Characterization of sheep lacrimal-gland peroxidase and its major physiological electron donor.
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.
Mazumdar
A
A
Department of Physiology, Indian Institute of Chemical Biology, Calcutta, India.
Chatterjee
R
R
Adak
S
S
Ghosh
A
A
Mondal
C
C
Banerjee
R K
RK
D000818:Animals; D015153:Blotting, Western; D002384:Catalysis; D002942:Circular Dichroism; D004583:Electrons; D004591:Electrophoresis, Polyacrylamide Gel; D007106:Immune Sera; D007700:Kinetics; D007765:Lacrimal Apparatus / Q000201:enzymology*; D008970:Molecular Weight; D010084:Oxidation-Reduction; D010544:Peroxidases / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D012756:Sheep; D013056:Spectrophotometry, Ultraviolet; D013861:Thiocyanates / Q000378:metabolism
D007106:Immune Sera; D013861:Thiocyanates; D010544:Peroxidases