Jewett S L, Cushing S, Gillespie F, Smith D, Sparks S
Department of Chemistry, California State University, Northridge 91330.
Eur J Biochem. 1989 Apr 1;180(3):569-75. doi: 10.1111/j.1432-1033.1989.tb14683.x.
The reaction of hydrogen peroxide with the copper-zinc bovine-liver superoxide dismutase at low molar ratios (0.2-20.0) of H2O2/active site between pH 7.3-10.0 leads to the loss of native enzyme as a distinct form monitored by electrophoresis. The pH dependence of the loss of native enzyme between 7.3 and 9.0 indicates the involvement of a conjugate base on the enzyme of pKa of 8.7 +/- 0.1. The rate of loss of the native enzyme is first order with respect to the concentration of both enzyme and hydrogen peroxide between pH 7.3 and 9.0 with no evidence for binding of peroxide. A second-order rate constant of 3.0 +/- 1.0 M-1 s-1 is obtained from these data. At pH 10.0 the reaction is first order with respect to enzyme concentration but saturable in H2O2. All data are consistent with the interpretation that H2O2 reacts with the enzyme at the lower pH where the reaction is dependent upon the conjugate base of a functional group on the enzyme. At the higher pH, the data are consistent with the reaction of HO2- and H2O2 with the dismutase. The dissociation constant for HO2- calculated from the kinetic data at pH 10.0 is between 25-50 microM and the rate constant for the breakdown of the HO2- dismutase complex is 1.10 + 0.05 x 10(-2) s-1. The change in the electrophoretic pattern at all pH values is accompanied by the loss of the ability of the enzyme to bind copper. Weakly bound or free copper can be detected using bathocuproine disulfonate. Furthermore copper-defficient forms of the enzyme can be detected by staining gels of the peroxide-treated dismutase with diethyldithiocarbamate.
在pH 7.3 - 10.0范围内，过氧化氢与铜锌牛肝超氧化物歧化酶以低摩尔比（H₂O₂/活性位点为0.2 - 20.0）反应，会导致天然酶以一种可通过电泳监测到的独特形式丧失。在7.3至9.0之间天然酶丧失的pH依赖性表明，酶上存在pKa为8.7 ± 0.1的共轭碱参与其中。在pH 7.3至9.0之间，天然酶丧失的速率对酶和过氧化氢的浓度均为一级反应，没有过氧化物结合的证据。从这些数据获得的二级速率常数为3.0 ± 1.0 M⁻¹ s⁻¹。在pH 10.0时，反应对酶浓度为一级反应，但对H₂O₂是可饱和的。所有数据都与以下解释一致：在较低pH下H₂O₂与酶反应，此时反应取决于酶上一个官能团的共轭碱。在较高pH下，数据与HO₂⁻和H₂O₂与歧化酶的反应一致。根据pH 10.0时的动力学数据计算出的HO₂⁻的解离常数在25 - 50 μM之间，HO₂⁻ - 歧化酶复合物分解的速率常数为1.10 + 0.05 × 10⁻² s⁻¹。在所有pH值下电泳图谱的变化都伴随着酶结合铜能力的丧失。使用双磺酸 bathocuproine可以检测到弱结合或游离的铜。此外，用过氧化物处理的歧化酶的凝胶用二乙基二硫代氨基甲酸盐染色，可以检测到酶的铜缺乏形式。
