Purification and characterization of selenium-glutathione peroxidase from hamster liver.
Hamster liver glutathione peroxidase was purified to homogeneity in three chromatographic steps and with 30% yield. The purified enzyme had a specific activity of approximately 500 mumol cumene hydroperoxide reduced/min/mg of protein at 37 degrees C, pH 7.6, and 0.25 mM GSH. The enzyme was shown to be a tetramer of indistinguishable subunits, the molecular weight of which was approximately 23,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A single isoelectric point of 5.0 was attributed to the active enzyme. Amino acid analysis determined that selenocysteine, identified as its carboxymethyl derivative, was the only form of selenium. One residue of cysteine was found to be present in each glutathione peroxidase subunit. The presence of tryptophan was colorimetrically determined. Pseudo-first-order kinetics of inactivation of the enzyme by iodoacetate was observed at neutral pH with GSH as the only reducing agent. An optimal pH of 8.0 at 37 degrees C and an activation energy of 3 kcal/mol at pH 7.6 were found. A ter-uni-ping-pong mechanism was shown by the use of an integrated-rate equation. At pH 7.6, the apparent second-order rate constants for reaction of glutathione peroxidase with hydroperoxides were as follows: k1 (t-butyl hydroperoxide), 7.06 X 10(5) mM-1 min-1; k1 (cumene hydroperoxide), 1.04 X 10(6) mM-1 min-1; k1 (p-menthane hydroperoxide), 1.2 X 10(6) mM-1 min-1; k1 (diisopropylbenzene hydroperoxide), 1.7 X 10(6) mM-1 min-1; k1 (linoleic acid hydroperoxide), 2.36 X 10(6) mM-1 min-1; k1 (ethyl hydroperoxide), 2.5 X 10(6) mM-1 min-1; and k1 (hydrogen peroxide), 2.98 X 10(6) mM-1 min-1. It is concluded that for bulky hydroperoxides, the more hydrophobic the substrate, the faster its reduction by glutathione peroxidase.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D006224:Cricetinae; D003545:Cysteine / Q000031:analogs & derivatives / Q000378:metabolism; D005979:Glutathione Peroxidase / Q000302:isolation & purification* / Q000378:metabolism; D006861:Hydrogen Peroxide; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008297:Male; D012643:Selenium / Q000378:metabolism; D017279:Selenocysteine; D013379:Substrate Specificity
D000596:Amino Acids; D017279:Selenocysteine; D006861:Hydrogen Peroxide; D005979:Glutathione Peroxidase; D012643:Selenium; D003545:Cysteine