Revisiting absorbance at 230 nm as a protein unfolding probe

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 (A230) 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 A230 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 A230. Significant changes in A230 were observed with both proteins on unfolding in the chemical denaturants. The global stabilities were successfully determined by measuring the change in A230 in varying concentrations of denaturants. Also, unfolding kinetics was investigated by monitoring the change in A230 under denaturing conditions. The results were quite consistent with those determined by CD. Unlike CD, A230 allowed us to monitor protein unfolding in a 96-well microtiter plate with a UV plate reader. Our finding suggests that A230 is a valid and convenient structural probe to determine thermodynamic stability and unfolding kinetics of proteins with many potential applications.