Continuous flow assay for the simultaneous measurement of the electrophoretic mobility, catalytic activity and its variation over time of individual molecules of Escherichia coli β-galactosidase
Continuous flow assay for the simultaneous measurement of the electrophoretic mobility, catalytic activity and its variation over time of individual molecules of Escherichia coli β-galactosidase
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DOI：
10.1002/elps.200800482
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发表时间：
2008-11-01
期刊：
影响因子：
2.9
通讯作者：
Nichols, Ellert R.
中科院分区：
文献类型：
--
作者：
Craig, Douglas B.;Nichols, Ellert R.
建立了一种基于CE-LIF检测的检测方法，用于研究大肠杆菌β-半乳糖苷酶的单个分子。该测定允许同时测量电泳迁移率、催化活性和单个酶分子随时间的活性变化。除了显示酶分子在流动性和活性方面的微观异质性之外，还证明了在升高的温度下酶活性随时间波动。在不同温度下的孵育表明，在孵育温度变化时，单个β-半乳糖苷酶分子的活性变化表现出差异。在一个温度下孵育，然后在升高的温度下孵育，随后在初始较低的温度下孵育，导致分子具有与最初不同的活性。此外，发现热变性导致活性的快速和完全丧失。
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.