CO2.- RADICAL INDUCED CLEAVAGE OF DISULFIDE BONDS IN PROTEINS - A GAMMA-RAY AND PULSE-RADIOLYSIS MECHANISTIC INVESTIGATION

Disulfide bond reduction by the CO2 •− radical was investigated in aponeocarzinostatin, aporiboflavin-binding protein, and bovine immunoglobulin. Protein-bound cysteine free thiols were formed under γ-ray irradiation in the course of a pH-dependent and protein concentration dependent chain reaction. The chain efficiency increased upon acidification of the medium, with an apparent pKa around 5, and decreased abruptly below pH 3.6. It decreased also at neutral pH as cysteine accumulated. From pulse radiolysis analysis, CO2 •− proved able to induce rapid one-electron oxidation of thiols and of tyrosine phenolic groups in addition to one-electron donation to exposed disulfide bonds. The bulk rate constant of CO2 •− uptake by the native proteins was 5- to 10-fold faster at pH 3 than at pH 8, and the protonated form of the disulfide radical anion, [formula omitted], appeared to be the major protein radical species formed under acidic conditions. The main decay path of [formula omitted] consisted of the rapid formation of a thiyl radical intermediate [formula omitted] in equilibrium with the closed, cyclic form. The thiyl radical was subsequently reduced to the sulfhydryl level SH HS on reaction with formate, generating 1 mol of the CO2 •− radical, thus propagating the chain reaction. The disulfide radical anion [formula omitted] at pH 8 decayed through competing intramolecular and/or intermolecular routes including disproportionation, protein–protein cross-linking, electron transfer with tyrosine residues, and reaction with sulfhydryl groups in prereduced systems. Disproportionation and cross-linking were observed with the riboflavin-binding protein solely. Formation of the disulfide radical cation [formula omitted], phenoxyl radical Tyr-O• disproportionation, and phenoxyl radical induced oxidation of preformed thiol groups should also be taken into consideration to explain the fate of the oxygen-centered phenoxyl radical. © 1990, American Chemical Society. All rights reserved.