Characterization of sheep lacrimal-gland peroxidase and its major physiological electron donor

Abstract

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.

Mechanism-based inactivation of lacrimal-gland peroxidase by phenylhydrazine: a suicidal substrate to probe the active site

“…LGP was purified as described previously [6]. Briefly, the soluble supernatant was treated with (NH % ) # SO % , and the precipitate at 30-60 % saturation was applied to a concanavalin A-Sepharose column.…”

“…Peroxidase activity has also been demonstrated in rat lacrimal gland [4], from which the enzyme has been purified and characterized [5]. We have also purified a soluble peroxidase from sheep lacrimal gland (LGP) and characterized its physical, catalytic and kinetic properties [6]. The enzyme is a single-subunit (82 kDa) glycoprotein of native molecular mass 75 kDa having similarity to LPO.…”

“…The enzyme can scavenge endogenous H # O # effectively through SCN − oxidation in the presence of glutathione, NADPH and glutathione reductase [6]. H # O # may be generated intracellularly from oxidative metabolism or extracellularly from microbial cell growth.…”

“…The haem of LGP is tightly bound to the apoenzyme and cannot be dissociated by standard methods [7,8]. The enzyme catalyses the oxidation of SCN − , I − and Br − but not Cl − [6]. Considering the physiological concentration of SCN − and its oxidation at physiological pH, SCN − is the plausible physiological electron donor of the enzyme Abbreviations used : PH, phenylhydrazine ; DETAPAC, diethylenetriaminepenta-acetic acid ; DMPO, 5,5-dimethyl-1-pyrroline N-oxide ; LGP, lacrimalgland peroxidase ; LPO, lactoperoxidase ; HRP, horseradish peroxidase.…”

“…Although the mechanism of inactivation of haemoglobin [21], catalase [22] and HRP [28] are available, very little is known about the detailed mechanism of inactivation of mammalian peroxidases. As LGP prevents oxidative damage and bacterial infection [3,4,6,11], it is of interest to investigate whether this enzyme is also inactivated by PH. We present evidence here to show that PH is a suicidal substrate of LGP which is inactivated via a mechanism-based mode of action.…”