Characterization of the major hydroperoxide-reducing activity of human plasma. Purification and properties of a selenium-dependent glutathione peroxidase

• Published in: The Journal of biological chemistry Vol. 262; no. 36; pp. 17398 - 17403
• Main Authors: Maddipati, K R, Marnett, L J
• Format: Journal Article
• Language: English
• Published: Bethesda, MD Elsevier Inc 25.12.1987; American Society for Biochemistry and Molecular Biology
• Subjects: Analytical, structural and metabolic biochemistry; Biological and medical sciences; Electrophoresis, Polyacrylamide Gel; Enzymes and enzyme inhibitors; Erythrocytes - enzymology; Fluorometry; Fundamental and applied biological sciences. Psychology; glutathione peroxidase; Glutathione Peroxidase - blood; Humans; Hydrogen Peroxide - blood; Kinetics; man; Oxidoreductases; plasma; Selenium - metabolism; Glutathione peroxidase; Human; Purification; Gel electrophoresis; Enzyme; Tetramer; Red blood cell; Gel filtration; Kinetics; Molecular weight; Ion exchange chromatography; Blood plasma
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)45392-6

We have recently characterized the major hydroperoxide-reducing enzyme of human plasma as a glutathione peroxidase (Maddipati, K. R., Gasparski, C., and Marnett, L. J. (1987) Arch. Biochem. Biophys. 254, 9-17). We now report the purification and kinetic characterization of this enzyme. The purification steps involved ammonium sulfate precipitation, hydrophobic interaction chromatography on phenyl-Sepharose, anion exchange chromatography, and gel filtration. The purified peroxidase has a specific activity of 26-29 mumol/min/mg with hydrogen peroxide as substrate. The human plasma glutathione peroxidase is a tetramer of identical subunits of 21.5 kDa molecular mass as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and is different from human erythrocyte glutathione peroxidase. The plasma peroxidase is a selenoprotein containing one selenium per subunit. Unlike several other glutathione peroxidases this enzyme exhibits saturation kinetics with respect to glutathione (Km for glutathione = 4.3 mM). The peroxidase exhibits high affinity for hydroperoxides with Km values ranging from 2.3 microM for 13-hydroperoxy-9,11-octadecadienoic acid to 13.3 microM for hydrogen peroxide at saturating glutathione concentration. These kinetic parameters are suggestive of the potential of human plasma glutathione peroxidase as an important regulator of plasma hydroperoxide levels.