A comparison of the effectiveness of protein denaturants for β-lactoglobulin and ribonuclease

Abstract

An improved method for comparing protein denaturants is described. Enough urea is added to a solution of protein so that the equilibrium constant for the native ⇌ denatured equilibrium is around 0.25. Small amounts of the denaturant of interest are then added to shift the equilibrium and this is monitored by measuring the optical rotation. The major advantages of this approach are, first, that the reaction being studied is approximately the same for each denaturant; and, second, a two-state mechanism can be assumed with some assurance so that the dependence of the free energy of denaturation, ΔG D, on denaturant concentration can be determined. Two proteins which differ markedly in polarity, β-lactoglobulin and ribonuclease, were used for the studies reported here. For most of the compounds tested, the dependence of ΔG D on denaturant concentration is linear and the slopes of these plots vary from −230 to −7430 cal/mol/ m. Alkyl ureas are better denaturants than amides which are better denaturants than alcohols and for each series the denaturing ability increases with increasing alkyl chain length, 1,1,3,3-tetramethylurea, 1,1′-ethylenediurea, and thiourea are all more potent denaturants than guandidine hydrochloride and may prove useful in protein research. Propylene carbonate is approximately equivalent to urea as a protein denaturant and has several other properties which should make it a useful solvent for proteins. On the basis of nonlinear plots of ΔG D vs denaturant, it is suggested that native β-lactoglobulin has specific binding sites for guanidinium and acetamidinium ions and for dichloro- and trifluoroacetate ions.