25 Steady State and Pulse Radiolysis of Aqueous Chloride Solutions of Nucleic
Downloaded by CORNELL UNIV on June 16, 2017 | http://pubs.acs.org Publication Date: January 1, 1968 | doi: 10.1021/ba-1968-0081.ch025
Acid Components J. F. WARD and I. KUO Laboratory of Nuclear Medicine and Radiation Biology of the Department of Biophysics and Nuclear Medicine, School of Medicine, University of California, Los Angeles, Calif.
The pulse radiolysis technique has been used to measure absolute rate constants for reactions of some nucleic acid constituents with Cl2 radicals (the species produced by reaction of O H radicals with chloride ions in acid aqueous solution). The rate of disappearance of the Cl2 absorption spectrum was measured in the absence and presence of the various solutes. Rate constants for the corresponding O H radical reactions are found to be 20 to 200 times greater than the rate constants for the Cl2 radical reactions. Steady state radiolysis showed that in some cases the radicals pro duced by reaction of these compounds with Cl2 radicals differ in their subsequent reaction from the corresponding O H radical adduct. -
-
-
-
T T y d r o x y l free r a d i c a l s f o r m e d i n w a t e r r a d i o l y s i s react w i t h c h l o r i d e X X
|
o
n
s
j
n
a c
j j s o l u t i o n ( I , 9 ) , to give a transient p r o d u c t w h i c h has (
b e e n i d e n t i f i e d as the C L " i o n r a d i c a l ( 1 ) . T h i s species differs f r o m the O H free r a d i c a l i n that it reacts r a p i d l y w i t h t h y m i n e b u t n o t w i t h e t h y l alcohol.
Previous work
( 9 ) u s e d this difference
to d e t e r m i n e
a rate
constant f o r the r e a c t i o n of O H r a d i c a l s w i t h c h l o r i d e ions ( r e l a t i v e to the rate constant f o r the r e a c t i o n of O H r a d i c a l s w i t h t h y m i n e ) .
Subse
q u e n t w o r k ( 8 ) has s h o w n that the presence of c h l o r i d e ions d u r i n g i r r a d i a t i o n of other p y r i m i d i n e s i n o x y g e n a t e d ,
a c i d , aqueous
solution
leads to a m a r k e d d e p a r t u r e f r o m the results o b t a i n e d i n t h e absence of c h l o r i d e — i . e . , c h l o r i d e ions increase the extent of u r a c i l a n d of cytosine d e s t r u c t i o n u p to s i x - f o l d , b u t decrease G ( - b a s e ) f o r p u r i n e s , nucleosides, 368 Hart; Radiation Chemistry Advances in Chemistry; American Chemical Society: Washington, DC, 1968.
25.
WARD AND KUO
Nucleic
Acid
369
Components
a n d nucleotides. T h e s e changes i n y i e l d s s h o w a p H d e p e n d e n c e w h i c h closely f o l l o w s that c a l c u l a t e d for C l ~ f o r m a t i o n . It seems that r e a c t i o n 2
of C l ~ w i t h the p y r i m i d i n e or p u r i n e is i m p o r t a n t i n d e t e r m i n i n g the 2
extent of base d e s t r u c t i o n . T h e present i n v e s t i g a t i o n was u n d e r t a k e n to d e t e r m i n e the rates of r e a c t i o n of some n u c l e i c a c i d d e r i v a t i v e s w i t h C l ~ 2
b y p u l s e r a d i o l y s i s a n d to attempt to correlate these w i t h o b s e r v e d steady state r a d i o l y s i s results.
