Laboratory of Cardiac Energetics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892-1061, USA.
Anal Biochem. 2012 Dec 1;431(1):30-9. doi: 10.1016/j.ab.2012.08.023. Epub 2012 Sep 10.
Native gel electrophoresis allows the separation of very small amounts of protein complexes while retaining aspects of their activity. In-gel enzymatic assays are usually performed by using reaction-dependent deposition of chromophores or light-scattering precipitates quantified at fixed time points after gel removal and fixation, limiting the ability to analyze the enzyme reaction kinetics. Herein, we describe a custom reaction chamber with reaction medium recirculation and filtering and an imaging system that permits the continuous monitoring of in-gel enzymatic activity even in the presence of turbidity. Images were continuously collected using time-lapse high-resolution digital imaging, and processing routines were developed to obtain kinetic traces of the in-gel activities and analyze reaction time courses. This system also permitted the evaluation of enzymatic activity topology within the protein bands of the gel. This approach was used to analyze the reaction kinetics of two mitochondrial complexes in native gels. Complex IV kinetics showed a short initial linear phase in which catalytic rates could be calculated, whereas Complex V activity revealed a significant lag phase followed by two linear phases. The utility of monitoring the entire kinetic behavior of these reactions in native gels, as well as the general application of this approach, is discussed.
天然胶电泳允许在保留其活性的同时分离非常少量的蛋白质复合物。胶内酶分析通常通过使用反应依赖性显色剂沉积或光散射沉淀物进行，在凝胶去除和固定后固定的时间点进行定量，从而限制了分析酶反应动力学的能力。在此，我们描述了一种带有反应介质再循环和过滤的定制反应室，以及一种成像系统，即使在存在浊度的情况下，也可以连续监测胶内酶活性。使用时移高分辨率数字成像连续采集图像，并开发了处理例程以获得胶内活性的动力学轨迹并分析反应时间过程。该系统还允许评估凝胶蛋白带中酶活性的拓扑结构。该方法用于分析天然凝胶中两种线粒体复合物的反应动力学。复合物 IV 动力学显示出一个短的初始线性阶段，在此阶段可以计算催化速率，而复合物 V 活性则显示出一个明显的滞后阶段，随后是两个线性阶段。讨论了监测这些反应在天然凝胶中的整个动力学行为以及该方法的一般应用。
