24 Pulse Radiolysis of D N A and Related Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on August 29, 2015 | http://pubs.acs.org Publication Date: January 1, 1968 | doi: 10.1021/ba-1968-0081.ch024
Pyrimidine Compounds: Reactions of the OH∙ Free Radical L. S. MYERS, JR., MARY LYNN HOLLIS, and L. M. THEARD Laboratory of Nuclear Medicine and Radiation Biology, Departments of Biophysics and of Radiology, University of California, Los Angeles, Calif. 90024 and Gulf General Atomic, Inc., San Diego, Calif.
Formation, decay, and absorption spectra of transient spe cies produced by reaction of OH∙ free radicals with D N A and nucleic acid constituents have been investigated by pulse radiolysis. Dilute aqueous N O saturated solutions were exposed to 2-500 nsec. pulses (750-1700 rads/pulse) of 10 Mev. electrons, and changes in optical density were measured. The rate constant for formation of the D N A transient (nucleotide M.W. 350) is 6 X 10 M sec. . The transient persists for more than 1 msec. with no significant change in spectrum. Thymine, uracil, deoxyribose, and thymidine transients have complex decay patterns which vary with pH. Comparisons of transient spectra confirm that the sites of attack by OH∙ on thymine, 5-methylcytosine, and thymidine depend on pH. Abstraction from 5,6-dihydrothymine is slower than addition to thymine. 2
8
A
-1
-1
p u l s e r a d i o l y s i s s t u d y of t h e f o r m a t i o n , decay, a n d a b s o r p t i o n spectra of transients p r o d u c e d b y reactions of D N A a n d of some of its c o n
stituents w i t h t h e O H - free r a d i c a l is p r e s e n t e d i n this p a p e r . w e r e saturated w i t h N
2
Solutions
0 so that t h e o n l y significant reactive species p r o
d u c e d b y r a d i a t i o n w a s t h e O H - free r a d i c a l . Rates of f o r m a t i o n w e r e d e t e r m i n e d b y d i r e c t o b s e r v a t i o n of the g r o w t h of transient a b s o r p t i o n . C o m p o u n d s a n d c o n d i t i o n s w e r e selected to p e r m i t i n v e s t i g a t i o n of ( a ) the sites of attack b y O H - o n p y r i m i d i n e bases, nucleosides, a n d D N A u n d e r various c o n d i t i o n s ; ( b ) charge effects o n r e a c t i o n rates; ( c ) c o m p a r a t i v e rates of a d d i t i o n to p y r i m i d i n e bases, a n d a b s t r a c t i o n 345 In Radiation Chemistry; Hart, E.; Advances in Chemistry; American Chemical Society: Washington, DC, 1968.
from
346
RADIATION CHEMISTRY
1
saturated analogs; ( d ) c o m p a r a t i v e rates of reactions of bases, pentoses, nucleosides, a n d D N A ; a n d ( e )
the r e a c t i v i t y of D N A , a n d s t a b i l i t y of
D N A r a d i c a l s f o r m e d b y O H - reactions.
T h e results p e r m i t a n u m b e r
of significant conclusions to be d r a w n . T h e c o m p l e x i t y of m a n y of the reactions is s u c h , h o w e v e r , that a major f u n c t i o n of this p a p e r is to o u t l i n e areas w h e r e f u r t h e r w o r k is n e e d e d . Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on August 29, 2015 | http://pubs.acs.org Publication Date: January 1, 1968 | doi: 10.1021/ba-1968-0081.ch024
A n i n v e s t i g a t i o n of aerated solutions of D N A a n d n u c l e i c acids c o n stituents b y p u l s e r a d i o l y s i s has b e e n p u b l i s h e d b y Scholes, S h a w , a n d W i l l s o n (12),
a n d a m o r e extensive s t u d y has b e e n r e p o r t e d b y W i l l s o n
i n his P h . D . thesis (18).
