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Radiolysis WARREN M. GARRISON, MICHAEL E. JAYKO, MICHAEL A. J. RODGERS, HARVEY A. SOKOL, and WINIFRED BENNETT-CORNIEA Lawrence Radiation Laboratory, University of California, Berkeley, Calif. 94720

Major chemical effects of γ-rays on simple peptides in the polycrystalline state, in the glassy state and in concentrated aqueous solutions lead to degradation of the peptide chain. Several different reaction modes are involved and these all yield amide-like products that readily hydrolyze to give ammonia. Major concomitant products include fatty acid, ketoacid and aldehyde. Studies of N-acetylalanine glass con taining added electron scavengers provide a measurement of the ionization yield, G 3. Conventional radical scav engers have relatively little effect on G(NH ) 3.5. How ever, certain aromatic compounds effectively quench a major fraction of the (amide) ammonia yield. The evidence is that keto acid and aldehyde are formed from positive-ion inter mediates while fatty acid and amide are derived through reactions involving excited (triplet) states. e-

3

A

major c h e m i c a l effect of y - r a y s o n s i m p l e p e p t i d e s s u c h as the Na c y l a m i n o acids u n d e r oxygen-free c o n d i t i o n s , b o t h i n the s o l i d state

a n d i n c o n c e n t r a t e d aqueous s o l u t i o n , leads to f o r m a t i o n of l a b i l e a m i d e l i k e c o m p o u n d s w h i c h are r e a d i l y d e g r a d e d o n m i l d h y d r o l y s i s to y i e l d a m m o n i a as a characteristic p r o d u c t . S e v e r a l classes of nitrogen-deficient p r o d u c t s are f o r m e d c o n c o m i t a n t l y w i t h t h e a m m o n i a . E a r l i e r c o m m u n i cations h a v e discussed c e r t a i n l i m i t e d aspects of t h e r a d i o l y t i c l a b i l i t y of s i m p l e peptides i n the s o l i d state a n d i n c o n c e n t r a t e d solutions ( 9 , 10, 18).

T h e r a d i a t i o n c h e m i s t r y of these systems is m o r e c o m p l e x t h a n that

i n v o l v e d i n the r a d i o l y s i s of s i m p l e p e p t i d e s i n d i l u t e oxygen-free

aqueous

s o l u t i o n u n d e r w h i c h c o n d i t i o n s m a i n - c h a i n d e g r a d a t i o n is of m i n o r i m p o r t a n c e (10).

I n this p a p e r w e report d e t a i l e d e x p e r i m e n t a l e v i d e n c e 384

Hart; Radiation Chemistry Advances in Chemistry; American Chemical Society: Washington, DC, 1968.

27.

GARRISON E T A L .

and

specific f o r m u l a t i o n s f o r a n u m b e r of d e g r a d a t i o n m o d e s that h a v e

Peptide

385

Radiolysis

b e e n f o u n d to be i n d u c e d d i r e c t l y t h r o u g h i o n i z a t i o n a n d excitation of p e p t i d e s i n the p o l y c r y s t a l l i n e state, i n the glassy state, a n d i n c o n c e n t r a t e d aqueous s o l u t i o n . Experimental M a t e r i a l s . T h e N - a c e t y l a m i n o acids w e r e of reagent grade or of the highest p u r i t y a v a i l a b l e c o m m e r c i a l l y ( C y c l o C h e m i c a l s , K a n d K L a b o ratories, M a n n R e s e a r c h L a b o r a t o r i e s , N u t r i t i o n a l B i o c h e m i c a l s ) a n d w e r e r e c r y s t a l l i z e d at least once f r o m d i s t i l l e d w a t e r . P o l y a l a n i n e ( Y e d a , M . W . 1700) w a s d i a l y z e d against d i s t i l l e d w a t e r a n d l y o p h i l i z e d . C h l o r acetic a c i d ( E a s t m a n ) w a s r e d i s t i l l e d in vacuo. O t h e r c h e m i c a l s w e r e of C P . grade. W a t e r f r o m a B a r n s t e a d still w a s r e d i s t i l l e d first f r o m a l k a l i n e p e r m a n g a n a t e a n d t h e n f r o m p h o s p h o r i c a c i d . T h e p H adjustments w e r e m a d e w i t h N a O H or H S 0 . 2

