Hanna P M, Tamilarasan R, McMillin D R
Department of Chemistry, Purdue University, West Lafayette, IN 47906.
Biochem J. 1988 Dec 15;256(3):1001-4. doi: 10.1042/bj2561001.
A simple colorimetric test for the Cu(I) content in blue copper proteins is described. The procedure is based on the formation of a complex between Cu(I) and 2,2'-biquinoline in an acetic acid medium. Analyses of spinach plastocyanin, Pseudomonas aeruginosa azurin and Rhus vernicifera stellacyanin show that the cysteine residue in the type 1 site does not induce Cu(II) reduction under our conditions. There is evidence in laccase samples for the presence of an endogenous reductant that can reduce 0.14 +/- 0.04 mol of Cu(II)/mol of protein; however, the addition of EDTA eliminates the interference. The analysis shows that 25 +/- 2% of the type 3 copper ions are in the reduced form in the resting enzyme and that 80 +/- 15% of the type 3 copper ions are reduced in preparations of type-2-depleted laccase. There is growing interest in the development of chemically modified forms of laccase, and our method should be very useful for establishing the valence state of the metal centres in the various derivatives.
本文描述了一种用于检测蓝色铜蛋白中Cu(I)含量的简单比色法。该方法基于在醋酸介质中Cu(I)与2,2'-联喹啉形成络合物。对菠菜质体蓝素、铜绿假单胞菌天青蛋白和漆树漆蓝蛋白的分析表明，在我们的实验条件下，1型位点的半胱氨酸残基不会诱导Cu(II)还原。有证据表明漆酶样品中存在一种内源性还原剂，其可将0.14±0.04 mol的Cu(II)/mol蛋白还原；然而，加入EDTA可消除干扰。分析表明，在静息酶中25±2%的3型铜离子呈还原态，在去除2型铜的漆酶制剂中80±15%的3型铜离子被还原。人们对开发化学修饰形式的漆酶越来越感兴趣，我们的方法对于确定各种衍生物中金属中心的价态应该非常有用。
