Tryptic Conversion of Cytochrome b5 Reductase to an Active Derivative Containing Two Peptide Chains

Abstract

Abstract Cytochrome b5 reductase has been converted to an active, lower molecular weight flavoprotein derivative by incubation with high concentrations of trypsin. A total of 47 amino acid residues is removed from the single polypeptide chain of this enzyme to form a flavoprotein of 28,400 daltons composed of two peptide chains and 1 molecule of FAD. Noncovalent interactions between the smaller peptide of 75 amino acid residues and the larger, 158 to 170 amino acid segment provide a conformation which binds flavin and retains the structural features essential for NADH cytochrome b5 reductase activity. The maximum velocity is 65% of that of the unmodified enzyme. Procedures have been described for the isolation of the separate peptide fragments, and recombination experiments show that both peptides are necessary for refolding to form a catalytically active flavoprotein. The larger peptide fragment includes both tryptophanyl residues present in the unmodified enzyme, and indirect evidence suggests that this larger peptide fragment may also contain the sulfhydryl group essential for pyridine nucleotide interaction and structures directly related to flavin binding.