4

S A M P L E P R E P A R A T I O N A N D IRRADIATION. T O o b t a i n a c e t y l a l a n i n e glass,

the p o l y c r y s t a l l i n e free a c i d w a s d i s s o l v e d to — 2 M i n r e d i s t i l l e d w a t e r a n d the s o l u t i o n w a s adjusted to p H 6.5 w i t h N a O H . T e n - m l . a l i q u o t s w e r e t r a n s f e r r e d to f l a t - b o t t o m e d c y l i n d r i c a l i r r a d i a t i o n cells (2.5 c m . d i a m . ) a n d w a t e r w a s s l o w l y r e m o v e d o n the v a c u u m line. T h e solutions w e r e k e p t at 0 ° C . d u r i n g the d e h y d r a t i o n . U n d e r these c o n d i t i o n s the w a t e r content g r a d u a l l y decreases over a p e r i o d of 24 h o u r s u n t i l a clear glass of c o m p o s i t i o n C H C O N H C H ( C H ) C O O N a • 2 H 0 is o b t a i n e d . A d d i t i o n a l f o u r to six hours p u m p i n g does not change the w a t e r m o l e f r a c t i o n a p p r e c i a b l y . T h e samples w e r e i r r a d i a t e d at 0 ° C . M o r e d i l u t e solutions w e r e p r e p a r e d i n the o r d i n a r y w a y . T h e p o l y c r y s t a l l i n e Na c e t y l a m i n o a c i d R C O N H C H ( R ) C O O H , a n d the p o l y a l a n i n e w e r e de gassed b y e v a c u a t i o n o n the v a c u u m l i n e f o r at least 24 hours p r i o r to irradiation. 3

8

2

A l l samples w e r e i r r a d i a t e d w i t h C o y - r a y s at a dose-rate of 1.2 X 1 0 e . v . / g r a m / m i n . as d e t e r m i n e d b y the F r i c k e dosimeter [ G ( F e ) = 15.5, e 5 = 2180 at 2 4 ° C . ] . A l l y i e l d s are expressed as G values ( m o l e cules p e r 100 e.v. of a b s o r b e d e n e r g y ) . E n e r g y d e p o s i t i o n i n the solids and c o n c e n t r a t e d solutions w a s t a k e n to b e p r o p o r t i o n a l to e l e c t r o n density. 6 0

18

3 +

80

A N A L Y T I C A L M E T H O D S . Gaseous p r o d u c t s w e r e p u m p e d off f o l l o w i n g c o m p l e t e d i s s o l u t i o n of the i r r a d i a t e d s o l i d i n degassed w a t e r o n the v a c u u m l i n e ; analysis w a s b y mass s p e c t r o m e t r y ( C o n s o l i d a t e d 120) a n d by gas-chromatography ( A e r o g r a p h A 9 0 - P 3 ) . F o r other analyses, the i r r a d i a t e d solids w e r e d i s s o l v e d i n w a t e r u n d e r n i t r o g e n i n a g l o v e box. Free ammonia and amide ammonia were determined b y modifying the m i c r o - d i f f u s i o n m e t h o d of C o n w a y ( 6 ) ; the diffusates w e r e assayed b y means of Nessler reagent. I n the m e a s u r e m e n t of free a m m o n i a , the samples w e r e d i l u t e d three times w i t h saturated K C 0 s o l u t i o n i n the outer c o m p a r t m e n t of the d i f f u s i o n c e l l ; r e c o v e r y of a m m o n i a i n the a c i d c o m p a r t m e n t (0.1IV H S 0 ) is c o m p l e t e i n three h o u r s . F o r t o t a l a m m o n i a ( f r e e p l u s a m i d e ) the s a m p l e was m a d e 2N i n N a O H ; the a m i d e 2

2

3

4


386

RADIATION CHEMISTRY

1

h y d r o l y s i s a n d a m m o n i a transfer is c o m p l e t e i n 24 h o u r s . T h e necessary b l a n k a n d s t a n d a r d runs w e r e m a d e i n p a r a l l e l . T h e f a t t y acids w e r e separated t h r o u g h l y o p h i l i z a t i o n of the s a m p l e s o l u t i o n after a c i d i f i c a t i o n to 2N w i t h H S 0 . A s s a y was b y v a p o r - p h a s e c h r o m a t o g r a p h y ( A e r o g r a p h , 600 C ) . T h e p o l y a l a n i n e w a s d i s s o l v e d i n 2N H S 0 ( u n d e r N ) a n d h y d r o l y z e d 18 hours p r i o r to l y o p h i l i z a t i o n . 2

2

4

4

2

C a r b o n y l p r o d u c t s w e r e i d e n t i f i e d b y p a p e r c h r o m a t o g r a p h y of the 2 , 4 - d i n i t r o p h e n y l h y d r a z o n e s (20). T h e i r r a d i a t e d N - a c e t y l a l a n i n e s h o w e d only p y r u v i c acid and acetaldehyde. These were determined quantita t i v e l y b y the m e t h o d of J o h n s o n a n d Scholes (13) w i t h m i n o r m o d i f i c a tions. C h l o r i d e i o n was d e t e r m i n e d b y the m e t h o d of L u c e et al. (15) after H a y o n a n d A l l e n (12). A c o l o r i m e t r i c m e t h o d (8) lactic acid. Results and

w a s u s e d to set a l i m i t o n the y i e l d of

Discussion

T h e 100 e.v. y i e l d f o r the r a d i o l y t i c d e g r a d a t i o n of the p e p t i d e b o n d , as m e a s u r e d i n terms of G ( N H ) after m i l d h y d r o l y s i s , has b e e n 3

deter

m i n e d for a v a r i e t y of a l i p h a t i c , a r o m a t i c a n d s u l f u r - c o n t a i n i n g a m i n o acids i n the IV-aeetyl f o r m . T h e s e d a t a are s u m m a r i z e d i n T a b l e I.

