Department of Biophysics and Biophysical Chemistry, Howard Hughes Medical Institute and the Johns Hopkins University School of Medicine, Baltimore, MD 21202, USA.
Anal Biochem. 2011 Nov 1;418(1):102-10. doi: 10.1016/j.ab.2011.06.034. Epub 2011 Jul 2.
Ubiquitination is a widely studied regulatory modification involved in protein degradation, DNA damage repair, and the immune response. Ubiquitin is conjugated to a substrate lysine in an enzymatic cascade involving an E1 ubiquitin-activating enzyme, an E2 ubiquitin-conjugating enzyme, and an E3 ubiquitin ligase. Assays for ubiquitin conjugation include electrophoretic mobility shift assays and detection of epitope-tagged or radiolabeled ubiquitin, which are difficult to quantitate accurately and are not amenable to high-throughput screening. We have developed a colorimetric assay that quantifies ubiquitin conjugation by monitoring pyrophosphate released in the first enzymatic step in ubiquitin transfer, the ATP-dependent charging of the E1 enzyme. The assay is rapid, does not rely on radioactive labeling, and requires only a spectrophotometer for detection of pyrophosphate formation. We show that pyrophosphate production by E1 is dependent on ubiquitin transfer and describe how to optimize assay conditions to measure E1, E2, and E3 activity. The kinetics of polyubiquitin chain formation by Ubc13-Mms2 measured by this assay are similar to those determined by gel-based assays, indicating that the data produced by this method are comparable to methods that measure ubiquitin transfer directly. This assay is adaptable to high-throughput screening of ubiquitin and ubiquitin-like conjugating enzymes.
泛素化是一种广泛研究的调节修饰，参与蛋白质降解、DNA 损伤修复和免疫反应。泛素通过涉及 E1 泛素激活酶、E2 泛素结合酶和 E3 泛素连接酶的酶级联反应连接到底物赖氨酸上。泛素缀合的测定包括电泳迁移率变动测定和表位标记或放射性标记泛素的检测，这些方法难以准确定量，并且不适用于高通量筛选。我们开发了一种比色测定法，通过监测在泛素转移的第一步中释放的焦磷酸来定量泛素缀合，即 E1 酶的 ATP 依赖性充电。该测定法快速，不依赖放射性标记，并且仅需要分光光度计即可检测焦磷酸的形成。我们表明，E1 的焦磷酸产生依赖于泛素转移，并描述了如何优化测定条件以测量 E1、E2 和 E3 活性。通过该测定法测量的 Ubc13-Mms2 多泛素链形成的动力学与基于凝胶的测定法相似，表明该方法产生的数据与直接测量泛素转移的方法相当。该测定法适用于泛素和泛素样连接酶的高通量筛选。
