o chains e n t w i n e d a b o u t e a c h other, some cross r e a c t i o n m i g h t b e expected.

I f t h e p r o t e i n s a r e d e s i c c a t e d t h e r e s h o u l d b e some u n r a v e l i n g

i n t h e s t r u c t u r e , m a k i n g r e a c t i o n e v e n less l i k e l y .

Significantly more

k i n e t i c i n f o r m a t i o n is n e e d e d b e f o r e these final steps i n t h e r a d i o l y t i c a l l y i n i t i a t e d s e q u e n c e o f reactions a r e u n d e r s t o o d . Summary M a n y of the reactions t h a t o c c u r i n t h e specific systems d e s c r i b e d a r e c o m m o n to most i r r a d i a t e d p r o t e i n s . T h e s e reactions a r e s u m m a r i z e d i n the g e n e r a l i z e d scheme g i v e n b e l o w .

N o a t t e m p t is m a d e t o s h o w a l l

p o s s i b l e p a t h w a y s f o r r e a c t i o n o r t o e x p l a i n a l l of t h e observations n o t e d f o r p r o t e i n s . F o r t h e sake of c l a r i t y , t h e p r o t e i n s t r u c t u r e is i d e a l i z e d a n d schematically represented b y J X J X J X J X J X J ,

w h i c h is e q u i v a -

l e n t t o ( 2 0 ) ( d e t a i l s i n t h e s t r u c t u r e a r e i n c l u d e d o n l y f o r specific cases) :



136

PROTEINS

H,.() - V W V

A T L O W TEMPERATURES

(21)

·• c ", O H ·, I T , H · s

ΛΛΛΪΛΛJ

(22a

>

ionization

JWVWvw-

excitation

(22b)

(ΛΛΛΛΛJ)*

+AJWW

attachment

abstraction

0H- +JVWW

AAJVVAJ cr J W v V V

+

(23)

H

(24a)

-'0

addition-elimination J V J V J V J V J V J -|- Η

0

124b)

~ 0 -Η-

JW\JW

charge transferdecarboxylation

- Λ Λ J W V

charge transferhydrolysis

θ;


(25a)

6.

TAUB E T

AL.

Irradiated

137

Proteins

deexcitation neutralization-


excitation

- Λ Λ Λ Λ Λ J . (26a)

-ΛΛΛΛν+Η·(26ΐ))

-(ΛΛΛΛΛJ)*

Η

Η deamination

Λ J W W ι 0"

ΝΗ

3

+

(27a)

V W W

deamidation (+Η*)

Η

JJJJ

(27b)

4

demethylation

ΛΛΛΛΛJ + C H CH,

(27c)

3

I

CH, I sW

+

Λ Λ Λ J W

a b s t r a c t i o n

* Λ Λ Λ J W

+

disproportionation

(28)

W

Q

^ΛΛΛ^ΛΛJ+ΛΛΛΛΑJ (29a)

ΛΛΛΛΛJ+ ΛΛΛΛΛJ Η

Ν

combination

V V W s A

(29b)

Λ Λ Λ Λ Λ J

A s is i m p l i e d i n this s c h e m e , there w i l l b e c e r t a i n m o d i f i c a t i o n s i n t h e p r o t e i n r e s u l t i n g f r o m i r r a d i a t i o n that d e p e n d o n specific c o n d i t i o n s . T h e i n d i r e c t effects of reactions of p r i m a r y w a t e r r a d i c a l , as w e l l as those r e s u l t i n g f r o m u n i m p e d e d d i f f u s i o n of s e c o n d a r y r a d i c a l s , w i l l b e m i n i m i z e d o r e l i m i n a t e d i n d r y systems o r f r o z e n a q u e o u s systems. F o r m a t i o n of t h e p e p t i d e r a d i c a l b y l o n g c h a i n , f a t t y a c i d - t y p e r a d i c a l s a b s t r a c t i n g hydrogen

could occur i n

fluid,

concentrated

solutions, b u t w o u l d

difficult i n rigid m e d i a , b e i n g l i m i t e d to either n e i g h b o r i n g m o l e c u l e s p r o x i m a l parts of t h e same m o l e c u l e .

Effects o n globular a n d


be or

fibrous

138

PROTEINS

p r o t e i n s w i l l differ f o r this reason.