In

the case of the a l i p h a t i c series, w e note that the l e n g t h of the s i d e - c h a i n has r e l a t i v e l y l i t t l e effect o n the y i e l d of m a i n - c h a i n d e g r a d a t i o n .

The

effect of the a r o m a t i c groups of a c e t y l p h e n y l a l a n i n e a n d of acetyltyrosine is to q u e n c h i n p a r t the y i e l d s of those reactions that l e a d to f o r m a t i o n of a m i d e a m m o n i a .

T h e s u l f u r m o i e t y of m e t h i o n i n e o n the other h a n d

appears to be r e l a t i v e l y ineffective i n q u e n c h i n g s u c h reactions.

Table I. N-acetyl

y-ray Induced Degradation of Solid N-aeetylamino Acids, C H 3 C O N H C H (R ) C O O H

Derivative

glycine alanine a-aminobutyric acid leucine glutamic acid phenylalanine tyrosine methionine a b

G(NH )

(R)

a

— H —CH

3

3

— CH9CH3 —CH" CH(CH

) —CH CH COOH —CH (C H ) —CH (C H OH) 2

2

3

2

2

6

5

2

6

4

CH0CH2SCH3

2

2.68 3.4 2.7 3.2 2.3 0.8 1.6 2.3

N-acetyl-DL-amino acids were used with the exception of N-acetyl-L-glutamic acid. After hydrolysis. A s a p r e l i m i n a r y step i n this i n q u i r y into the n a t u r e of the r a d i o l y t i c

processes that l e a d to d e g r a d a t i o n of the p e p t i d e c h a i n , w e h a v e c o m p l e t e d a d e t a i l e d s t u d y of the r e a c t i o n p r o d u c t s f o r m e d i n the y - r a d i o l y s i s


27.

GARRISON E T A L .

Peptide

387

Radiolysis

of s i m p l e p e p t i d e d e r i v a t i v e s of alanine, viz p o l y - D L - a l a n i n e a n d a c e t y l D L - a l a n i n e p o l y c r y s t a l l i n e . T h e s e d a t a are s u m m a r i z e d i n T a b l e I I .

We

find t h a t the major o r g a n i c p r o d u c t s i n the o r d e r of d e c r e a s i n g y i e l d are propionic acid, acetaldehyde,

p y r u v i c acid, and lactic acid.

ammonia f r o m acetylalanine, G = ide, G =

2.8, p l u s a s m a l l a m o u n t of free a m m o n i a , G =

Table I I .

The

labile

3.4, is d e r i v e d p r i m a r i l y f r o m acetam0.6.

Product Yields in the y-Radiofysis of N - a c e t y l - D L - a l a n i n e and Poly-DL-alanine

Product

Polyalanine

N-acetylalanine

ammonia (total) amide free propionic acid pyruvic acid acetaldehyde lactic acid acrylic acid hydrogen

Table III.

3.6 3.1 0.5 1.8 —1 -0.4

3.4 2.8 0.6 1.4 0.4 0.8 R C O C H R 2

(1)

2

R C O N H C H R -> R C O N = C R + H ( 2 H )

(2)

RCONHCHR -» RCON=CHR + RH

(3)

2

2

2

2

T h e r a d i a t i o n - i n d u c e d N - O shift represented

b y E q u a t i o n 1 leads

to

f o r m a t i o n of a l a b i l e i m i n o ester w h i c h species is r e a d i l y h y d r o l y z e d to y i e l d a m m o n i a a n d the h y d r o x y a c i d , l a c t i c a c i d NH

II

RCOCHR

+ H 0 -> R C O O H + N H + R C H O H

2

2

3

T h e unsaturated products (dehydropeptides)

(4)

2

of E q u a t i o n s 2 a n d 3 are

l a b i l e a n d r e a d i l y h y d r o l y z e to y i e l d a m i d e p l u s p y r u v i c a c i d a n d acetal dehyde respectively

(11)

RCON=CR, + H 0 -» RCONH 2

+ R CO

2

RCON=CHR + H 0 -» RCONH 2

(5)

2

2

+ RCHO

(6)

A c e t y l a-aminobutyric acid yields a-ketobutyric acid and propionaldehyde i n a c c o r d w i t h the above f o r m u l a t i o n . T h e f o r m a t i o n of p r o p i o n i c a c i d as the p r i n c i p a l organic p r o d u c t of the r a d i o l y s i s of a c e t y l a l a n i n e i m p l i e s that direct m a i n - c h a i n cleavage is i n v o l v e d as the major d e c o m p o s i t i o n m o d e .

