Purification and characterization of human biliverdin reductase

Abstract

Conversion of biliverdin to bilirubin is catalyzed by the cytosolic enzyme biliverdin reductase. We have purified and characterized the human liver reductase and find it to differ extensively from the previously described rat enzyme (H. Fakhrai and M. D. Maines, 1992, J. Biol. Chem. 267, 4023-4029) in its primary structure/composition, yet share kinetic properties. The human enzyme is substantially larger than the rat enzyme (approximately 41,000-42,000 versus 33,000-34,000), is dual cofactor and dual pH dependent, and requires free-SH groups. At pH 6.0-7.0 the NADH was the more effective cofactor, whereas at pH 8.5-8.75 NADPH was the preferred cofactor. The activity was inhibited by-SH reagents, 5'-dithiobis(2-nitrobenzoic acid) and p-chloromercuribenzoic acid, and protected from these reagents by cofactors and substrate. On two-dimensional electrophoresis, the purified protein resolved into four distinct isoelectric zones (pI 6.03, 5.83, 5.68, and 5.55) and two molecular weight forms (approximately 40,700 and approximately 39,600). Variants with similar pI values were detected in the purified human kidney reductase, although their relative tissue abundance varied. The tryptic map, amino acid composition, and sequence of NH2 terminus and four tryptic peptides of human reductase were compared with those of the rat. The HPLC profile and amino acid composition of the human and the rat enzymes differed vastly, and two tryptic peptides were present in the human that could not be detected in the predicted amino acid sequence of the rat enzyme. At the same time, the first 21 amino acids of the NH2 terminus of rat and human, except for the substitution of glutamic acid in human for lysine (amino acid 4) in the rat, were found identical and two peptides with 78-87% similarity to the rat reductase were found in the human reductase. Of the seven cysteine residues present in the human, four or five were titratable with 5'-dithiobis(2-nitrobenzoic acid).