PROTEIN STABILITY - UREA-INDUCED VERSUS GUANIDINE-INDUCED UNFOLDING OF METMYOGLOBIN

We have studied the denaturation of metmyoglobin at pH 6.0 and 25 degr ees C by urea and guanidine hydrochloride (GdnHCl) which are known to unfold the protein to the same extent. It has been observed that estim ates of protein stability (Delta G(N-U)(0)) from urea-induced and GdnH Cl-induced denaturations do not agree with one another; the linear ext rapolation method gave Delta G(N-U)(0) values of 7.59 +/- 0.33 and 5.3 5 +/- 0.10 kcal mol(-1) for urea and GdnHCl denaturations, respectivel y. Measurements of the effect of the addition of KCl in the concentrat ion range 0.1-1.0 M to urea denaturation have suggested that this disa greement is not due to the nonionic and ionic characters of urea and G dnHCl, respectively. The functional dependence of the free energy chan ge of unfolding (Delta G(N-U)) on [denaturant], the molar concentratio n of the denaturant, has been investigated for understanding the cause (s) of the disagreement between the two estimates of Delta G(N-U)(0) o f metmyoglobin. For this purpose, we have studied the GdnHCl-induced d enaturation of the protein in the presence of different urea concentra tions at pH 6.0 and 25 degrees C and vice versa. These measurements yi eld Delta G(N-U) values in the full concentration range [Ahmad et al. (1994) J. Biochem. 115, 322-327], and these results provide strong evi dence that the Delta G(N-U) dependence on [urea] is linear (linear fre e energy model of denaturation) and the relation between Delta G(N-U) and [GdnHCl] is curved (binding model of denaturation). It has been ob served that the extrapolated value of Delta G(N-U) in urea using the l inear free energy model becomes identical to the extrapolated value of Delta G(N-U) in GdnHCl using the binding model.