W e t e n t a t i v e l y define

the

s t o i c h i o m e t r y of this cleavage i n terms of RCONHCHR

2

-> R C O N H + C H R

RCONH + RCONHCHR CHR

2

+ RCONHCHR

-» RCONH

~* C H R

2

w h e r e the r a d i c a l s R C O N H C R

2

2

2

2

(7)

2

2

+ RCONHCR

+ RCONHCR

(8)

2

(9)

2

are l o n g - l i v e d a n d c o r r e s p o n d to

the

r a d i c a l species o b s e r v e d at r o o m t e m p e r a t u r e b y E S R measurements

(4).

O n d i s s o l u t i o n i n w a t e r ( o x y g e n - f r e e ) the a-carbon r a d i c a l s R C O N H C R u n d e r g o d i m e r i z a t i o n to y i e l d are stable e n d - p r o d u c t s (10).

ttja'-diaminosuccinic

2

a c i d derivatives w h i c h

T h e f o r m u l a t i o n of E q u a t i o n s 7, 8, a n d 9 is

i n t e n d e d o n l y to c o n v e y the nature of the o v e r a l l s t o i c h i o m e t r y . a n d / o r e x c i t e d species are p r e s u m a b l y i n v o l v e d as a c t u a l

Ionic

intermediates

since c a g i n g effects i n the s o l i d phase w o u l d l e a d to p r e f e r e n t i a l r e c o m b i n a t i o n of the r a d i c a l p a i r of E q u a t i o n 7.

It is this q u e s t i o n of

the

nature of the intermediates i n v o l v e d i n the f o r m a t i o n of a m i d e a n d f a t t y a c i d that w e n o w consider.


27.

Peptide

GARRISON E T A L .

389

Radiolysis

T h e i n i t i a l r a d i a t i o n - i n d u c e d step w e represented i n terms of the ionization RCONHCHR

2

—

M

— » (RCONHCHR ) 2

+

+ e~

(10)

S i m p l e charge r e c o m b i n a t i o n m a y b e e n v i s a g e d as l e a d i n g to d i s s o c i a t i o n of the N - C b o n d e~ + ( R C O N H C H R ) -> R C O N H * + C H R +

2

(11)

2

to g i v e r a d i c a l s w i t h excess energy w h i c h w o u l d t e n d to f a v o r the abstrac t i o n Reactions 8 a n d 9 i n c o m p e t i t i o n w i t h r a d i c a l r e c o m b i n a t i o n .

Alter

n a t i v e l y , the i o n - m o l e c u l e r e a c t i o n (RCONHCHR ) 2

+

+ RCONHCHR - » (RCONHoCH CHR ) 2

2

2

+

+ RCONHCR (12)

w h i c h is of the t y p e o b s e r v e d i n other p o l a r o r g a n i c systems

(16,

2

21)

m a y o c c u r p r i o r to the n e u t r a l i z a t i o n e~ + ( R C O N H C H R ) 2

RCONH + CHR

2

2

(13)

2

T h e other p o s s i b i l i t y of course is that the electron escapes the p o s i t i v e charge a n d reacts at a distance—e.g., e~ + ( R C O N H C H C H R ) -> R C O N H " + C H R 2

2

2

+

(14)

2

W e note that the r a d i a t i o n - i n d u c e d Step 10 f o l l o w e d b y e l e c t r o n r e m o v a l via a n y of Reactions 11, 13, a n d 14 leads to s t o i c h i o m e t r y of E q u a t i o n s 7-9. It is of interest to consider at this p o i n t the effects of a d d e d electron scavengers o n the y i e l d of m a i n - c h a i n r u p t u r e . F o r t u n a t e l y , for this p u r pose w e have b e e n able to p r e p a r e N - a c e t y l a l a n i n e i n the f o r m of a clear glassy

solid

at

room

temperature.

CH CONHCH(CH )COONa 3

3

The

glass

has

the

composition

• 2 H 0 , a n d gives p r o d u c t y i e l d s that are 2

essentially the same as those o b t a i n e d w i t h the p o l y c r y s t a l l i n e s o l i d — e.g.,

G(NH ) 3

Chloracetate

^

3.4,

G(propionic)

i o n w h i c h has

been

=

1.6,

G(acetaldehyde)

s h o w n to b e

a n effective

=

0.8

electron

scavenger e~ + R C 1 - » R + CI"

