Abstract
A new method has been described for the study of the denaturation of proteins by guanidine·HCl. The pH of an unbuffered solution of protein is monitored as Gu·HCl solution at a constant rate is run into the protein solution. As the protein undergoes the transition from the native to denatured state a significant and fairly abrupt change in the pH is observed. The behaviour of 14 proteins has been explored. Lysozyme, pepsin, α‐lactalbumin, lima bean trypsin inhibtor, and insulin failed to yield satisfactory transitions by this method. The method is entirely satisfactory with the other proteins. The maximum stabilities of the proteins in respect to Gu·HCl concentration and pH, and at zero rate (by extrapolation) of delivery of Gu·HCl have been studied. The relative stabilities of the proteins differ greatly. The stabilities are not related in a simple way to any known parameter.
To supplement the pH change method outlined, the viscosities of nine proteins have been studied as a function of Gu·HCl concentration. A useful empirical equation relating the viscosity of a Gu·HCl solution to its molar concentration has been developed. The linear relationship between the relative fluidity and protein concentration has been confirmed and the intrinsic viscosities of the proteins have been calculated. The viscosities of the proteins vary significantly in respect to Gu·HCl concentration. All of the proteins undergo transitions from the native to the denatured state as shown by increases in the viscosities of their solutions. These transitions coincide closely with those found by the pH change method in respect to Gu·HCl concentration and pH. After the initial transition, the proteins continue to expand (viscosity increases) as the Gu·HCl concentration increases, indicating that the denatured state consists of many molecular configurations likely differing little in their energy content. The presence of disulfide bonds tends to limit the expansion of the protein molecules.