Radiation Chemistry - ACS Publicationspubs.acs.org/doi/pdf/10.1021/ba-1968-0081.ch027SimilarSeveral different reaction m...
0 downloads
123 Views
1MB Size
27 Ionization and Excitation in Peptide Downloaded via TUFTS UNIV on July 10, 2018 at 23:46:40 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
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
Hart; Radiation Chemistry Advances in Chemistry; American Chemical Society: Washington, DC, 1968.
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
Hart; Radiation Chemistry Advances in Chemistry; American Chemical Society: Washington, DC, 1968.
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.
Hart; Radiation Chemistry Advances in Chemistry; American Chemical Society: Washington, DC, 1968.
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
Hart; Radiation Chemistry Advances in Chemistry; American Chemical Society: Washington, DC, 1968.
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
Hart; Radiation Chemistry Advances in Chemistry; American Chemical Society: Washington, DC, 1968.
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
Hart; Radiation Chemistry Advances in Chemistry; American Chemical Society: Washington, DC, 1968.
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
Hart; Radiation Chemistry Advances in Chemistry; American Chemical Society: Washington, DC, 1968.
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 ) .
Hart; Radiation Chemistry Advances in Chemistry; American Chemical Society: Washington, DC, 1968.
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
Hart; Radiation Chemistry Advances in Chemistry; American Chemical Society: Washington, DC, 1968.
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
Hart; Radiation Chemistry Advances in Chemistry; American Chemical Society: Washington, DC, 1968.
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.
Hart; Radiation Chemistry Advances in Chemistry; American Chemical Society: Washington, DC, 1968.
