The effect of free cysteine (in different concentrations) on the thermal aggregation of calcium-saturated (Ca-sat) and -depleted (Ca-dep) α-lactalbumin (α-LA) was investigated at 25, 50, and 70 °C. The temperatures chosen were below the denaturation temperature ( Td) of Ca-dep and Ca-sat α-LA (25 °C), above the Td of Ca-dep α-LA and below that of Ca-sat α-LA (50 °C), and above the Td of Ca-sat α-LA (70 °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 Ca-sat α-LA even at extended holding times. Aggregates of Ca-sat α-LA were larger than those developed for Ca-dep α-LA. The addition of cysteine, a low-molecular-mass free thiol, resulted in increased aggregation of both Ca-sat and Ca-dep α-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 α-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 α-LA when the cysteine concentration was increased. This study showed that it was possible to control the aggregation size of α-LA by manipulating the incubation temperature and the cysteine concentration.
Keywords: aggregation; calcium; cysteine; kinetics; thiol−disulfide exchange; α-lactalbumin.