CONFORMATION AND AGGREGATION OF BOVINE BETA-CASEIN-A .2. THERMODYNAMICS OF THERMAL ASSOCIATION AND THE EFFECTS OF CHANGES IN POLAR AND APOLAR INTERACTIONS ON MICELLIZATION

A sedimentation analysis has been used to determine the proportion of protein present as monomer and aggregate in 0.5 and 1.0 g/dl solutions of β‐casein A in pH 7 phosphate buffer over the temperature range 10–40°C. The amount and molecular weight of the aggregate increase with temperature; under the conditions used, the aggregation number (n) of β‐casein is given approximately by n = 0.6t + 2 with t in degrees centigrade. The concentration of β‐casein in monomeric and aggregated states at different temperatures is used to calculate the standard enthalpy of aggregation ΔH° (Van't Hoff) by assuming that β‐casein undergoes a cooperative, two‐state, micellization process; aggregation is an endothermic process and ΔH° = 66.0 ± 2.6 kJ mol−1. Combination of this ΔH° with the amount of protein calculated to dissociate when 1 g/dl solutions are diluted isothermally to 0.5 g/dl gives the heat of dilution at various temperatures. These calculated heats of dilution are compared with the experimental values obtained by carrying out the same dilutions in a microcalorimeter. The heat of dilution decreases linearly with β‐casein concentration, but the extrapolated zero‐concentration values of 65.8 ± 1.6 kJ mol−1 is the same as the Van't Hoff enthalpy. This agreement in the enthalpy values indicates that the micellization of β‐casein occurs cooperatively. The effect of modifying the hydrophobic/hydrophilic balance of the system on the micellization of β‐casein A has been investigated. The hydrophobic interaction between the protein molecules is decreased by removing the three C‐terminal residues (Ileu Ileu Val) with carboxypeptidase‐A. This modification drastically reduces the ability of the β‐casein molecule to form micelles. Substitution of 2H2O for H2O at constant temperature perturbs the monomer–micelle equilibrium in favor of micelles because of enhanced hydrophobic interactions in the former solvent. The results are consistent with β‐casein micellization involving a delicate balance of the hydrophobic forces favoring aggregation and electrostatic forces opposing it. Copyright © 1979 John Wiley & Sons, Inc.