(15)

i n other p o l a r glasses ( 3 ) is s o l u b l e (as the s o d i u m salt) i n a c e t y l a l a n i n e glass w h e n p r e p a r e d as d e s c r i b e d i n the e x p e r i m e n t a l p a r t of this p a p e r . We

find

that G ( C 1 " )

increases

w i t h chloracetate

c o n c e n t r a t i o n range 1 to 10 m o l e percent

c o n c e n t r a t i o n i n the

as s h o w n i n F i g u r e 1;

the

r e c i p r o c a l - y i e l d p l o t of F i g u r e 2 gives a l i m i t i n g v a l u e of G ( C 1 " )

—

3 w h i c h v a l u e p r o v i d e s a measure of the y i e l d f o r i o n - p a i r p r o d u c t i o n via R e a c t i o n 10 i n the present system. A t the same t i m e there is b u t a s m a l l effect of a d d e d chloracetate o n G ( N H ) even u n d e r the c o n d i t i o n 3


390

RADIATION CHEMISTRY

i n w h i c h G ( C 1 " ) is m a x i m a l . T h e e v i d e n c e is t h e n that the

1

electron-

c a p t u r e reactions of t y p e 11, 13, a n d 14 d o not represent major

paths

f o r cleavage of the N - C b o n d .

Mole %

Chloracetate

Figure I . Ammonia (O) and chloride-ion (%) yields as a function of chloracetate concentration in acetylalanine glass, RCONHCH(R)COONa 2H 0 2

T h e r e w o u l d a p p e a r to be t w o r e m a i n i n g p o s s i b i l i t i e s ; ( a ) the p o s i t i v e ions f o r m e d i n R e a c t i o n 10 u n d e r g o f r a g m e n t a t i o n , for e x a m p l e (RCONHCHR ) 2

RCONHCHR

2

-> R C O N H

+ (RCONHCHR ) 2

CHR and/or (b)

+

2

+

+ RCONHCR

-f C H R ,

+

-> R C O N H

(16) 2

+

+ (16a)

2

n e u t r a l excited species are f o r m e d t h r o u g h a process

other

t h a n that of charge r e c o m b i n a t i o n a n d s u b s e q u e n t l y u n d e r g o u n s p e c i f i e d c h e m i s t r y to y i e l d a m i d e a n d p r o p i o n i c a c i d . T o o b t a i n i n f o r m a t i o n o n the possible i m p o r t a n c e of these r e a c t i o n modes, w e h a v e t a k e n a d v a n tage of the fact that the a c e t y l a l a n i n e glass C H C O N H C H ( C H ) C O O N • a

3


a

27.

GARRISON E T A L .

Peptide

391

Radiolysis

2 H 0 is m i s c i b l e w i t h w a t e r i n a l l p r o p o r t i o n s . If d i r e c t 2

energy-absorp

t i o n i n the p e p t i d e via R e a c t i o n 10 f o l l o w e d b y the d i s s o c i a t i o n R e a c t i o n 16 is of i m p o r t a n c e , w e c o u l d expect that the d e g r a d a t i o n y i e l d s w o u l d decrease w i t h i n c r e a s i n g w a t e r content of the system.

T h e possibility

that i o n i z a t i o n i n w a t e r H 0 — —> H 0 M

2

2

+ e~

+

(17)

is f o l l o w e d b y charge transfer—e.g., H 0

+

+ R C O N H C H R -> R C O N H

H 0

+

+ R C O N H C H R -> R C O N H

2

+ CHR

+

2

2

+ H 0

(18)

+ OH

(18a)

2

or 2

2

2

+ CHR

+

2

w o u l d also b e e x c l u d e d at the h i g h e r w a t e r concentrations b y v i r t u e of the fast c o m p e t i n g R e a c t i o n 14 H 0 2

0

+

+ H 0 -> H 0 2

10

3

+

+ OH.

(19)

20

30

( A c e t y l a l a n i n e ) / (Chloracetate)

Figure 2. Reciprocal chloride-ion as a function of (acetylalanine)/(chloracetate) concentration ratio in the systems RCONHCH(R)COONa • 2H 0, (O); RCONHCH(R)COONa • 8H 0, (•) 2

2


392

RADIATION CHEMISTRY

T h e effects of a d d e d w a t e r o n G ( N H )

1

a n d G ( p r o p i o n i c ) f r o m the

3

s o d i u m salt of a c e t y l a l a n i n e are s u m m a r i z e d i n F i g u r e 3. T h e a m m o n i a y i e l d w h i c h as w e h a v e n o t e d is d e r i v e d f r o m a n u m b e r of r e a c t i o n m o d e s shows b u t a s m a l l decrease, A G ( N H ) ^

