Thiocyanate, a plausible physiological electron donor of gastric peroxidase

Abstract

Gastric peroxidase (GPO) was purified to apparent homogeneity to characterize its major physiological electron donor. The enzyme (RZ = 0.7), with a subunit molecular mass of 50 kDa, is a glycoprotein, with a relative abundance of aspartic and glutamic acid over arginine and lysine. It has a Soret maximum at 412 nm, which is shifted to 426 nm by H₂O₂ due to formation of compound II. Although the physiological electron donors I^-, Br^- and SCN^-, but not Cl^-, are oxidized by GPO optimally at acid pH, only I^- and SCN^- are oxidized appreciably at physiological pH. Considering that the I^- concentration in stomach is less than 1 microM, whereas the SCN^- concentration is about 250 μM, SCN^- may act as a major electron donor for GPO. Moreover, SCN^- oxidation remains unaltered in the presence of physiological concentrations of other halides. The second-order rate constant for the reaction of GPO with H₂O₂ (k1) and compound I with SCN^- (k₂) at pH 7 was found to be 8 × 10⁷ M^-1.s^-1 and 2 × 10(5) M^-1.s^-1 respectively. GPO has significant pseudocatalase activity also in the presence of I^- or Br^-, but it is blocked by SCN^-. The SCN^- oxidation product OSCN^- may be reduced back to SCN^- by cellular GSH, and GSSG may be reduced back to GSH by glutathione reductase and NADPH. In a system reconstituted with pure glutathione reductase, NADPH, GSH, SCN^- and H₂O₂. GPO-catalysed SCN^- oxidation could be coupled to NADPH oxidation. This system where GPO utilizes SCN^- as the major physiological electron donor may operate efficiently to scavenge intracellular H₂O₂.