Metmyoglobin reductase. Identification and purification of a reduced nicotinamide adenine dinucleotide-dependent enzyme from bovine heart which reduces metmyoglobin.
Beef heart muscle has been found to contain an enzyme which will rapidly and directly reduce metmyoglobin in vitro. Reduction rates are far greater than any previously reported for nonspecific or nonenzymatic systems. The enzyme is NADH-dependent and requires the presence of ferrocyanide ion for in vitro assay. The artificial electron carriers, dichlorophenolindophenol and methylene blue, are not required. Nonenzymatic reduction of metmyoglobin, which has previously been reported, was not encountered under the assay conditions described herein. Demonstration of enzymatic activity is dependent on a suitable myoglobin substrate, NADH, and ferrocyanide. An equimolar amount of cytochrome b5 was more effective than ferrocyanide in the enzymatic reduction of metmyoglobin. The methods for preparation of beef heart myoglobin and for purification of the enzyme are presented. The enzyme has been purified over 2000-fold. The enzyme has a pH optimum about 6.5 and a Km of 5.0 x 10(-5) M, and is unaffected by the absence of O2. Sodium dodecyl sulfate-gel electrophoresis revealed a molecular weight around 30,000. Purified enzyme does not react with lipoamide. The reaction is markedly influenced by the composition of the buffering milieu. Enzyme activity is inhibited by p-chloromercuriphenyl sulfonic acid, quinacrine dihydrochloride, and N-ethyl-maleimide. Activity was slightly stimulated by FMN. The characteristics of the enzymatic activity and the assay system are similar to those reported by Hegesh et al. (J. Lab. Clin. Med. 72, 339-344, 1968) for erythrocyte methemoglobin reductase.
D000818:Animals; D002417:Cattle; D004912:Erythrocytes / Q000201:enzymology; D006454:Hemoglobins; D007700:Kinetics; D008786:Metmyoglobin; D009132:Muscles / Q000201:enzymology; D009206:Myocardium / Q000201:enzymology*; D009247:NADH, NADPH Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D013053:Spectrophotometry; D013379:Substrate Specificity
D006454:Hemoglobins; D008786:Metmyoglobin; D009247:NADH, NADPH Oxidoreductases; C021794:metmyoglobin reductase