Redox equilibrium between guanyl radicals and thiocyanate influences base damage yields in gamma irradiated plasmid DNA. Estimation of the reduction potential of guanyl radicals in plasmid DNA in aqueous solution at physiological ionic strength
Jr. Milligan et al., Redox equilibrium between guanyl radicals and thiocyanate influences base damage yields in gamma irradiated plasmid DNA. Estimation of the reduction potential of guanyl radicals in plasmid DNA in aqueous solution at physiological ionic strength, INT J RAD B, 77(12), 2001, pp. 1195-1205
Purpose: Gamma irradiation of an aqueous solution containing thiocyanate io
ns produces the strongly oxidizing intermediate (SCN)(2)(.-) Reaction of th
is species with plasmid DNA produces damage that is revealed as strand brea
ks after incubation with the Escherichia coli base excision repair endonucl
ease formamidopyrimidine- DNA N-glycosylase (FPG). It has been previously r
eported that the yield of damage is highly sensitive to the experimental co
nditions, leading to the suspicion that electron transfer between DNA and (
SCN)(2)(.-) is reversible. In principle this makes it possible to determine
the oxidation potential for plasmid DNA (more formally the reduction poten
tial of one-electron oxidized plasmid DNA), a fundamental parameter describ
ing the reactivity of DNA towards electron transfer reactions.
Materials and methods: Aqueous solutions of plasmid DNA and thiocyanate ion
s were subjected to Cs-137 gamma -irradiation. After irradiation, the plasm
id was incubated with the E. coli base excision repair endonuclease formami
dopyrimidine-DNA N-glycosylase (FPG). The yield of this damage was quantifi
ed by using agarose gel electrophoresis to identify the fraction of the pla
smid population that contains strand breaks.
Results: The yield of FPG-sensitive sites decreases with increasing thiocya
nate concentration, decreasing DNA concentration, and increasing dose rate.
By making some simple assumptions about the chemical reactions that produc
e DNA damage, it is possible to derive a quantitative mathematical model fo
r the yield of FPG-sensitive sites. A good agreement was found between this
model and the experimental observations over a wide range of conditions (t
hiocyanate concentrations, DNA concentrations, and dose rates that vary by
20-, 40-, and 150-fold respectively).
Conclusions : It was possible to assign a value to the equilibrium constant
for the one electron transfer reaction between the two radical species (SC
N)(2)(.-) and DNA-GV(.+). This leads to an estimate of the reduction potent
ial at pH 7 for the couple DNA-G(.+)/DNA of E-7 = +1.39+/-0.01 V.