Mueller S, Riedel H D, Stremmel W
Department of Internal Medicine IV, University of Heidelberg, Germany.
Anal Biochem. 1997 Feb 1;245(1):55-60. doi: 10.1006/abio.1996.9939.
A method for the determination of catalase activity (EC 1.11.1.6.) in homogenates and cell suspensions is described by following the decomposition of H2O2 at physiological H2O2 levels. This first chemiluminescence assay for catalase activity is based on the reaction of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) and NaOCl. The chemiluminescence of this reaction specifically depends on the H2O2 concentration and shows fast kinetics of less than 2 s. Using a flow technique, the exponential decay of H2O2 in the presence of catalase is followed down to 10(-8) M H2O2 at pH 7.4 over three orders of magnitude. At these very low H2O2 concentrations neither oxygen is liberated in gaseous form nor enzyme inactivation or loss of cell viability is observed. Addition of the catalase inhibitor NaN3 completely inhibits H2O2 decomposition. Since the method is not influenced by sulfhydryl and amino group containing compounds, it is especially suited for crude tissue homogenates and suspensions of intact cells. Interestingly, application to cell suspensions shows that intact human erythrocytes and rat hepatocytes exhibit only 5.8 and 1.9% of catalase activity when compared to homogenized cells. These data suggest that the diffusion of H2O2 through membranes is lower than that assumed so far.
本文描述了一种通过跟踪生理过氧化氢水平下过氧化氢的分解来测定匀浆和细胞悬液中过氧化氢酶活性（EC 1.11.1.6.）的方法。这种用于过氧化氢酶活性的首次化学发光测定基于鲁米诺（5-氨基-2,3-二氢-1,4-酞嗪二酮）与次氯酸钠的反应。该反应的化学发光特别取决于过氧化氢浓度，并且显示出小于2秒的快速动力学。使用流动技术，在pH 7.4条件下，在过氧化氢酶存在下过氧化氢的指数衰减可跟踪至10^(-8) M过氧化氢，跨越三个数量级。在这些非常低的过氧化氢浓度下，既没有以气态形式释放氧气，也没有观察到酶失活或细胞活力丧失。添加过氧化氢酶抑制剂叠氮化钠可完全抑制过氧化氢的分解。由于该方法不受含巯基和氨基化合物的影响，因此特别适用于粗组织匀浆和完整细胞悬液。有趣的是，应用于细胞悬液时发现，与匀浆细胞相比，完整的人类红细胞和大鼠肝细胞仅表现出5.8%和1.9%的过氧化氢酶活性。这些数据表明，过氧化氢通过膜的扩散比迄今为止假设的要低。
