Kinetics of Thermal Denaturation of β-Lactoglobulin at pH 2.5

Abstract

Thermal denaturation of β-lactoglobulin at pH 2.5 was studied at temperatures ranging from 60 to 130C. Changes were followed by measurements of specific optical rotation [α]58925 and solubility at pH 4.5. Denaturation, as determined by both methods, was irreversible and was evident only at 75C or above, a higher temperature than 65C reported for denaturation at neutral pH. Also, the cooperative transition temperatures were higher (83 to 84C) at pH 2.5 than at pH 6.7 (68 to 69C).Changes in specific rotation indicated that at all temperatures above 75C denaturation proceeded in two stages. The first stage (up to 5min) was faster than the subsequent stage (5 to 60min). Specific rotations of the pH 4.5-insoluble fraction resolubilized at pH 2.5 and the pH 4.5-soluble fraction, readjusted to pH 2.5, increased steadily with duration and temperature of heat treatment, indicating progressively increased unfolding of protein molecules. Stable [α]58925 were not reached after heating for 60min. The final [α]58925 was much lower than that (∼100) achieved in the presence of concentrated urea or guanidine hydrochloride. Changes in optical rotation could not be fitted into a simple kinetic scheme.The extent of denaturation measured by loss of solubility at pH 4.5 indicated it followed pseudo first-order or two consecutive first-order reactions. Denaturation rate constants were determined from the slope of the lines. The Arrhenius plot of the logarithm of the apparent denaturation rate constant as a function of the reciprocal of the temperature (°K) enabled calculation of the activation energy of denaturation (∼43 Kcal/mol) for β-lactoglobulin heated at pH 2.5. The kinetics of thermal denaturation at pH 2.5 have been compared with those at neutral pH.