R e c e n t r e v i e w s of r e l a t e d stationary state studies
h a v e b e e n p u b l i s h e d b y W e i s s (17)
a n d b y Scholes
(11).
Experimental T h e electron linear accelerator a n d associated a p p a r a t u s for k i n e t i c s p e c t r o s c o p y at G u l f G e n e r a l A t o m i c , Inc. h a v e b e e n d e s c r i b e d b r i e f l y (14). D e t a i l s w i l l b e p u b l i s h e d elsewhere. I n the present w o r k 10 M e v . electron pulses of 2 to 500 nsec. d u r a t i o n w e r e u s e d . R a d i a t i o n doses w e r e m e a s u r e d f o r each p u l s e b y a b e a m collector w h i c h was c a l i b r a t e d b y e l e c t r o n a b s o r p t i o n t a k i n g g(e\ ) = 2.6 a n d e ( e " ) = 10,600 at 578 n . m . T h e y r a n g e d f r o m 750 to 1700 rads/pulse, a n d d e p e n d e d o n m a c h i n e c o n f i g u r a t i o n as w e l l as p u l s e d u r a t i o n a n d current. L i g h t for the s p e c t r o p h o t o m e t r i c analyses was passed f o u r times t h r o u g h the i r r a d i a t i o n c e l l of 2 c m . p a t h l e n g t h . A x e n o n flash l a m p w a s u s e d f o r f o r m a t i o n studies; m e r c u r y - x e n o n or x e n o n arc l a m p s w e r e u s e d for spectra a n d d e c a y curves. Solutions w e r e p r e p a r e d f r o m t r i p l y d i s t i l l e d w a t e r w i t h the purest c h e m i c a l s u s u a l l y a v a i l a b l e c o m m e r c i a l l y . M e a s u r e m e n t s of p H w e r e m a d e w i t h B e c k m a n Z e r o m a t i c a n d M o d e l G p H meters, a n d electrodes suitable for a l k a l i n e solutions. Solutions w i t h p H greater t h a n 11.4 w e r e m a d e i m m e d i a t e l y p r i o r to the experiment. A l l solutions w e r e saturated w i t h M a t h e s o n N 0 ( m i n i m u m p u r i t y 9 8 % ) b y b u b b l i n g for at least 20 m i n u t e s . T h e N 0 was p r e - s a t u r a t e d w i t h w a t e r v a p o r to m i n i m i z e c o n c e n t r a t i o n changes d u r i n g b u b b l i n g . q
aq
2
2
Results and
Discussion
N a O H Solutions. S p e c t r a of transients o b s e r v e d i n the r a d i o l y s i s of N a O H solutions saturated w i t h N 0 w e r e o b t a i n e d as controls for e x p e r i 2
ments w i t h p y r i m i d i n e bases.
T h e t i m e r e q u i r e d to r e a c h m a x i m u m
a b s o r b a n c e (rise t i m e ) w a s m o r e t h a n 35 /xsec. D e c a y w a s m u c h slower. A b s o r p t i o n is n e g l i g i b l e at w a v e l e n g t h s b e t w e e n 350 a n d 450 n . m . , at p H 9.9-12.4, b u t becomes a p p r e c i a b l e at h i g h e r w a v e l e n g t h s ( O D
=
0.025 at A 575 n . m . , a n d l i g h t p a t h 8 c m . ) . T h e shape of the spectra suggests that the a b s o r p t i o n is c a u s e d b y C0 ~ (I). 3
T h i s c a n be f o r m e d b y r e a c t i o n of O H - w i t h C 0
as a n i m p u r i t y i n the N a O H .
8
2
present
Its rate of f o r m a t i o n is s l o w c o m p a r e d w i t h
In Radiation Chemistry; Hart, E.; Advances in Chemistry; American Chemical Society: Washington, DC, 1968.
24.
DNA
MYERS E T A L .
and Pyrimidine
347
Compounds
other reactions r e p o r t e d i n this p a p e r (rise times
