Mazumdar A, Chatterjee R, Adak S, Ghosh A, Mondal C, Banerjee R K
Department of Physiology, Indian Institute of Chemical Biology, Calcutta, India.
Biochem J. 1996 Mar 1;314 ( Pt 2)(Pt 2):413-9. doi: 10.1042/bj3140413.
A soluble sheep lacrimal-gland peroxidase was purified to apparent homogeneity. It had a native molecular mass of 75 kDa with a subunit molecular mass of 82 kDa and an isoelectric point of 6.5. Western blotting showed that it shares some of the enzyme antigenic determinants in common with other soluble peroxidases. The enzyme exhibits a Soret peak at 410 nm which is shifted to 431 nm by 5 equiv. of H2O2 due to the formation of compound II. The latter is, however, unstable and gradually returns to the native state. The enzyme forms complexes with CN- and N3- and is reduced by dithionite showing a characteristic reduced peroxidase spectrum. Although the enzyme oxidizes I-, SCN- and Br- optimally at pH 5.5., 5.25 and 5.0 respectively, at physiological pH, it oxidizes I- and SCN- only. Since extracellular SCN- concentration is much higher than I-, SCN- may act as the major electron donor to the enzyme. The second-order rate constants for the reaction of the enzyme with H2O2 (k+1) and of compound I with SCN- (k+2) were 4 X 10(7) M-1 X s-1 and 8.1 X 10(5) M-1 X s-1 respectively. A plot of log Vmax against pH yields a sigmoidal curve consistent with a single ionizable group on the enzyme with a pK(a) value of 5.75, controlling thiocyanate oxidation. In a coupled system with the peroxidase, H2O2, SCN-, GSH, NADPH and glutathione reductase, peroxidase-catalysed SCN- oxidation by H2O2 could be coupled to NADPH consumption. The system is proposed to operate in vivo for the efficient elimination of endogenous H2O2.
一种可溶性绵羊泪腺过氧化物酶被纯化至表观均一。其天然分子量为75 kDa，亚基分子量为82 kDa，等电点为6.5。蛋白质免疫印迹显示，它与其他可溶性过氧化物酶有一些共同的酶抗原决定簇。该酶在410 nm处有一个Soret峰，由于化合物II的形成，在加入5当量的H2O2后该峰会移至431 nm。然而，后者不稳定，会逐渐恢复到天然状态。该酶能与CN-和N3-形成复合物，并被连二亚硫酸盐还原，呈现出特征性的还原过氧化物酶光谱。尽管该酶在pH 5.5、5.25和5.0时分别对I-、SCN-和Br-有最佳氧化作用，但在生理pH下，它仅氧化I-和SCN-。由于细胞外SCN-的浓度远高于I-，SCN-可能是该酶的主要电子供体。该酶与H2O2反应的二级速率常数（k+1）以及化合物I与SCN-反应的二级速率常数（k+2）分别为4×10(7) M-1×s-1和8.1×10(5) M-1×s-1。以log Vmax对pH作图得到一条S形曲线，这与该酶上一个pK(a)值为5.75的单一可电离基团一致，该基团控制着硫氰酸盐的氧化。在一个与过氧化物酶、H2O2、SCN-、谷胱甘肽（GSH）、烟酰胺腺嘌呤二核苷酸磷酸（NADPH）和谷胱甘肽还原酶组成的偶联体系中，过氧化物酶催化的H2O2对SCN-的氧化可以与NADPH的消耗相偶联。该体系被认为在体内发挥作用，以有效消除内源性H2O2。
