Bolann B J, Henriksen H, Ulvik R J
Laboratory of Clinical Biochemistry, University of Bergen, Haukeland Hospital, Norway.
Biochim Biophys Acta. 1992 Dec 8;1156(1):27-33. doi: 10.1016/0304-4165(92)90091-8.
The kinetic behaviour of O2.- during spontaneous dismutation and in the presence of Cu,Zn-superoxide dismutase and other compounds, was studied by monitoring the decrease in absorbance (A250nm-A360nm) on a time-scale of > or = 1 min, at pH 9.5. O2.- was generated from KO2, and calculations were performed between 25 and 4 microM of O2.-. An algorithm for the simultaneous calculation of the 1st and 2nd-order rate constants from the decay curve, was evolved. The respective fractions of O2.- which interacted with catalysts or disappeared spontaneously, in various experimental situations, could be estimated. Substances could be classified as inert, catalysts or scavengers. The high assay pH excluded examination of the effect of alkali sensitive substances, e.g., Mn-superoxide dismutase. However, the high pH minimized the interfering effect of trace amounts of Cu(II). Therefore a metal chelator was superfluous and even the effect of metals and metal complexes could be tested. The extremely high sensitivity of the method allowed minute concentrations of reagents to be used, including proteins absorbing in the UV-region. The rate constants found by this simple method, agreed with those obtained by more sophisticated and inaccessible techniques like pulse radiolysis and stopped-flow spectrophotometry.
在pH 9.5条件下，通过监测吸光度下降（A250nm - A360nm），在≥1分钟的时间尺度上研究了超氧阴离子（O2.-）在自发歧化过程中以及在存在铜锌超氧化物歧化酶和其他化合物时的动力学行为。O2.-由超氧化钾生成，在25至4 microM的O2.-浓度范围内进行计算。开发了一种从衰变曲线同时计算一级和二级速率常数的算法。可以估计在各种实验情况下，与催化剂相互作用或自发消失的O2.-的各自比例。物质可分为惰性、催化或清除剂。高测定pH排除了对碱敏感物质（如锰超氧化物歧化酶）作用的研究。然而，高pH使痕量铜（II）的干扰作用最小化。因此，金属螯合剂是多余的，甚至可以测试金属和金属配合物的作用。该方法的极高灵敏度允许使用微量试剂，包括在紫外区域有吸收的蛋白质。通过这种简单方法获得的速率常数与通过脉冲辐射分解和停流分光光度法等更复杂且难以实现的技术获得的速率常数一致。
