Craig Douglas B, Nichols Ellert R
Chemistry Department, University of Winnipeg, Winnipeg, Manitoba, Canada.
Electrophoresis. 2008 Nov;29(21):4298-303. doi: 10.1002/elps.200800482.
A CE-LIF detection-based assay was developed for the study of individual molecules of Escherichia coli beta-galactosidase. The assay allows for the simultaneous measurement of the electrophoretic mobility, catalytic activity and the variation in activity over time of individual enzyme molecules. In addition to showing the microheterogeneity of the enzyme molecules with respect to mobility and activity, it was demonstrated that at elevated temperatures the enzyme activity fluctuates over time. Incubation at varying temperatures showed that individual beta-galactosidase molecules exhibit differences in their change in activity upon a change in incubation temperature. Incubation at one temperature, followed by an incubation at an elevated temperature and subsequent incubation back at the initial lower temperature caused the molecules to have a different activity than they had initially. Additionally, thermal denaturation was found to cause a rapid and complete loss of activity.
开发了一种基于毛细管电泳激光诱导荧光检测（CE-LIF）的分析方法，用于研究大肠杆菌β-半乳糖苷酶的单个分子。该分析方法能够同时测量单个酶分子的电泳迁移率、催化活性以及活性随时间的变化。除了显示酶分子在迁移率和活性方面的微观异质性外，还证明在升高温度时酶活性会随时间波动。在不同温度下孵育表明，单个β-半乳糖苷酶分子在孵育温度变化时其活性变化存在差异。在一个温度下孵育，随后在升高的温度下孵育，然后再回到初始较低温度下孵育，导致分子具有与最初不同的活性。此外，发现热变性会导致活性迅速且完全丧失。
