Resolution of sulphydryl oxidase from gamma-glutamyltransferase in bovine milk by covalent chromatography on cysteinylsuccinamidopropyl-glass.
1. Sulphydryl oxidase from bovine milk was purified by covalent affinity chromatography on cysteinylsuccinamidopropyl-glass. Selective immobilization of the oxidase occurs through formation of a mixed disulphide between the enzyme and the substrate cysteinyl-glass matrix. Reductive elution of the bound protein can be accomplished with small thiols such as reduced glutathione (GSH), dithiothreitol or cysteine. This method leads to approx. 4000-fold purification of the enzyme from whey. Furthermore, complete resolution of sulphydryl oxidase from gamma-glutamyltransferase was achieved with this procedure. 2. Antibodies prepared against this purified enzyme quantitatively precipitated 95% of the GSH-oxidative activity from detergent-solubilized skim-milk membranes, whereas 100% of the transferase activity remained in the supernatant fraction; these findings confirmed the distinction between these two enzymes. 3. Reverse-phase high-pressure-liquid-chromatographic analyses of assay mixtures containing both enzymes revealed an array of GSH derivatives generated by a combination of the oxidative and hydrolytic activities. However, purified sulphydryl oxidase yielded only GSSG with concomitant stoichiometric loss of GSH. 4. The chromatographic method described is simple and reproducible, and may be applicable to isolation of sulphydryl oxidase from other tissues.
D000818:Animals; D002417:Cattle; D055598:Chemical Phenomena; D002621:Chemistry; D002846:Chromatography, Affinity / Q000379:methods; D003545:Cysteine; D003611:Dairy Products / Q000032:analysis; D005260:Female; D005898:Glass; D005980:Glutathione Reductase; D007785:Lactose / Q000032:analysis; D008892:Milk / Q000201:enzymology*; D010088:Oxidoreductases / Q000276:immunology / Q000302:isolation & purification*; D011233:Precipitin Tests; D013386:Succinates; D005723:gamma-Glutamyltransferase / Q000302:isolation & purification*; D050862:Oxidoreductases Acting on Sulfur Group Donors
D003545:Cysteine; D005980:Glutathione Reductase; D007785:Lactose; D010088:Oxidoreductases; D013386:Succinates; D005723:gamma-Glutamyltransferase; D050862:Oxidoreductases Acting on Sulfur Group Donors