Favaudon V, Tourbez H, Houée-Levin C, Lhoste J M
Unité 219 INSERM, Institut Curie-Biologie, Centre Universitaire, Orsay, France.
Biochemistry. 1990 Dec 11;29(49):10978-89. doi: 10.1021/bi00501a016.
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 gamma-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, [symbol: see text], appeared to be the major protein radical species formed under acidic conditions. The main decay path of [symbol: see text] consisted of the rapid formation of a thiyl radical intermediate [symbol: see text] in equilibrium with the closed, cyclic form. The thiyl radical was subsequently reduced to the sulfhydryl level [symbol: see text] on reaction with formate, generating 1 mol of the CO2.- radical, thus propagating the chain reaction. The disulfide radical anion [symbol: see text] 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 [symbol: see text], 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.
研究了CO₂⁻自由基对脱辅基羧菌素、脱辅基核黄素结合蛋白和牛免疫球蛋白中二硫键的还原作用。在γ射线辐照下，在pH和蛋白质浓度依赖性链反应过程中形成了与蛋白质结合的半胱氨酸游离硫醇。随着培养基酸化，链效率增加，表观pKa约为5，在pH 3.6以下急剧下降。在中性pH下，随着半胱氨酸积累，链效率也会下降。通过脉冲辐解分析表明，CO₂⁻除了能向暴露的二硫键提供单电子外，还能诱导硫醇和酪氨酸酚基的快速单电子氧化。天然蛋白质对CO₂⁻的总体摄取速率常数在pH 3时比在pH 8时快5至10倍，二硫自由基阴离子的质子化形式[符号：见原文]似乎是在酸性条件下形成的主要蛋白质自由基物种。[符号：见原文]的主要衰变途径包括快速形成与闭环形式处于平衡的硫自由基中间体[符号：见原文]。硫自由基随后在与甲酸反应时被还原为巯基水平[符号：见原文]，生成1摩尔CO₂⁻自由基，从而使链反应得以传播。pH 8时的二硫自由基阴离子[符号：见原文]通过竞争性的分子内和/或分子间途径衰变，包括歧化、蛋白质-蛋白质交联、与酪氨酸残基的电子转移以及与预还原系统中巯基的反应。仅在核黄素结合蛋白中观察到歧化和交联。为了解释以氧为中心的苯氧自由基的归宿，还应考虑二硫自由基阳离子[符号：见原文]、苯氧自由基Tyr-O·歧化以及苯氧自由基诱导的预先形成的硫醇基团氧化。
