Parker V D, Roddick A, Seefeldt L C, Wang H, Zheng G
Department of Chemistry and Biochemistry, Utah State University, Logan 84322-0300, USA.
Anal Biochem. 1997 Jul 1;249(2):212-8. doi: 10.1006/abio.1997.2176.
Redox proteins undergo measurable charge transfer at electrodes only under special circumstances, while they readily take part in electron transfer reactions with mediators in solution. Advantage was taken of the latter fact to develop a new method to study the kinetics and equilibria of protein-mediator electron transfer reactions. It was shown that rate and equilibrium constants for the electron exchange between electron transfer mediator and the protein can be obtained from the analysis of the perturbation of the linear sweep voltammetry (LSV) response of the mediator due to the presence of the protein. The experiments were carried out under conditions where the protein does not interact with the electrode. Theoretical data obtained by digital simulation are presented to show the conditions under which rate and equilibrium constants are accessible by the LSV technique. The electron transfer reactions between ferri- and ferrocytochrome c and N,N,N',N'-tetramethylphenylenediamine and the corresponding radical cation in phosphate-buffered saline (0.04 M phosphate, pH 7.4, 0.1 M NaCl) buffer were selected to demonstrate the technique. These studies resulted in an equilibrium constant equal to 1.0 and forward and reverse rate constants equal to 1.6 x 10(4) M-1 s-1. The data available from this method include forward and reverse rate constants for electron transfer and the formal potential for the protein redox couple.
氧化还原蛋白仅在特殊情况下才会在电极上发生可测量的电荷转移，而它们很容易与溶液中的介质发生电子转移反应。利用后一个事实开发了一种新方法来研究蛋白质 - 介质电子转移反应的动力学和平衡。结果表明，电子转移介质与蛋白质之间电子交换的速率常数和平衡常数可以通过分析由于蛋白质的存在而引起的介质线性扫描伏安法（LSV）响应的扰动来获得。实验是在蛋白质不与电极相互作用的条件下进行的。通过数字模拟获得的理论数据表明了通过LSV技术可获得速率常数和平衡常数的条件。选择铁氰化细胞色素c和亚铁氰化细胞色素c与N，N，N'，N'-四甲基对苯二胺以及相应的自由基阳离子在磷酸盐缓冲盐水（0.04 M磷酸盐，pH 7.4，0.1 M NaCl）缓冲液中的电子转移反应来证明该技术。这些研究得出平衡常数等于1.0，正向和反向速率常数等于1.6×10⁴ M⁻¹ s⁻¹。该方法可获得的数据包括电子转移的正向和反向速率常数以及蛋白质氧化还原对的形式电位。
