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

Citation
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
Citations number
48
Categorie Soggetti
Experimental Biology
Journal title
INTERNATIONAL JOURNAL OF RADIATION BIOLOGY
ISSN journal
09553002 → ACNP
Volume
77
Issue
12
Year of publication
2001
Pages
1195 - 1205
Database
ISI
SICI code
0955-3002(200112)77:12<1195:REBGRA>2.0.ZU;2-X
Abstract
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.