Revisiting absorbance at 230 nm as a protein unfolding probe
Section snippets
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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