DETERMINING GLOBULAR PROTEIN STABILITY - GUANIDINE-HYDROCHLORIDE DENATURATION OF MYOGLOBIN

The guanidine hydrochloride (Gdn .cntdot. HCl) denaturation of horse myoglobin (Mb) was investigated at several pH values using absorbancy measurements at 409 nm. The free energy of denaturation, .DELTA.GD, was calculated and .DELTA.GD values were measured to zero concentrations of denaturant. The dependence of .DELTA.GD on Gdn .cntdot. HCl concentration, d(.DELTA.GD)/d(Gdn .cntdot. HCl), increases markedly as the denaturant concentration decreases. An increase in the number of Gdn .cntdot. HCl binding sites on unfolding is probably the major driving force for denaturation by Gdn .cntdot. HCl. An equation based on denaturant binding which fits the experimental data for Mb at pH 7 is .DELTA.GD = .DELTA.GDH2O - .DELTA.nRT 1n (1 + ka.+-.), where .DELTA.GDH2O (= 10.1 kcal/mol) is the free energy of denaturation in the absence of denaturant, .DELTA.n (= 42.8) is the difference in the number of Gdn .cntdot. HCl binding sites on the native and denatured states of the protein, k (= 0.6) is the average denaturant binding constant and a.+-. is the mean ion activity of Gdn .cntdot. HCl. Several lines of evidence show that k = 0.6 should be used in the analysis of Gdn .cntdot. HCl denaturation curves rather than k = 1.2, is currently in widespread use. For Mb and 4 other proteins [lysozyme, .alpha.-chymotrypsin, RNase and .beta.-lactoglobulin], an equivalent and more convenient method of analyzing Gdn .cntdot. HCl denaturation curves is to use Gdn .cntdot. HCl molarities rather than mean ion activities and a denaturant binding constant of 0.8.