The influence of temperature and pH upon the rate of denaturation of ricin.

For many years distinction has been made between acid denaturation, alkali denaturation, and the heat denaturation of proteins. In a recent review (24) heat is classified as a “physical” denaturing agent and hydrogen and hydroxyl ions as “chemical” denaturing agents. Nevertheless, it is clear that the intensity factors associated with acidity and alkalinity (the pH) and with heat (the temperature) are continuous and not mutually exclusive functions in aqueous solutions. The rate of denaturation of a protein in solution is a simultaneous function of temperature and pH. The temperature function has been handled in terms of the Arrhenius equation or some interpretation of it (cf. Eyring and Stearn (9)), but the pH function has been dealt with rationally only in limited situations. Steinhardt (27) in a paper on the inactivation of pepsin showed inverse proportionality of the rate constant to the fifth power of the hydrogen ion activity. By assigning five successive ionizations of hydrogen ion to pepsin and ascribing to the product of the fifth of these a unique instability he derived an equation which accommodated the facts. Later Neurath et al. (24) demonstrated the applicability of the Steinhardt method to some data on the denaturation of hemoglobin (Cubin (7)) over a pH range of 0.5 unit in which the rate was proportional to the square of the hydrogen ion activity. Lewis (21) suggested that protein denaturation was catalyzed by hydrogen and hydroxyl ions. The rate should then be directly proportional to the hydrogen ion activity on the acid side of a minimum and inversely proportional to it on the alkaline side. Since these expectations are not fulfilled, the suggestion need not be considered further. From the postulates of Steinhardt (27), suitably generalized and modified, we have developed equations which satisfactorily describe data on the denaturation of ricin from pH 1 to 12 and over a range of rates changing