Calorimetric Study of Thermal Denaturation of (3-Lactoglobulin

Effects of pH and milk constituents (milk ultrafiltrate and ~:-casein) on denaturation of fl-lactoglobulin were investigated by a dynamic method based on differential scanning calorimetry. The apparent reaction order of /Ltactoglobulin denaturation by the dynamic method was 2.0 over the pH range of 4.0 to 9.0, which is in fair agreement with results by other investigators using more classical methods./3-Lactoglobulin was more stable in the neutral pH region 5.0 to 7.0. The activation energy for thermal denaturation of/3-1actoglobulin was not dependent on pH from 4.0 to 9.0. Activation energy was estimated to be about 125 kcal/mol. Heat of denaturation is larger for pH 5.0 to 8.0 (4.6 cal/g) than at pH 4.0 and 9.0 (4.1 cal/g). Lactose stabilized fl-lactoglobulin against thermal denaturation, and lactose-free milk ultrafil trate reduced the thermal stability of /3-1actoglobulin. As compared to buffer at pH 6.6, activation energy of fl-lactoglobulin denaturation in milk ultrafiltrate was constant, but heat of denaturation was larger. The rate of thermal denaturation of /Llactoglobulin increased as K-casein content increased. However, activation energy remained constant, suggesting that the enhanced rate was entropy driven. Because the natural helix order of some proteins in solution is changed to a random coil by heat treatment, the enthalpy associated with this helix-coil transition also can be observed as a function of time, temperature, and solvent composit ion by DSC (18). The use of DSC to investigate protein denaturation has certain advantages over other methods. However, few calorimetric investigations of protein denaturation have included kinetic analysis of the DSC thermograms (2, 8). The usual isothermal method of obtaining kinetic measures on protein denaturation generally involves a series of experiments in which the protein solution is subjected to combinations of various time constant temperature treatments. However, this method is laborious. Dynamic methods that measure the heat evolution of a temperatureprogrammed sample provide a more rapid technique for the same information. There is no agreement on kinetic measures of rates of/3-1actoglobulin (fl-lg) denaturation. Gough and Jenness (9) reported that denaturation of fi-lg followed a first order model, and other workers found that /3-1g followed either second order kinetics (13, 19) or t w o consecutive first order reactions (11, 26). Moreover, denaturation of t3-1g has not been observed over a wide range of pH. In our studies, we measured t3-1g denaturation in the pH range 4.0 to 9.0, in solutions of simulated milk ultrafil trate (SMUF), and in the presence of ~:-casein. I N T R O D U C T I O N Differential scanning calorimetry (DSC) has been used to study kinetics of a number of chemical reactions and physical changes (27). Received September 30, 1983. M A T E R I A L S A N D METHODS Materials and Solutions Commercial fl-lg (three times crystallized and lyophilized, Sigma Chemical Co., St. Louis, MO) was used without further purification. Buffer solutions were .05 M potassium biphthalate for 1984 J Dairy Sci 67:1699-1706 1699