猪肝胆绿素还原酶的纯化及某些性质的研究

通过高速离心(105,000×g),硫酸铵分级分离、NADP—agaxose层析和SephadexG—100层析,从猪肝的胞浆中分离获得胆绿素还原酶.经SDS—聚丙烯酰胺凝胶电泳表明,该酶达到均一程度,分子量约为47,000道尔顿.酶被纯化4200倍,回收率为38%.这种酶利用NADPH和NADH作为电子供体,对巯基试剂特别敏感.如DTNB、NEM、pCMB及IDA等,都能不同程度地抑制该酶的活性.由HgCl_2处理,该酶活性的抑制是不可逆的.该酶用NADPH或NADH作电子供体时的最适pH分别是pH7.4和pH7.0.

Purification and Characterization of Biliverdin Reductase from Pig Liver

Biliverdin reductase from pig liver cytosol was purified to apparent homogeneity by high -speed centrifuge (105, 000 ×g). fractionate with (NH4)2SO4 NADP - agarose chromatography and Sephadex G-100 chrornatography. The purified biliverdin reductase preparation appeared a single protein band on sodium dodecyl sulfate -polyacrylamide gel electrophoresis which the molecular weight of is about 47,000 dalton. The specific activity of purified enzyme increased 4200 folds the crude perpartion, and the extent of recovery was 38%. Amino acid analysis showed that purified enzyme consists of about 408 amino acid residues. The reductase utilized NADPH and NADH as eletron donors. The NADPH -dependent biliverdin reductase activity was extremly Senective to SH reagents, Such as 5. 5' -dithiobis(2 - nitrobenzoic acid),N- ethylmaleimide, p-chloromercurivenzoic acid, and iodoacetamide. The enzyme activity was irreversibly inhibited by HgCl2- The enzyme exhibited different pH optima for activity with NADPH(pH7. 4) and NADH(pH7. 0).