CHARACTERIZATION OF SHEEP LACRIMAL-GLAND PEROXIDASE AND ITS MAJOR PHYSIOLOGICAL ELECTRON-DONOR

A soluble sheep lacrimal-gland peroxidase was purified to apparent hom ogeneity. It had a native molecular mass of 75 kDa with a subunit mole cular mass of 82 kDa and an isoelectric point of 6.5. Western blotting showed that it shares some of the enzyme antigenic determinants in co mmon 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 fo rmation of compound II. The latter is, however, unstable and gradually returns to the native state. The enzyme forms complexes with CN- and N-3(-) and is reduced by dithionite showing a characteristic reduced p eroxidase spectrum. Although the enzyme oxidizes I-, SCN- and Br- opti mally at pH 5.5, 5.25 and 5.0 respectively, at physiological pH, it ox idizes I- and SCN- only. Since extracellular SCN- concentration is muc h higher than I-, SCN- may act as the major electron donor to the enzy me. The second-order rate constants for the reaction of the enzyme wit h H2O2 (k(+1)) and of compound I with SCN- (k(+2)) were 4 x 10(7) M(-1 ) . s(-1) and 8.1 x 10(5) M(-1) . s(-1) respectively. A plot of log V- max against pH yields a sigmoidal curve consistent with a single ioniz able group on the enzyme with a pK(a) value of 5.75, controlling thioc yanate oxidation. In a coupled system with the peroxidase, H2O2, SCN-, GSH, NADPH and glutathione reductase, peroxidase-catalysed SCN- oxida tion by H2O2 could be coupled to NADPH consumption. The system is prop osed to operate in vivo for the efficient elimination of endogenous H2 O2.