Regeneration of full enzymatic activity by reoxidation of reduced pancreatic phospholipase A2.
Reduction of porcine pancreatic phospholipase A2 with 2-mercaptoethanol in the presence of 6M guanidinium hydrocholoride or 8M urea resulted in the complete disruption of all seven disulfide bridges and the concomitant loss of all enzymatic activity. Through aerobic oxidation of the reduced protein at pH 8.0 in the presence of 5 mM cysteine and 0.9M guanidinium hydrochloride up to 90% of the specific activity of the native enzyme was restored. After purification of the reoxidized phospholipase A2 by Sephadex G-75 gel filtration and chromatography on CM-cellulose pure phospholipase A2 was obtained in 80% yield. This purified reoxidized phospholipase A2 was found to be indistinguishable from the native enzyme as judged by its enzymatic activity, lipid and Ca2+ binding properties. Similar results were obtained using S-sulfonated phospholipase A2, prophospholipase A2 or Nepsilon-amidinated phospholipase A2. For the latter two proteins reoxidation was slower than that of the reduced phospholipase A2.
D000818:Animals; D004220:Disulfides / Q000032:analysis; D006146:Guanidines / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008623:Mercaptoethanol / Q000494:pharmacology; D010084:Oxidation-Reduction; D010179:Pancreas / Q000201:enzymology*; D010740:Phospholipases / Q000378:metabolism*; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D011487:Protein Conformation; D013552:Swine
D004220:Disulfides; D006146:Guanidines; D008623:Mercaptoethanol; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2