Control of alpha-Lactalbumin Aggregation by Modulation of Temperature and Concentration of Calcium and Cysteine

摘要

The effect of free cysteine (in different concentrations) on the thermal aggregation of calcium-saturated (Ca-sat) and -depleted (Ca-dep) alpha-lactalbumin (alpha-LA) was investigated at 25, 50, and 70 degrees C. The temperatures chosen were below the denaturation temperature (T-d) of Ca-dep and Ca-sat alpha-LA (25 degrees C), above the T-d of Ca-dep alpha-LA and below that of Ca-sat alpha-LA (50 degrees C), and above the T-d of Ca-sat alpha-LA (70 degrees C). Size-exclusion chromatography coupled to multiangle light scattering showed that no aggregation or only minor aggregation was obtained at the investigated temperatures for both Ca-dep and Casat alpha-LA even at extended holding times. Aggregates of Ca-sat alpha-LA were larger than those developed for Ca-dep alpha-LA. The addition of cysteine, a low-molecular-mass free thiol, resulted in increased aggregation of both Ca-sat and Ca-dep alpha-LA. Comparisons of SDS-PAGE run under reducing and nonreducing conditions showed that the formed cross-links were primarily disulfide bonds, but Western blots also showed small contributions from dityrosine cross-link formation. The aggregation kinetics related to monomer loss during heat treatment were determined by RP-UPLC and showed that the addition of cysteine increased the rate of aggregation. The activation energies for Ca-dep alpha-LA with 0.35 and 0.7 mM cysteine were found to be 59 +/- 1 and 46 +/- 4 kJ/mol, respectively, which showed that less energy was needed for the enhanced thermal aggregation of alpha-LA when the cysteine concentration was increased. This study showed that it was possible to control the aggregation size of alpha-LA by manipulating the incubation temperature and the cysteine concentration.