1, as the a c e t y l a l a n i n e c o n c e n

3

t r a t i o n is decreased to 1 M . A n d , e v e n m o r e s t r i k i n g is the fact that the y i e l d of the m a j o r o r g a n i c p r o d u c t , p r o p i o n i c a c i d , is essentially i n d e p e n d e n t of a c e t y l a l a n i n e c o n c e n t r a t i o n over the entire r a n g e of F i g u r e 3. O u r tentative c o n c l u s i o n is t h e n that cleavage of the N - C b o n d to y i e l d p r o p i o n i c a c i d does not arise i n the m a i n f r o m the p o s i t i v e - i o n c h e m i s t r y of R e a c t i o n s 16, 16a, 18, a n d 18a.

n

H0 2

Figure 3. Effect of increasing water content on ammonia (O) and propionic acid (%) yields from acetylalanine in the system RCONHCH(R)COONa • nH^O. Values at n = o are for polycrystalline acetylalanine, RCONHCH(R)COOH On

l o w e r i n g the

acetylalanine

concentration

f r o m 1 M to

0.1M,

G ( p r o p i o n i c ) d r o p s to zero a n d G ( N H ) decreases to 0.5; the c h e m i s t r y 3

of these d i l u t e solutions is of a different n a t u r e as w e h a v e d e s c r i b e d elsewhere (2,

10).

N o w , w e h a v e a l r e a d y n o t e d o n the basis of the chloracetate d a t a of F i g u r e 1, that e l e c t r o n c a p t u r e via

R e a c t i o n s 11, 13, a n d 14 does

not

represent a major p a t h f o r cleavage of the N - C b o n d i n the case of the a c e t y l a l a n i n e glass, C H C O N H C H ( C H ) C O O N 3

find

3

that the q u a n t i t a t i v e s c a v e n g i n g of e~

m

a

• 2H 0. 2

Similarly, we

b y chloracetate i o n i n 2 M

a c e t y l a l a n i n e s o l u t i o n has r e l a t i v e l y l i t t l e effect o n G ( N H ) as s h o w n b y 3

the d a t a of F i g u r e 4. A n d , w e also observe f r o m F i g u r e 4 that the prefer e n t i a l r e m o v a l of O H r a d i c a l s b y f o r m a t e i o n has essentially n o effect o n G ( N H ) f r o m the 2 M s o l u t i o n . 3


27.

GARRISON E T A L .

Peptide

393

Radiolysis

150 (Chloracetate)

Figure 4. Ammonia (O) and chloride-ion (%) yields as a junction of chloracetate concentration in 2M acetylalanine, pH 7. The points (U) represent ammonia yields in the presence of 0.5M and 1.0M formate ion l ~aa chloracetate, k = 1.2 X I 0 M sec.' ; e~ + acetylamine (pH 7), k = 1.1 X lO'M seer ; OH + formate, k = I 0 M " seer ; OH + acetylalanine (pH 7), k = 2.5 X W'M seer (Refs. 1, 18, 23)] 9

e

_ i 1

9

J

1

aa

1

1

1

1

B y a process o f e l i m i n a t i o n , t h e n , w e c o m e t o t h e p o s s i b i l i t y that a m i d e f o r m a t i o n i n these c o n c e n t r a t e d p e p t i d e solutions i n v o l v e s reac tions o f n e u t r a l e x c i t e d species.

N o w , a r o m a t i c c o m p o u n d s are, of course,

k n o w n t o b e effective scavengers o f e x c i t e d states p r o v i d i n g t h e e n e r g y levels of the e x c i t e d species are h i g h e r t h a n those of t h e q u e n c h e r ( 2 2 ) . We

d o find that c e r t a i n a r o m a t i c solutes s u c h as n a p h t h a l e n e s u l f o n i c

a c i d , b e n z a l d e h y d e , a n d b e n z o i c a c i d at m i l l i m o l a r concentrations a r e r e m a r k a b l y effective i n q u e n c h i n g t h e f o r m a t i o n of a m i d e a m m o n i a i n 2 M acetylalanine.

T y p i c a l data for naphthalenesulfonic acid are shown

i n F i g u r e 5. W e also

find

that p h e n o l a n d b e n z e n e s u l f o n i c a c i d a r e

essentially w i t h o u t effect e v e n at t h e h i g h e r concentrations.

T h e r e is,

of course, the p o s s i b i l i t y that the q u e n c h i n g of G ( N H ) b y n a p h t h a l e n e H

s u l f o n i c a c i d , b e n z a l d e h y d e , a n d b e n z o i c a c i d i n v o l v e s s i m p l y t h e scav e n g i n g of a n a m i d e p r e c u r s o r via r a d i c a l - a d d i t i o n t o t h e b e n z e n e

ring,

for example

C H 3 C O N H + O H -> C H 3 C O N H O H

(20)

H o w e v e r , i f this w e r e t h e case b o t h p h e n o l a n d b e n z e n e s u l f o n i c a c i d s h o u l d also b e effective.

I n fact, rates f o r r a d i c a l a d d i t i o n t o p h e n o l a r e

e v e n faster t h a n those f o r a d d i t i o n to b e n z a l d e h y d e a n d b e n z o i c a c i d ( I ) .


