Abstract
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.
Highlights
Zymeshowed3700-foldincrease in specificactivity (12).the significance of heme oxygenase activity when compared with the crude preparation, and the in regulating heme-dependent cellular functions is becoming extent of recovery was 30-35%.The molecular weight increasinglyevident.withthe exception of the was estimated at 34,000-36,000
NADPH-dependentbiliverdinreductase activity was extremely sensitive to SH reagents, including 5,5’-dithiobis(2-nitrobenzoic acid),N-ethylmaleimide,p-chloromercuribenzoic acid, and iodoacetamide
It was established that thienclusion of 2.0 M KC1 The column was washed with the following solutions: buffer B con- in bufferB used forthe preparation anwdashing of the affinity taining 2.0 M KC1 (50 ml), buffer B containing 1.0 mM NADH and 2.0 column greatly enhanced thseelectivity of the gel for biliver
Summary
(Received for publication, July 28, 1980,and in revised form, December 19, 1980). From the Departmentof Pharmacology, University of Illinois College of Medicine, Chicago, Illinois 60680. Biliverdin reductasein a stable formwas purified to substrate the heme moiety of hemoglobin ( l l ) ,but homogeneity from rat liver cytosol. 10.4 I11”’cm” (l),has encouraged investigators to resort to the pretreatment of the enzyme withNADPH and bili- the measurement of bilirubin as the indicator of heme oxyverdin fully protected the reductase from inactivation genase activity. It follows that, due to thelack of availability by these reagents.The enzyme activitywas irreversibly of a procedure for thepurification of biliverdin reductase in a inhibited byHgC12. Theaddition of dithiothreitolto the stable homogeneous form, thceytosol fraction of the ratliver enzyme inhibited by5,5’-dithiobis(2-nitrobenzoicacid) isroutinely utilized as thesource of biliverdin reductase.
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