Downloaded by CORNELL UNIV on June 16, 2017 | http://pubs.acs.org Publication Date: January 1, 1968 | doi: 10.1021/ba-1968-0081.ch025
Experimental P u l s e r a d i o l y s i s experiments w e r e c a r r i e d out i n c o o p e r a t i o n w i t h L . M . T h e a r d at G u l f G e n e r a l A t o m i c , Inc., S a n D i e g o , C a l i f . , u s i n g apparatus p r e v i o u s l y d e s c r i b e d ( 7 ) . T y p i c a l l y a 10-nsec. p u l s e g i v i n g a dose of 2 0 0 - 3 0 0 rads w a s u s e d . T h e f o r m a t i o n a n d d e c a y of the C l ~ transient was f o l l o w e d at 360 n . m . T h e e x t i n c t i o n coefficient of this species ( I ) at 360 n . m . is m u c h h i g h e r t h a n those q u o t e d f o r p y r i m i d i n e a n d p u r i n e transients ( 5 ) . Results o b t a i n e d u s i n g other w a v e l e n g t h s of the C l " a b s o r p t i o n s p e c t r u m , s h o w e d close agreement w i t h those o b t a i n e d at 360 n . m . y - I r r a d i a t i o n s w e r e c a r r i e d out i n a C o source at a dose rate of 6 X 10 e.v. m l . m i n . " ( c a l i b r a t e d b y ferrous sulfate d o s i m e t r y ) . G values ( G = n u m b e r of molecules c h a n g e d p e r 100 e.v. a b s o r b e d ) w e r e c a l c u l a t e d f r o m y i e l d dose plots. Solutions f o r y - i r r a d i a t i o n w e r e satu rated w i t h oxygen. M a t e r i a l s . A l l n u c l e i c a c i d c h e m i c a l s w e r e A grade ( C a l b i o c h e m ) i r r a d i a t e d d i s s o l v e d i n t r i p l y d i s t i l l e d water. O t h e r c h e m i c a l s w e r e re agent grade. F o r p u l s e r a d i o l y s i s , solutions w e r e e q u i l i b r a t e d w i t h the r e q u i r e d gas b e f o r e i n t r o d u c t i o n to the r a d i a t i o n c e l l , the same gas b e i n g u s e d to d r i v e the s o l u t i o n i n the s a m p l e c h a n g i n g arrangement. U n l e s s o t h e r w i s e stated a r g o n was u s e d . 2
2
6 0
16
- 1
Results and
1
Discussion
P r e v i o u s w o r k (1,9)
has s h o w n that the r e a c t i o n of O H free r a d i c a l s
w i t h c h l o r i d e ions shows a
first
order d e p e n d e n c e
on hydrogen ion
concentration and chloride ion concentration. O H ' + CI" + H 0 3
+
-» Cr + 2H 0
(1)
2
( T h e c h l o r i n e a t o m a p p a r e n t l y reacts i m m e d i a t e l y w i t h a c h l o r i d e i o n to g i v e C l " i n a r e a c t i o n w h i c h is not rate l i m i t i n g ( 1 ) . ) 2
T h e t h i r d order
r e a c t i o n rate constant for R e a c t i o n 1 o b t a i n e d i n this l a b o r a t o r y u s i n g a steady state m e t h o d is o n l y h a l f that f o u n d b y A n b a r a n d T h o m a s u s i n g p u l s e r a d i o l y s i s (1).
U s i n g p u l s e r a d i o l y s i s w e o b t a i n results close
those of the latter w o r k e r s .
to
T h e rate w e o b t a i n e d for R e a c t i o n 1 w a s
1.5 ± 0.3 X 1 0 M " sec." . W e f o u n d no consistent v a r i a t i o n of this v a l u e 1 0
2
1
w i t h c h a n g i n g p H (0.8 to 3.4) to 1 0 " ) . 1
T h e rate constant
or c h a n g i n g c h l o r i d e m o l a r i t y ( 3 X
10"
4
d e t e r m i n e d i n the steady state w o r k w a s
Hart; Radiation Chemistry Advances in Chemistry; American Chemical Society: Washington, DC, 1968.
370
RADIATION
CHEMISTRY-
o b t a i n e d f r o m a m e a s u r e d r e l a t i v e rate constant, u s i n g a p u b l i s h e d rate constant as a reference. T h e m e t h o d u s e d i n d e t e r m i n i n g rate constants for C l " reactions is 2
essentially the same as that u s e d to d e t e r m i n e h y d r a t e d e l e c t r o n rate constants: T h e rate of d i s a p p e a r a n c e of the C l ~ a b s o r p t i o n w a s m e a s u r e d 2
i n the presence of v a r i o u s concentrations of solute.
T h e d e c a y of C l ~ 2
i n the absence of r e a c t i n g solute seemed to be second order.
Increasing
the dose to 850 rads a n d r e m o v i n g h y d r a t e d electrons b y saturation of the s o l u t i o n w i t h nitrous o x i d e at p H 3.1 gave a g o o d second o r d e r p l o t for C l " decay. T h i s suggests that C l " is d e c a y i n g b y R e a c t i o n 2 : Downloaded by CORNELL UNIV on June 16, 2017 | http://pubs.acs.org Publication Date: January 1, 1968 | doi: 10.1021/ba-1968-0081.ch025
2
2
C l " + C l " -> (2C1- + C l ) 2
2
(2)
2
T h e rate constant for this r e a c t i o n was f o u n d to be 1.4 ± sec."
