Complete Amino Acid Sequence of NADH-Cytochrome b5 Reductase Purified from Human Erythrocytes1

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Abstract
The complete amino acid sequence of soluble NADH–cytochrome b 5 reductase punfled from human erythrocytes was determined. The enzyme, which contained 8 methionine residues, was cleaved by cyanogen bromide. The resulting nine peptides were separated by gel filtration and punfled further by high-performance liquid chromatography. The purified peptides were sequenced by automated Edman degradation. Three large CNBr peptides, residues 1–101, 109–151, and 169–231, were further fragmented with trypsin, Staphylococcus aureus V8 protease or a lysyl endopeptidase of Achromobacter lyticus . The peptides obtained from the tryptic digest of citraconylated FAD-depleted apoprotein completed the alignments of the other peptides. The enzyme was composed of 275 amino acid residues. The 4 functionally important cysteine residues were located in the COOH-terminal portion. The molecular weight of the protein was calculated to be 31,260 without FAD. A prediction of the secondary structure was made by the method of Chou and Fasman. The protein was hydrophilic as a whole (43 % polarity), but some regions were rich in hydrophobic residues. From the sequence homology of this enzyme with the pyridine nucleotide-binding sites of other flavoproteins, three candidates for the FAD and NADH-binding domains were suggested.