Experimental approach to controllably vary protein oxidation while minimizing electrode adsorption for boron-doped diamond electrochemical surface mapping applications.
Experimental approach to controllably vary protein oxidation while minimizing electrode adsorption for boron-doped diamond electrochemical surface mapping applications.
复制标题
可控地改变蛋白质氧化的实验方法，同时最大限度地减少硼掺杂金刚石电化学表面测绘应用的电极吸附。
DOI：
10.1021/ac302418t
复制
发表时间：
2013
影响因子：
7.4
通讯作者：
Hettich,RobertL
中科院分区：
文献类型：
--
作者：
McClintock,CarleeS;Hettich,RobertL
氧化蛋白质表面图谱已成为衡量折叠蛋白质结构的溶剂可及性的有力手段。存在多种用于产生关键试剂的技术（即，羟基自由基）;然而，这些方法在其操作的复杂性和费用方面有很大的差异。这项研究扩展了早期的工作，以提高硼掺杂金刚石（BDD）电化学的可控性，作为一种易于使用的工具，用于产生羟基自由基，以氧化一系列完整的蛋白质。调节氧化水平同时最小化蛋白质对电极的吸附的努力涉及使用相对高的流速以减少蛋白质在电化学流动室内的停留时间。此外，使用可变电压提供受控电流的不同细胞激活方法使我们能够以蛋白质依赖性方式精确调节氧化程度。为了获得关于蛋白质吸附到电极表面上的水平的观点，进行研究以监测电解期间的蛋白质浓度和在细胞活化事件之间电极表面中的计量变化。本报告证明了成功使用BDD电化学更精确地产生目标数量的氧化事件后，完整的蛋白质。
Oxidative protein surface mapping has become a powerful approach for measuring the solvent accessibility of folded protein structures. A variety of techniques exist for generating the key reagent (i.e., hydroxyl radicals) for these measurements; however, these approaches range significantly in their complexity and expense of operation. This research expands upon earlier work to enhance the controllability of boron-doped diamond (BDD) electrochemistry as an easily accessible tool for producing hydroxyl radicals in order to oxidize a range of intact proteins. Efforts to modulate the oxidation level while minimizing the adsorption of protein to the electrode involved the use of relatively high flow rates to reduce protein residence time inside the electrochemical flow chamber. Additionally, a different cell activation approach using variable voltage to supply a controlled current allowed us to precisely tune the extent of oxidation in a protein-dependent manner. In order to gain perspective on the level of protein adsorption onto the electrode surface, studies were conducted to monitor protein concentration during electrolysis and gauge changes in the electrode surface between cell activation events. This report demonstrates the successful use of BDD electrochemistry for greater precision in generating a target number of oxidation events upon intact proteins.