Bonini Marcelo G, Fernandes Denise C, Augusto Ohara
Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, CP 26077, CEP 05513-970, São Paulo, SP, Brazil.
Biochemistry. 2004 Jan 20;43(2):344-51. doi: 10.1021/bi035606p.
The peroxidase activity of Cu,Zn-superoxide dismutase (Cu,Zn-SOD) has been extensively studied in recent years due to its potential relationship to familial amyotrophic lateral sclerosis. The mechanism by which Cu,Zn-SOD/hydrogen peroxide/bicarbonate is able to oxidize substrates has been proposed to be dependent on an oxidant whose nature, diffusible carbonate radical anion or enzyme-bound peroxycarbonate, remains debatable. One possibility to distinguish these species is to examine whether protein targets are oxidized to protein radicals. Here, we used EPR methodologies to study bovine serum albumin (BSA) oxidation by Cu,Zn-SOD/hydrogen peroxide in the absence and presence of bicarbonate or nitrite. The results showed that BSA oxidation in the presence of bicarbonate or nitrite at pH 7.4 produced mainly solvent-exposed and -unexposed BSA-tyrosyl radicals, respectively. Production of the latter was shown to be preceded by BSA-cysteinyl radical formation. The results also showed that hydrogen peroxide/bicarbonate extensively oxidized BSA-cysteine to the corresponding sulfenic acid even in the absence of Cu,Zn-SOD. Thus, our studies support the idea that peroxycarbonate acts as a two-electron oxidant and may be an important biological mediator. Overall, the results prove the diffusible and radical nature of the oxidants produced during the peroxidase activity of Cu,Zn-SOD in the presence of bicarbonate or nitrite.
近年来，由于铜锌超氧化物歧化酶（Cu,Zn-SOD）的过氧化物酶活性与家族性肌萎缩侧索硬化症可能存在关联，因此受到了广泛研究。有人提出，Cu,Zn-SOD/过氧化氢/碳酸氢盐氧化底物的机制取决于一种氧化剂，但其性质，即可扩散的碳酸根自由基阴离子或酶结合的过氧碳酸盐，仍存在争议。区分这些物质的一种可能性是检查蛋白质靶点是否被氧化成蛋白质自由基。在此，我们使用电子顺磁共振（EPR）方法研究了在不存在和存在碳酸氢盐或亚硝酸盐的情况下，Cu,Zn-SOD/过氧化氢对牛血清白蛋白（BSA）的氧化作用。结果表明，在pH 7.4时，存在碳酸氢盐或亚硝酸盐时BSA的氧化分别主要产生溶剂暴露和未暴露的BSA-酪氨酸自由基。结果表明，后者的产生之前会形成BSA-半胱氨酸自由基。结果还表明，即使在没有Cu,Zn-SOD的情况下，过氧化氢/碳酸氢盐也会将BSA-半胱氨酸广泛氧化为相应的亚磺酸。因此，我们的研究支持过氧碳酸盐作为双电子氧化剂起作用且可能是一种重要生物介质的观点。总体而言，结果证明了在存在碳酸氢盐或亚硝酸盐的情况下，Cu,Zn-SOD过氧化物酶活性过程中产生的氧化剂具有可扩散性和自由基性质。
