Fried M G, Liu G
Department of Biochemistry and Molecular Biology, Pennsylvania State University College of Medicine, Hershey 17033.
Nucleic Acids Res. 1994 Nov 25;22(23):5054-9. doi: 10.1093/nar/22.23.5054.
The gel electrophoresis mobility shift assay is widely used for qualitative and quantitative characterization of protein complexes with nucleic acids. Often it is found that complexes that are short-lived in free solution (t1/2 of the order of minutes) persist for hours under the conditions of gel electrophoresis. We have investigated the influence of polyacrylamide gels on the pseudo first-order dissociation kinetics of complexes containing the E.coli cyclic AMP receptor protein (CAP) and lactose promoter DNA. Within the gel matrix, kdiss decreased with increasing [polyacrylamide] and the order of the reaction was changed. In free solution, kdiss was proportional to [DNA]2, while in 5% gels, kdiss was proportional to [DNA]0.3. In gels of [polyacrylamide] > or = 10%, kdiss was nearly independent of [DNA] until fragment concentrations exceeded 0.1 microM. Even in the absence of competing DNA, kdiss(gel) < kdiss(solution). These results suggest that the lifetime of CAP-DNA complexes in free solution is limited by their encounter frequency with molecules of DNA or with protein-DNA complexes; some or all of the stabilization observed in gels may be due to a reduction in this frequency.
凝胶电泳迁移率变动分析广泛用于对蛋白质与核酸复合物进行定性和定量表征。经常会发现，在自由溶液中寿命较短（半衰期约为几分钟）的复合物在凝胶电泳条件下能持续数小时。我们研究了聚丙烯酰胺凝胶对含有大肠杆菌环磷酸腺苷受体蛋白（CAP）和乳糖启动子DNA的复合物的准一级解离动力学的影响。在凝胶基质中，解离常数kdiss随聚丙烯酰胺浓度的增加而降低，并且反应级数发生了变化。在自由溶液中，kdiss与DNA浓度的平方成正比，而在5%的凝胶中，kdiss与DNA浓度的0.3次方成正比。在聚丙烯酰胺浓度≥10%的凝胶中，直到片段浓度超过0.1微摩尔，kdiss几乎与DNA浓度无关。即使在没有竞争性DNA的情况下，kdiss（凝胶）＜kdiss（溶液）。这些结果表明，CAP-DNA复合物在自由溶液中的寿命受其与DNA分子或蛋白质-DNA复合物的相遇频率限制；在凝胶中观察到的部分或全部稳定性可能是由于这种频率降低所致。
