MULTIPLE FORMS OF METHEMOGLOBIN REDUCTASE

Three pyridine nucleotide-dependent methemoglobin reductases (I, II, and III) have been purified from human erythrocytes by a procedure that involves batchwise treatment of the hemolyzate with DEAE-cellulose to remove most of the hemoglobin, fractionation with ammonium sulfate and acetone, and chromatography on DEAE-cellulose. Under optimal assay conditions, each enzyme exhibits both DPNH- and TPNH-methemoglobin reductase activities in a ratio of about 4:1. I is colorless, II contains a small amount of heme (λmax at 405 mμ) and a nucleotide-like material (λmax at 268 mμ), and III contains a relatively large amount of heme. In the presence of methylene blue as a carrier, each enzyme can utilize methemoglobin, cytochrome c or oxygen as the terminal acceptor. 2,6-Dichlorophenolindophenol can be used as a terminal acceptor in the absence of methylene blue. The enzymes have also been characterized with respect to: Km values for DPNH, TPNH, methylene blue, and methemoglobin; sensitivity toward p-chloromercuriphenylsulfonate; and heat stability. The pyridine nucleotide-linked reduction of methemoglobin is discussed in terms of possible electron transport sequences. © 1969.