AT LOW

TEMPERATURES

F i n a l reaction of the large peptide

r a d i c a l s , i n t u r n , w o u l d b e affected b y the constraints o f t h e m e d i u m o n t h e i r flexing a n d d i f f u s i o n a l m o t i o n s . fibrous

S u c h reactions i n f r o z e n , h y d r a t e d ,

p r o t e i n s m i g h t i n v o l v e o n l y n e i g h b o r i n g r a d i c a l s b r o u g h t close

e n o u g h together b y flexing a n d t o r s i o n a l m o d e s o f m o t i o n . I n d r y systems, t h e i n t e r a c t i o n of these w o u l d b e m o r e difficult b e c a u s e of a less f a v o r a b l e d i s p o s i t i o n o f the c h a i n s .


S i n c e the proteins i n f o o d p r e s e r v e d b y i r r a d i a t i o n t o a p p r o x i m a t e l y 4 0 k G y at — 4 0 ° C are h y d r a t e d a n d fixed i n a r i g i d m e d i u m , t h e o b s e r v a t i o n (6) that these p r o t e i n s are m i n i m a l l y affected i s consistent w i t h t h e i m p l i c a t i o n s o f this scheme.

T h e m a j o r free r a d i c a l s d e r i v e d f r o m t h e

p r o t e i n are of t h e p e p t i d e b a c k b o n e t y p e , a n d t h e y d o not persist i n t h e t h a w e d product. These radicals apparently undergo reconstitutive recom b i n a t i o n reactions, so n o significant d e g r a d a t i o n o r c o v a l e n t of t h e l o n g m o l e c u l a r c h a i n is o b s e r v e d .

aggregation

O t h e r free r a d i c a l s f r o m s i d e

c h a i n groups represent a s m a l l c o n t r i b u t i o n t o t h e t o t a l , a n d c o n s e q u e n t l y , n o n e o f t h e a m i n o acids i s d i s c e r n i b l y affected.

T h e l i m i t e d extent t o

w h i c h changes o c c u r i n t h e p r o t e i n s , as w e l l as i n the e q u a l l y i m p o r t a n t l i p i d components

(65,66,67),

explains t h e h i g h q u a l i t y of t h e l o w -

t e m p e r a t u r e i r r a d i a t e d m e a t , fish, a n d p o u l t r y p r o d u c t s . Acknowledgment T h e authors are g r a t e f u l t o D r . M i c h a e l G . S i m i c , D r . J a m e s J . S h i e h , M r . J o h n E . W a l k e r , a n d M r . James B . D ' A r c y f o r h e l p f u l discussions o f this subject a n d f o r m a k i n g a v a i l a b l e d a t a i n a d v a n c e o f p u b l i c a t i o n . Literature Cited 1. Spinks, J. W. T.; Woods, R. J. "An Introduction to Radiation Chemistry"; 2nd ed.; John Wiley: New York, 1976. 2. Hart, E . J.; Anbar, M. "The Hydrated Electron"; Wiley-Interscience: New York, 1970. 3. Anbar, M.; Bambenek, M.; Ross, A. B. "Selected Specific Rates of Reac tions of Transients from Water in Aqueous Solution. Hydrated Elec tron," Natl. Bur. Stand. (U.S.) Rep. 1973, NSRD-NBS 43. 4. Taub, I. Α.; Kaprielian, R. A. Abstr. First Int. Cong. Eng. Food, Aug 9-13, 1976, Boston, ΜΑ; p 82. 5. Taub, I. Α.; Kaprielian, R. Α.; Halliday, J. W. IAEA Proc. Int. Symp. Food Preserv. Irradiation, Nov. 21-25, 1977 1978, 1, 371. 6. Taub, Ι. Α.; Robbins, F. M.; Simic, M. G.; Walker, J. E . ; Wierbicki, E . Food Tech. 1979, 33, 184. 7. Rustgi, S.; Joshi, A; Friedberg, F.; Riesz, P. Int. J. Radiat.Biol.and Relat. Stud. Phys. Chem. Med. 1977, 32, 533. 8. Matheson, M. S.; Dorfman, L . M. "Pulse Radiolysis"; MIT: Cambridge, 1969. 9. Taub, I. Α.; Hurwitz, P. Α.; Tocci, J. Abstr. Fifth Int. Cong. of Radiat. Res., 1974, Seattle, WA, p 196.



6.

TAUB E T A L .

Irradiated Proteins

139

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Chemical Reactions in Proteins Irradiated at Subfreezing

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