Thermal denaturation of beta-lactoglobulin: effect of protein concentration at pH 6.75 and 8.05

Previous work on the thermal denaturation of beta-lactoglobulin at about neutral pH and concentrations generally above 50 mg/ml has shown that the temperature of the maximum in the thermogram increases only slightly with concentration. Likewise, there is little if any concentration dependence at acid pH over a wide concentration range. However, so far as we are aware, no work has been described on the thermal denaturation of beta-lactoglobulin in the physiological range of protein concentration and pH appropriate to milk. We report measurements at pH 6.75 and 8.05 in the concentration range 2-120 mg/ml and show that below about 50 mg/ml the position of the maximum becomes strongly dependent on concentration, passing through a minimum near 25 mg/ml and increasing towards the lowest concentrations where measurements were practicable. Moreover, the narrow, well defined and nearly symmetrical thermal transition observed at high protein concentrations contrasts with a broader and more asymmetric curve at lower concentrations. An explanation for the behaviour seen at the lower protein concentrations is suggested, based on the temperature- and concentration-dependent dissociation of the beta-lactoglobulin dimer and an associated conformational transition. The position of the maximum in the thermogram has a marked dependence on the rate of heating down to the lowest rate investigated of 10 degrees C per hour, showing the importance of slow kinetic effects in the denaturation of this protein.