Christgen Shelbi L, Becker Sophia M, Becker Donald F
Department of Biochemistry, Redox Biology Center, University of Nebraska-Lincoln, Lincoln, NE, United States.
Department of Biochemistry, Redox Biology Center, University of Nebraska-Lincoln, Lincoln, NE, United States.
Methods Enzymol. 2019;620:1-25. doi: 10.1016/bs.mie.2019.03.004. Epub 2019 Mar 29.
A key factor for flavoenzyme activity is the reduction potential of the bound flavin. The reduction potentials of protein-bound flavins span approximately a 500-mV range consistent with flavoenzymes having critical roles in metabolism and a variety of biological processes. Redox potentials of flavoenzymes have traditionally been determined using an electrode-based system with either direct or indirect electrochemical coupling between the protein and the working electrode. An electrode independent method, however, is also now commonly used and involves calculating the unknown flavin reduction potential of the protein from the known reduction potential of a reference or indicator dye. Here, the "classic" potentiometric method and the xanthine/xanthine oxidase methods are described. Both methods rely on equilibrium between protein-bound flavin and redox dyes. The potentiometric method measures the equilibrated redox potential of the protein-dye mixture whereas the xanthine/xanthine oxidase technique relies on slow continuous enzymatic reduction to maintain a constant equilibrium between the protein and the dyes. Because electrochemical equipment is not required, the xanthine/xanthine oxidase method is more accessible and convenient for researchers seeking to determine reduction potentials of flavoproteins or other biological redox centers such as hemes. The xanthine/xanthine oxidase method has been used to determine flavin reduction potentials from +132 to -417mV, demonstrating it is suitable for characterizing the redox properties of most flavoproteins.
黄素酶活性的一个关键因素是结合黄素的还原电位。蛋白质结合黄素的还原电位跨度约为500毫伏，这与黄素酶在代谢和各种生物过程中发挥关键作用相一致。传统上，黄素酶的氧化还原电位是使用基于电极的系统来测定的，该系统在蛋白质和工作电极之间具有直接或间接的电化学耦合。然而，现在也普遍使用一种不依赖电极的方法，该方法涉及根据参考染料或指示染料的已知还原电位来计算蛋白质未知的黄素还原电位。在此，将描述“经典”电位法和黄嘌呤/黄嘌呤氧化酶法。这两种方法都依赖于蛋白质结合黄素和氧化还原染料之间的平衡。电位法测量蛋白质-染料混合物的平衡氧化还原电位，而黄嘌呤/黄嘌呤氧化酶技术则依赖于缓慢的连续酶促还原，以维持蛋白质和染料之间的恒定平衡。由于不需要电化学设备，黄嘌呤/黄嘌呤氧化酶法对于寻求测定黄素蛋白或其他生物氧化还原中心（如血红素）还原电位的研究人员来说更容易获得且更方便。黄嘌呤/黄嘌呤氧化酶法已被用于测定从+132到-417毫伏的黄素还原电位，表明它适用于表征大多数黄素蛋白的氧化还原特性。
