Cascading transfers of free radical centres, involving sulphur and aromatic protein functions, have been studied in further detail. The disulphide radical anion appears to be an important terminus of both oxidative and reductive radical transfer. In deaerated solutions of cysteine (20 mmol dm-3) the yield of Cys2/SS.- closely resembles the yield of all primary free radicals generated by water radiolysis (.OH, H. and eaq-). The alanyl Ala/C beta., formed by electron addition to cysteine and subsequent SH- elimination, oxidizes cysteine with a rate constant of k8 = 5.0 x 10(6)dm3mol-1s-1 at pH 6 to 7 and 3.6 x 10(6)dm3mol-1s-1 at pH 9 to 10. In the case of glutathione (GSH) the eaq--induced carbon-centred radical oxidizes the parent thiol with rate constants k(G. + GSH) of 7.0 x 10(6) and 1.3 x 10(6)dm3mol-1s-1 at pH 8 and pH 10, respectively; and with dithiothreitol (D(SH)2) the corresponding reaction rate is k(.DSH + D(SH)2) = 5.5 x 10(6)dm3mol-1s-1 at pH 7.0. The decarboxylated methionyl Met/C. alpha, formed by reaction of .OH with methionine, is capable of electron transfer to cystine, indicating a reduction potential for decarboxylated methione more negative than -1.6 V. The ring-closed methionyl radical cation Met/SN.+, formed by reaction of .OH with Met-Gly, oxidizes azide via equilibration, Met/SN.+ + H+ + N3- in equilibrium Met + N3., which enables an estimate to be given for the one-electron reduction potential: E degrees (Met/SN.+ + H+; Met) = +1.42 +/- 0.3 V (pH 6.8). Some further reactions of oxidizing dimeric Met2/SS.+ species in neutral solution have been demonstrated. The direction and nature of the transfers can be expressed by the scheme: (formula; see text).
The fluorescent intercalation complex of ethidium bromide with DNA was used as a probe to demonstrate damage in the base-pair region of DNA, due to the action of superoxide radicals. The O.2- radical itself, generated by gamma-radiolysis of oxygenated aqueous Na-formate solutions, is rather ineffective with respect to impairment of DNA. Copper(II) ions, known to interact with DNA by coordinate binding at purines, enhance the damaging effect of O.2-. Addition of H2O2 to the DNA/Cu(II) system gives rise to further enhancement, so that DNA impairment by O.2- becomes comparable to that initiated by .OH radicals. These results suggest that the modified, Cu(II)-catalysed, Haber-Weiss process transforms O.2- into .OH radicals directly at the target molecule, DNA-Cu2+ + O.2-----DNA-Cu+ + O2 DNA-Cu+ + H2O2----DNA...OH + Cu2+ + OH- in a "site-specific" mechanism as proposed for other systems (Samuni et al. 1981; Aronovitch et al. 1984). Slow DNA decomposition also occurs without gamma-irradiation by autocatalysis of DNA/Cu(II)/H2O2 systems. In this context we observed that Cu(II) in the DNA-Cu2+ complex (unlike free Cu2+) is capable of oxidizing Fe(II) to Fe(III), thus the redox potential of the Cu2+/Cu+ couple appears to be higher than that of the Fe3+/Fe2+ couple when the ions are complexed with DNA. Metal-catalysed DNA damage by O.2- also occurs with Fe(III), but not with Ag(I) or Cd(II) ions. It was also observed that Cu(II) ions (but neither Ag(I) nor Cd(II] efficiently quench the fluorescence of the intercalation complex of ethidium bromide with DNA.
gamma-Radiolysis, with doses less than 1 kGy, of aqueous solutions of disulphides, disulphide-proteins or thiols leads to the generation of stable products, capable of stimulating the catalytic reduction of Fe(III)-cytochrome-c by unirradiated glutathione, and by other thiols. The stimulatory activity fades within 20-60 min in the case of irradiated thiols, but there was little loss of this activity when irradiated solutions of disulphides or disulphide-proteins were stored at 4 degrees C for days. Disulphides (e.g. cystamine) are mainly activated by .OH radicals, disulphide-proteins (e.g. alpha-chymotrypsin) mainly by e-aq, and thiols (e.g. cysteine) by virtually all water radicals. The radiolytic activation, which is only partially prevented by oxygen, can be attributed to the generation of trace amounts of higher sulphides and persulphides (RSSSR, RSSSSR and RSSH). Such species are known to stimulate Fe(III)-cytochrome-c reduction by glutathione in a chain reaction (Massey et al. 1971). The radiolytic stimulation of reductive catalytic activity of thiols and disulphides may play a role in irradiated biological systems, and might be exploited to identify irradiated proteins with Fe(III)-cytochrome-c as detector.
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