Shkumatov V M, Chashchin V L, Akhrem A A, Iuvonen R O, Lang M A
Biokhimiia. 1988 Mar;53(3):394-405.
Coumarin-specific cytochrome P-450 (P-450Coh) has been isolated from liver microsomes of DBA/2N mice induced with pyrazole. The induction effect was accompanied by a 5.8-5.9-fold increase in the P-450Coh content which made up to 14.4-17% of the total cytochrome P-450 pool in the microsomes. At the final step of P-450Coh purification, variously substituted Sepharoses (hydroxyphenyl-, cholate-, aminooctyl- and t-cytochrome-b5-) were used. The optimal scheme involved solubilization of microsomes with sodium cholate, hydrophobic chromatography on octyl-Sepharose, adsorption on calcium-tartrate gel and hydrophobic ion-exchange chromatography on aminooctyl-Sepharose. According to SDS gel electrophoresis data, the purity of P-450Coh was 95% and Mr was 50,000 Da. The amino acid composition of the protein includes 445 residues. At saturating concentrations of coumarin, more than 90% of P-450Coh are represented by the high spin form. The catalytic activity of P-450Coh was studied in reactions of xenobiotics oxidation.
香豆素特异性细胞色素P-450（P-450Coh）已从用吡唑诱导的DBA/2N小鼠肝脏微粒体中分离出来。诱导作用伴随着P-450Coh含量增加5.8至5.9倍，其占微粒体中细胞色素P-450总量的14.4%至17%。在P-450Coh纯化的最后一步，使用了各种取代的琼脂糖（羟苯基-、胆酸盐-、氨基辛基-和细胞色素b5-）。最佳方案包括用胆酸钠溶解微粒体、在辛基琼脂糖上进行疏水色谱、在酒石酸钙凝胶上吸附以及在氨基辛基琼脂糖上进行疏水离子交换色谱。根据SDS凝胶电泳数据，P-450Coh的纯度为95%，相对分子质量为50,000道尔顿。该蛋白质的氨基酸组成包括445个残基。在香豆素饱和浓度下，超过90%的P-450Coh以高自旋形式存在。在异生物素氧化反应中研究了P-450Coh的催化活性。
