Purification and characterization of biliverdin reductase from rat liver.
Biliverdin reductase in a stable form was purified to homogeneity from rat liver cytosol. The purified enzyme showed 3700-fold increase in specific activity when compared with the crude preparation, and the extent of recovery was 30-35%. The molecular weight was estimated at 34,000-36,000. The amino acid analysis of the purified preparation revealed the presence of 3 cysteine residues/mol of enzyme. The reductase utilized NADPH and NADH as electron donors. The NADPH-dependent biliverdin reductase activity was extremely sensitive to SH reagents, including 5,5'-dithiobis(2-nitrobenzoic acid), N-ethylmaleimide, p-chloromercuribenzoic acid, and iodoacetamide. However, the pretreatment of the enzyme with NADPH and biliverdin fully protected the reductase from inactivation by these reagents. The enzyme activity was irreversibly inhibited by HgCl2. The addition of dithiothreitol to the enzyme inhibited by 5,5'-dithiobis(2-nitrobenzoic acid) promoted the full reversal of inhibition. The enzyme exhibited different pH optima for activity with NADPH (pH 8.7) and NADH (pH 7.0). The apparent Km for biliverdin was established to be 5.0 microM with NADH and 3.0 microM with NADPH. The apparent Km for NADPH was 3.0 microM, while that of NADH was 270 microM. The enzyme activity was inhibited by the substrate when the concentration exceeded 4.0-5.0 microM. The product, bilirubin, inhibited the enzyme activity in a competitive manner. In addition, the reductase was inhibited by hematin and zinc-protoporphyrin. Dilution produced instability in the enzyme, but the presence of exogenous proteins, such as serum albumin, beta-lactoglobulin, and lysozyme, stabilized the enzyme protein.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001663:Bilirubin / Q000302:isolation & purification / Q000378:metabolism; D004355:Drug Stability; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008970:Molecular Weight; D010088:Oxidoreductases / Q000302:isolation & purification* / Q000378:metabolism; D044925:Oxidoreductases Acting on CH-CH Group Donors; D051381:Rats; D013438:Sulfhydryl Compounds / Q000494:pharmacology; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D000596:Amino Acids; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; C019789:biliverdin reductase; D001663:Bilirubin