一种用于测定超氧化物反应性的闪光光度法：应用于超氧化物歧化酶测定。

A flash-photometric method for determination of reactivity of superoxide: application to superoxide dismutase assay.

作者信息

Takahashi M, Asada K

出版信息

J Biochem. 1982 Mar;91(3):889-96. doi: 10.1093/oxfordjournals.jbchem.a133777.

DOI:10.1093/oxfordjournals.jbchem.a133777

Abstract

Pulse-generation of O2- by a flash was used to determine the reactivity of O2-, O2- was produced within 10 ms by a flash of light through the excitation of FMN in the presence of N,N,N',N'-tetramethylethylenediamine and oxygen. Kinetic analysis of cytochrome c reduction by O2- generated by flash yielded the reaction rate constant between cytochrome c and O2- and the spontaneous disproportionation rate constant of O2-. We applied it for superoxide dismutase assay using a linear relation between superoxide dismutase concentration and the apparent rate constant of cytochrome c reduction by O2-. The catalytic rate constant and activation energy at pH 7.3 of bovine liver Cu,Zn-superoxide dismutase were found to be 1.75 x 10(9) M-1 . s-1 at 25 degrees C and 26.9 kJ . M-1, respectively. The kinetics of O2- decay can be also monitored at 240 nm in this flash-photometric system and gave the spontaneous disproportionation rate constant of O2- and the catalytic rate constant of superoxide dismutase.

摘要

通过闪光产生O₂⁻来测定O₂⁻的反应活性，在N,N,N',N'-四甲基乙二胺和氧气存在下，通过闪光激发黄素单核苷酸，在10毫秒内产生O₂⁻。对闪光产生的O₂⁻还原细胞色素c的动力学分析得出细胞色素c与O₂⁻之间的反应速率常数以及O₂⁻的自发歧化速率常数。我们利用超氧化物歧化酶浓度与O₂⁻还原细胞色素c的表观速率常数之间的线性关系，将其应用于超氧化物歧化酶测定。发现在25℃时，牛肝铜锌超氧化物歧化酶在pH 7.3时的催化速率常数和活化能分别为1.75×10⁹ M⁻¹·s⁻¹和26.9 kJ·M⁻¹。在该闪光光度系统中，也可以在240 nm处监测O₂⁻的衰减动力学，从而得到O₂⁻的自发歧化速率常数和超氧化物歧化酶的催化速率常数。