Kinetics of Thermal Denaturation of β-Lactoglobulin at pH 2.5

Abstract

Thermal denaturation of .beta.-lactoglobulin at pH 2.5 was studied at temperatures ranging 60-130.degree. C. Changes were followed by measurements of specific optical rotation .**GRAPHIC**. and solubility at pH 4.5. Denaturation, as determined by both methods, was irreversible and was evident only at 75.degree. C or above; a higher temperature than 65.degree. C reported for denaturation at neutral pH. The cooperative transition temperatures were higher (83-84.degree. C) at pH 2.5 than at pH 6.7 (68-69.degree. C). Changes in specific rotation indicated that at all temperatures > 758% C denaturation proceeded in 2 stages. The first stage (up to 5 min) was faster than the subsequent stage (5-60 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.sbd.indicating progressively increased unfolding of protein molecules. Stable .**GRAPHIC**. were not reached after heating for 60 min. The final .**GRAPHIC**. was much lower than that (.apprx. 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 2 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 (.degree. K) enabled calculation of the activation energy of denaturation (.apprx. 43 Kcal/mol) for .beta.-lactoglobulin heated at pH 2.5. The kinetics of thermal denaturation at pH 2.5 were compared with those at neutral pH.