Thermal Denaturation of Ovine β-Lactoglobulin

Abstract

Ovine b-lactoglobulin ( b-LG) genetic variants A and B were purified by molecular sieving of acidic whey from homozygous ewes.Heat denaturation of both variants was studied by differential scanning calorimetry in phosphate buffers (0.05 M and 0.5 M, pH 6.6) at different heating rates.Ovine b-LG is more thermostable than its bovine homologue, and variant A is more heat-resistant than variant B. The denaturation temperatures ranged from 71.4°C (variant B in 0.05 M phosphate buffer at a heating rate of 1°C/min) to 89.9°C (variant A in 0.5 M buffer at a heating rate of 20°C/min).Values that were extrapolated to zero heating rate ranged from 70.5°C (variant B, 0.05 M buffer) to 81°C (variant A, 0.5 M buffer).There were no significant differences between variants in enthalpy change or cooperativity in denaturation, but activation energy was higher for variant B. Denaturation of ovine b-LG follows an apparent reaction order that depends on heating rate and buffer molarity, but was always >1. Van't Hoff enthalpies and the ratio of calorimetric enthalpy to Van't Hoff enthalpy were dependent on heating rate, revealing that, at low heating rates, important secondary reactions could take place while the protein was being unfolded.