394

RADIATION CHEMISTRY

1

W e c a n o n l y c o n c l u d e f r o m the present observations in toto that ( a )

a

major f r a c t i o n of the N - C f r a g m e n t a t i o n i n these c o n c e n t r a t e d solutions does i n d e e d arise f r o m reactions of e x c i t e d species a n d that ( b ) reactions

c a n be

effectively q u e n c h e d

such

t h r o u g h e x c i t a t i o n transfer

to

solutes s u c h as n a p h t h a l e n e s u l f o n i c a c i d b e n z a l d e h y d e a n d b e n z o i c a c i d . T h a t b e n z e n e s u l f o n i c a c i d a n d p h e n o l are ineffective quenchers i n the present system is not inconsistent w i t h the fact that the energy levels ( s i n g l e t and

t r i p l e t ) of these t w o b e n z e n e

h i g h e r t h a n those of the effective quenchers.

d e r i v a t i v e s are

somewhat

W e note that the singlet-

state levels of a l l the aromatics s t u d i e d here ( 5 ) are w e l l b e l o w the singletstate l e v e l of the p e p t i d e b o n d (19).

H e n c e , the fact that n a p h t h a l e n e

s u l f o n i c a c i d quenches w h i l e p h e n o l does not w o u l d suggest w e are d e a l i n g w i t h a triplet-state of the p e p t i d e c o n f i g u r a t i o n . T h e r e c i p r o c a l - y i e l d p l o t of F i g u r e 5 gives G ( N H ) =

G(propionic) =

8

1.6 as the e x c i t a t i o n

y i e l d i n 2 M acetylalanine. E x c i t a t i o n of the p e p t i d e b o n d b y l o w - e n e r g y electrons e + RCONHCHR w o u l d be

consistent

w i t h the

via

RCONHCHR/ + e

2

present

(21)

experimental requirements.

A

t h e o r e t i c a l treatment of this m o d e of e x c i t a t i o n f o r the g e n e r a l case has b e e n g i v e n b y P l a t z m a n (17).

W e envisage the c h e m i s t r y of the e x c i t e d

state b o t h i n c o n c e n t r a t e d s o l u t i o n a n d i n the s o l i d systems to b e of the form: O

O

II

R—C—N—CHR

II

R—C—N—CHR

2

2

H

(22)

:

->

o R—C—N—CHR *

H

o

R—C=N +

2

H where R C O N C H R

2

CHR

2

H

rearranges

instantaneously to g i v e the l o n g - l i v e d

r a d i c a l R C O N H C R . S i n c e the r a d i c a l p r o d u c t s of R e a c t i o n 22 are f o r m e d 2

at a distance, the effects of c a g i n g w i l l b e m i n i m a l . T h e o v e r - a l l energy r e q u i r e m e n t f o r R e a c t i o n 22 is essentially that r e q u i r e d for d i s s o c i a t i o n of the a l i p h a t i c N - C b o n d — i . e . , — 3 e.v. ( 7 ) .

W e note that b e n z o i c a c i d

w h i c h is a n effective q u e n c h e r of R C O N H C H R * has a t r i p l e t l e v e l at 2

3.4 e.v. If

(22). solid acetylalanine

nitrogen, then

the

is i r r a d i a t e d at the

p r o p i o n i c a c i d is

almost

temperature wholly

of

quenched


liquid from

27.

GARRISON E T A L .

G ( p r o p i o n i c ) 9O°K. = 2

Peptide

395

Radiolysis

1.6 to G ( p r o p i o n i c ) ° K . ^ 0.1. T h e a m m o n i a y i e l d 7 7

shows a c o r r e s p o n d i n g d r o p f r o m G ( N H ) 9 O ° K . 3

=

3.2 to G ( N H ) 77 K.

=

2

a

1.3. W e i n t e r p r e t this as e v i d e n c e that a n energy of a c t i v a t i o n is

i n v o l v e d i n R e a c t i o n 22. T h e y i e l d s of c a r b o n y l p r o d u c t s are u n c h a n g e d at t h e l o w e r t e m p e r a t u r e w i t h G ( c a r b o n y l ) 7°K. =

G(NH

7

8

)77 . = 0

K

1.3.

W e suggest that t h e c a r b o n y l p r o d u c t s arise f r o m p o s i t i v e i o n precursors

which

RCONHCHR

2

— —> (RCONHCHR)

RCONHCHR

2

— —> (RCONHCR )

M

M

2

+

+ R + e~

+

+ H + g"

(23) (24)

species u n d e r g o p r o t o n s t r i p p i n g to y i e l d t h e d e h y d r o p e p t i d e

d e r i v a t i v e s r e f e r r e d to i n E q u a t i o n s 2 a n d 3.

