血红素加氧酶系的共固定化及部分性质研究  

Studies on Coimmobilization of Heme Oxygenase System and Some Properties

作　　者：龚兴国[1] 曾冬云[1] 

机构地区：[1]浙江大学生物科学与技术系

出　　处：《中国生物化学与分子生物学报》1998年第6期726-731,共6页Chinese Journal of Biochemistry and Molecular Biology

基　　金：浙江省自然科学基金;浙江大学曹光彪高科技发展基金

摘　　要：为了检测血红素加氧酶系分子间的相互作用和共固定化酶系的反应动力学，利用２′，５′－ＡＤＰ－Ｓｅｐｈａｒｏｓｅ４Ｂ柱对血红素加氧酶、ＮＡＤＰＨ－细胞色素ｃ还原酶和胆绿素还原酶进行非膜重组，再以纤维素为载体，用重氮化法固定此重组的酶复合物和共固定非重组的３种酶，发现固定化的重组酶系比非重组酶系能更好地发挥协同作用，在室温条件下可催化血红素一步合成胆红素．共固定化酶的最适ｐＨ为７．２，最适温度为３８℃，Ｋｍ值为０．９３μｍｏｌ／Ｌ．巯基试剂和金属卟淋对固定化酶有抑制作用，共固定化酶比游离酶系稳定性提高，３８℃下的操作半寿期可延长至４２０ｈ，在０～４℃保存两个月其酶活力无明显变化．Heme is oxidatively cleaved and converted to bilirubin Ⅸ α by the combined actions of microsomal heme oxygenase system consisting of heme oxygenase,NADPH cytochrome c reductase,and biliverdin reductase.Molecular interactions among these enzymes were examined using purified enzyme preparations from pig sources.Heme oxygenase,NADPH cytochrome c reductase and biliverdin reductase were successfully reconstituted to form a ternary enzyme complex by 2′,5′ ADP Sepharose 4B column.The complex or the three dissociated enzymes mixture could be coimmobilized on cellulose by using diazo method.It was found that coimmobilized complex had higher activity compared with the coimmobilized three dissociated enzymes mixture,and hemin could be directly converted into bilirubin Ⅸ α,under lower temperature by coimmobilized enzyme complex.The coimmobilized enzymes exhibited optimum pH of 7 4 and temperature of 38℃. K m for heme of 0 93 μmol/L,a specific activity of 1 190 units/mg of protein. SH inactivating reagents and metalloporphyrins inhibited the reaction catalyzed by the coimmobilized heme oxygenase system.The half life of the coimmobilized enzymes operating at 38℃ was more than 420 h..

关 键 词：血红素加氧酶 性质 非膜重组 共固定化 

分 类 号：Q592.1[生物学—生物化学] Q554.03