=
1
Reference
1).
c e n t l y m e a s u r e d rate constants 2k
2
M
_ 1
L a n g m u i r and H a y o n (3)
h a v e re
of C l " reactions b y a flash p h o t o l y s i s 2
T h e y m e a s u r e d the rate constant for R e a c t i o n 2 a n d f o u n d
1.25 — 1.51
=
1 0
It was also s h o w n that this d e c a y rate was
i n d e p e n d e n t of p H f r o m 0.9 to 3.2. method.
10
2
2
10,000 (see
0.3 X
( T h e e x t i n c t i o n coefficient used for C l " at 360 n . m . was
2k
X
10
1 ( )
M
_ 1
sec." , w h i c h is i n g o o d agreement 1
with
the v a l u e o b t a i n e d here. A l l of the c o m p o u n d s e x a m i n e d for r e a c t i o n w i t h C l " react r a p i d l y 2
w i t h O H free r a d i c a l s ( 5 ) . sec."
T h u s , rate constants l o w e r t h a n 5 X
c o u l d not be d e t e c t e d :
1
10 M 6
I n c r e a s i n g the solute c o n c e n t r a t i o n
c i e n t l y to c o m p e t e w i t h R e a c t i o n 2 for the C l ~ , scavenges
_ 1
suffi
O H free
2
r a d i c a l s f r o m r e a c t i n g to f o r m C l ~ . 2
C h l o r i d e ions are u s e d i n the F r i c k e dosimeter as h y d r o x y l r a d i c a l scavengers (2).
Since a d d i n g c h l o r i d e ions does not decrease the y i e l d of
f e r r i c i o n ( e x c e p t i n the presence
of o r g a n i c i m p u r i t i e s ) , it c a n
be
a r g u e d that c h l o r i n e atoms a n d hence C l " r a d i c a l ions react to o x i d i z e 2
ferrous i o n . U s i n g the present t e c h n i q u e w e h a v e m e a s u r e d the effect of ferrous ions o n the rate of d e c a y of the C l " transient. 2
F e r r o u s ions
i n c r e a s e d this rate, a n d a rate constant for R e a c t i o n 3 was d e t e r m i n e d fc = 3
3.8 ±
0.3 X 1 0 W "
1
Fe
sec." at p H 2.1 1
2 +
+ C l " -> F e 2
3 +
+ 2C1"
T h e effect of salt c o n c e n t r a t i o n ( s o d i u m p e r c h l o r a t e ) of C l " transient i n the presence of 1 0 " M F e 3
2
2 +
(3) o n the d e c a y rate
was e x a m i n e d . A m a r k e d
salt effect was o b s e r v e d , the p s e u d o first o r d e r rate constant b e i n g r e d u c e d f r o m 3.8 X
10
strength 0.25.
4
sec." at i o n i c strength 0.04, to 2.5 X 1
10
4
sec.
-1
at i o n i c
R e a c t i o n 3 w o u l d be e x p e c t e d to s h o w a negative
salt
effect, b u t the o b s e r v e d decrease i n rate constant w i t h i n c r e a s i n g i o n i c strength is s o m e w h a t less t h a n e x p e c t e d .
Hart; Radiation Chemistry Advances in Chemistry; American Chemical Society: Washington, DC, 1968.
25.
WARD AND KUO
Nucleic
Acid
371
Components
T a b l e I shows rate constants for r e a c t i o n of some n u c l e i c a c i d c o m ponents w i t h C l ~ a n d w i t h O H radicals. 2
m i n e d b y Scholes et al. (5).
T h e latter are the data deter
A l s o s h o w n are our results f r o m steady state
w o r k i n w h i c h the G - v a l u e for base d e s t r u c t i o n , G ( - b a s e ) , was m e a s u r e d for i r r a d i a t i o n s c a r r i e d out i n the presence a n d i n the absence of
10 M l
s o d i u m c h l o r i d e at p H 2.7. Table I. Second Order Rate Constants for Reactions of C l ~ with Pyrimidines, Purines and Deoxynucleotides at p H 2.7 in 1 0 M Sodium Chloride Solution 2
Downloaded by CORNELL UNIV on June 16, 2017 | http://pubs.acs.org Publication Date: January 1, 1968 | doi: 10.1021/ba-1968-0081.ch025
_1
Rate Constant X lO'M seer 1
Cli
+ Base
Steady State Value G(-Base)
1
OH + Base
With Cl~
a
Without
PYRIMIDINES Thymine Uracil Cytosine
12 ± 1.0 4.1 ± 0.3 9.1 ± 0.7
310 290 185
2.4 9.5 5.6
2.4 2.6 2.7
PURINES Adenine Guanine
