Elsevier

Analytical Biochemistry

Volume 389, Issue 2, 15 June 2009, Pages 165-170
Analytical Biochemistry

Revisiting absorbance at 230 nm as a protein unfolding probe

https://doi.org/10.1016/j.ab.2009.03.028Get rights and content

Abstract

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.

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Materials

MBP and RNase H were expressed and purified as previous reported [10], [11]. Concentrations of proteins were determined spectrophotometrically in 20 mM sodium phosphate buffer (pH 6.5) containing 6.0 M guanidinium chloride (GdmCl) with extinction coefficients calculated with amino acid compositions [12]. Urea and GdmCl were from Shelton Scientific. A UV transparent disposable 96-well plate was obtained from BD Bioscience (San Jose, CA). CaCl2 and KCl were from J.T. Baker (Phillipsburg, NJ).

Difference spectra between folded and unfolded proteins in the UV region

Change in A230 on unfolding of MBP and RNase H

The change in A230 on protein unfolding is known to be dependent on the change in the number of solvent-exposed aromatic amino acids, especially tryptophan. MBP and RNase H have eight and six tryptophan residues, respectively, which make them good candidates for application of A230. To show that the proteins exhibit detectable changes in A230 on unfolding, we have determined the different spectra in the far UV region between folded and unfolded proteins. For comparison with circular dichroism,

Discussion

The chromophores for UV absorbance in proteins are divided into two classes: peptide bonds and aromatic side chains [3]. Absorbance at 280 nm by aromatic side chains has been commonly used to determine protein concentrations and also to monitor conformational changes in proteins. Absorbance of amide bonds near 190 nm has significant sensitivity to conformational changes [15]. However, the use of 190 nm is not practical due to strong influence from buffer and salt components. A230 is a relatively

Acknowledgments

We thank Jonathan Schlebach and Youngil Chang for helpful comments on the manuscript.

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