The isolation and purification of a specific "protector" protein which inhibits enzyme inactivation by a thiol/Fe(III)/O2 mixed-function oxidation system.
Mixed-function oxidation systems comprised of Fe3+, O2, and electron donors such as thiol compounds, ascorbate, NAD(P)H/NAD(P)H oxidase, and xanthine oxidase/hypoxanthine, catalyze the inactivation of many enzymes. This report describes the isolation and purification of a soluble protein from Saccharomyces cerevisiae, which specifically inhibits the inactivation of various enzymes by a nonenzymatic Fe3+/O2/thiol mixed-function oxidase system. When thiol is replaced with another electron donor (e.g. ascorbate), this specific protein no longer protects against iron (or copper)/O2-dependent radical-induced enzyme inactivation. Purification steps included a polyethylene glycol precipitation followed sequentially by a chromatography on DE52 and high pressure liquid chromatography on phenyl, DEAE, and gel-filtrated columns. The final gel filtration step yielded two protein peaks exhibiting protector activity and possessing a Mr of 500,000 and 90,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of these two fractions gave a single band at 27 kDa suggesting that these protein species simply represent different oligomeric structures. The protector protein did not possess catalase, glutathione peroxidase, superoxide dismutase, or iron chelation activities. Since the protection activity reported herein is specific for mixed-function oxidation systems containing thiols, we propose that the protector protein functions as a sulfur radical scavenger.
D000975:Antioxidants / Q000302:isolation & purification*; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D005656:Fungal Proteins / Q000302:isolation & purification* / Q000494:pharmacology; D005974:Glutamate-Ammonia Ligase / Q000037:antagonists & inhibitors*; D007501:Iron / Q000378:metabolism / Q000494:pharmacology*; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D009363:Neoplasm Proteins; D010100:Oxygen / Q000494:pharmacology*; D010544:Peroxidases; D054464:Peroxiredoxins; D010751:Phosphopyruvate Hydratase / Q000037:antagonists & inhibitors*; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000494:pharmacology*; D013482:Superoxide Dismutase / Q000378:metabolism
D000975:Antioxidants; D005656:Fungal Proteins; D009363:Neoplasm Proteins; D013438:Sulfhydryl Compounds; D007501:Iron; D006899:Mixed Function Oxygenases; D010544:Peroxidases; D054464:Peroxiredoxins; D013482:Superoxide Dismutase; D010751:Phosphopyruvate Hydratase; D005974:Glutamate-Ammonia Ligase; D010100:Oxygen