Figure 5. Effect of naphthalenesulfonic ammonia yields from 2M acetylalanine,

acid on pH 7

W e c o n c l u d e , t h e n , f r o m these studies of e l e m e n t a r y processes i n p e p t i d e r a d i o l y s i s that ( a ) a n u m b e r of r e a c t i o n m o d e s are of i m p o r t a n c e i n t h e r a d i o l y t i c d e g r a d a t i o n of the p e p t i d e c h a i n , ( b ) charge r e c o m b i n a t i o n does n o t a p p e a r to b e i n v o l v e d as a step i n a n y of the m a j o r r e a c t i o n sequences that l e a d to m a i n - c h a i n d e g r a d a t i o n , ( c ) t h e e l e c t r o n escapes t h e p o s i t i v e i o n a n d c a n b e c h e m i c a l l y t r a p p e d b y a p p r o p r i a t e e l e c t r o n scavengers,

( d ) p o s i t i v e - i o n c h e m i s t r y leads to t h e f o r m a t i o n of

c a r b o n y l p r o d u c t s , ( e ) n e u t r a l e x c i t e d species a p p e a r to b e major i n t e r mediates i n t h e r a d i a t i o n - i n d u c e d cleavage

of the N - C b o n d to y i e l d

a m i d e a n d f a t t y a c i d . A l t h o u g h this is n o t the p l a c e to speculate o n t h e r a d i a t i o n - b i o l o g i c a l i m p l i c a t i o n s of p a r t i c u l a r s

(a)

to ( e ) , w e w o u l d ,

nevertheless, p o i n t o u t that finding ( e ) suggests that e x c i t a t i o n scavengers


396

RADIATION CHEMISTRY

1

as w e l l as r a d i c a l scavengers c a n b e of i m p o r t a n c e i n m i t i g a t i n g t h e b i o l o g i c a l effects of i o n i z i n g r a d i a t i o n s .

Literature Cited (1) Anbar, M., Neta, P., Intern. J. Appl. Radiation Isotopes 17, 493 (1967). (2) Atkins, H. L., Bennett-Corniea, W., Garrison, W. M.,J.Phys. Chem. 71, 772 (1967). (3) Ayscough, P. B., Collins, R. G., Dainton, F. S., Nature 205, 965 (1965). (4) Box, N. C., Freund, H. G., Lilga, K., "Free Radicals in Biological Sys tems," M. Blois etal.,ed.,Academic Press, New York, Ν. Y., 1961. (5) Calvert, J. G., Pitts Jr., J. N., "Photochemistry," John Wiley and Sons, New York, Ν. Y., 1967. (6) Conway, E. J., "Microdiffusion Analysis," Crosby Lockwood and Sons, Ltd., London, 1962. (7) Cottrell, T. L., "The Strengths of Chemical Bonds," Butterworths Scien tific Publications, London, 1954. (8) Feigel, F., "Spot Tests in Organic Analysis," Elsevier Publishing Co., New York, Ν. Y., 1956. (9) Garrison, W. M., Jayko, M. E., Weeks, Β. M., Sokol, Η. Α., BennettCorniea, W., J. Phys. Chem. 71, 1546 (1967). (10) Garrison, W. M., Weeks, Β. M., Radiation Res. Suppl. 4, 148 (1964). (11) Greenstein, J. P., Winitz, M., "Chemistry of Amino Acids," John Wiley and Sons, Inc., New York, Ν. Y., 1961. (12) Hayon, Ε., Allen, A. O.,J.Phys. Chem. 65, 2181 (1961). (13) Johnson, G. R. Α., Scholes, G., Ind. Eng. Chem. 79, 217 (1954). (14) Lampe, F. W., Field, F. H., Franklin, J. L.,J.Am. Chem. Soc. 79, 6132 (1957). (15) Luce, N. E., Denice, E . C., Akerlund, F. E., Ind. Eng. Chem. 15, 365 (1943). (16) Myron, J. J., Freeman, G. R., Can. J. Chem. 43, 381 (1965). (17) Platzman, R. L., Radiation Res. 21 (1955). (18) Rogers, M. A. J., Garrison, W. M., U. S. At. Energy Commission UCRL 17886, October 1967(J.Phys. Chem. 72, 758 (1968)). (19) Saidel, L. J., Arch. Biochem. Biophys. 54, 184 (1955). (20) Weeks, B. M., Garrison, W. M., Radiation Res. 9, 291 (1958). (21) Ward, J. Α., Harrill, W. H.,J.Am. Chem. Soc. 87, 1853 (1965). (22) Wilkinson, F., Adv. Photochem. 3, 241 (1964). (23) Willix, R. L. S., Garrison, W. M., U. S. At. Energy Commission UCRL17285, November 1966 (Radiation Res. 32, 452 (1967)). RECEIVED December 26, 1967. This work was performed under the auspices of the U. S. Atomic Energy Commission.


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