醌蛋白——猪肾二胺氧化酶的提取和性质

醌蛋白——猪肾二胺氧化酶的提取和性质 QUINOPROTEIN PURIFICATION AND CHARACTERIZATION OF DIAMINE OXIDASE FROM PIG KIDNEY 黄健 Huang Jian 1 first-author 赵永芳 Zhao Yongfang 1 武汉大学生命科学学院 武汉大学生命科学学院 College of Life Sciences,Wuhan University College of Life Sciences,Wuhan University 新鲜猪肾匀浆后，经热处理、盐析、羟基磷灰石和ＤＥＡＥ－纤维素柱层析等纯化步骤，二胺氧化酶的收得率为２３．９％，纯化倍数为４２５．该酶进行凝胶电泳后，经氨基黑、酶活性和ＮＢＴ溶液染色分别得到相对应的蛋白质、酶活性及醌辅基谱带．该酶可与腐胺、尸胺、己二胺、组胺等底物作用，释放出Ｈ２Ｏ２，当底物为腐胺时，最适浓度为５ｍｍｏｌＬ－１，Ｋｍ值为０．５ｍｍｏｌＬ－１，最适ｐＨ为７．０，最适温度为４０°Ｃ．在非酶系统中，该酶可与ＮＢＴ溶液反应，呈现紫色，并随时间延长而加深．经ＳＤＳ板状不连续聚丙烯酰胺凝胶电泳，得该酶亚基分子量为８．３×１０４． Diamine oxidase was prepared from fresh pig kidney by a procedure involving homogenization,heat denaturation,ammonium sulfate fractionation,sequential chromatography on hydroxyapatite and DEAE cellulose with 425 fold purification and 23.9% recovery.The purified enzyme was applied to 6% polyacrylamide gel and gave a single band of protein which coincided with the single band of enzyme activity and quinoprotein. The enzyme catalyzed the oxidation of putrescine,cadaverine,1,6 diaminohexane and histamine with the production of H 2O 2.When the substrate was putrescine,the optimum concentration of the enzyme was 5 mmol·L -1 , K m value was 0.5 mmol·L -1 ,the optimum pH was 7.0,the optimum temperature was 40℃.The enzyme could react with nitro blue tetrazolium and the color would change from light purple to heavy purple gradually. 猪肾 二胺氧化酶 6－羟多巴醌 pig kidney diamine oxidase 6 hydroxydopa quinone〖HT S858.28 国家自然科学基金(61672102) 1996-04-01 2021-04-01 4