Kinetics of Thermal Denaturation of β-Lactoglobulin at pH 2.5

Abstract

Thermaldenaturation of /~-lactoglobulin at pH 2.5 was studied at temperatures ranging from 60 to 130 C. Changes were followed by measurements of specific optical rotation [0d2ss9 and solubility at pH 4.5.Denaturation, as determined by both methods, was irreversible and was evident only at 75 C or above, a higher temperature than 65 C reported for denaturation at neutral pH.Also, the cooperative transition temperatures were higher (83 to 84 C) at pH 2.5 than at pH 6.7 (68 to 69 C).Changes in specific rotation indicated that at all temperatures above 75 C denaturation proceeded in two stages.The first stage (up to 5 min) was faster than the subsequent stage (5 to 60 min).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 [a] 2s 5 8 9 were not reached after heating for 60 min.The final [a]~s~9 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