Denaturation behavior of α-chymotrypsinogen A in urea and alkylurea solutions: Fluorescence studies

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Abstract
The solvent denaturation ofα-chymotrypsinogen (α-ctg A) in aqueous solution of urea, methyl-,N,N′-dimethyl-, ethyl-, propyl- and butylurea was studied by fluorescence measurements. Data were analyzed on the assumption of a two-state approximation to obtain the apparent equilibrium constant,K ∪ and the apparent Gibbs free energy of transition ΔG ∪ 0 . It has been observed that alkylsubstitution of urea significantly lowers the denaturant concentration needed to denatureα-ctg A at 25°C. Denaturation was accompanied by the red shift of emission maxima, the increase of the half-width of the fluorescence spectra, the increase of the fluorescence intensity, and the decrease of the fluorescence polarization. The differences of these fluorescence parameters observed forα-ctg A in alkylureas and urea can be ascribed to different unfolded states of the protein in different denaturant solutions. Minor differences in the extent of unfolding were confirmed by size-exclusion chromatography.