期刊
ANALYTICAL BIOCHEMISTRY
卷 389, 期 2, 页码 165-170出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2009.03.028
关键词
Protein stability; UV absorbance; 230 nm; Conformational change; Unfolding kinetics
Thermodynamic stability and unfolding kinetics of proteins are typically determined by monitoring protein unfolding with spectroscopic probes, such as circular dichroism (CD) and fluorescence. UV absorbance at 230 nm (A(230)) is also known to be sensitive to protein conformation. However, its feasibility for quantitative analysis of protein energetics has not been assessed. Here we evaluate A(230) as a structural probe to determine thermodynamic stability and unfolding kinetics of proteins. By using Escherichia coli maltose binding protein (MBP) and E. coli ribonuclease H (RNase H) as our model proteins, we monitored their unfolding in urea and guanidinium chloride with A(230). Significant changes in A(230) were observed with both proteins on unfolding in the chemical denaturants. The global stabilities were successfully determined by measuring the change in A(230) in varying concentrations of denaturants. Also, unfolding kinetics was investigated by monitoring the change in A(230) under denaturing conditions. The results were quite consistent with those determined by CD. Unlike CD, A(230) allowed us to monitor protein unfolding in a 96-well microtiter plate with a UV plate reader. Our finding suggests that A(230) is a valid and convenient structural probe to determine thermodynamic stability and unfolding kinetics of proteins with many potential applications. (C) 2009 Elsevier Inc. All rights reserved.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据