A calorimetric study of the influence of calcium on the stability of bovine alpha-lactalbumin

Bovine alpha-lactalbumin has been studied by differential scanning calorime try with various concentrations of calcium to elucidate the effect of this ligand on its thermal properties. In the presence of excess calcium, alpha- lactalbumin unfolds upon heating with a single heat-absorption peak and a s ignificant increase of heat capacity. Analysis of the observed heat effect shows that this temperature-induced process closely approximates a two-stat e transition. The transition temperature increases in proportion with the l ogarithm of the calcium concentration, which results in an increase in the transition enthalpy as expected from the observed heat capacity increment o f denaturation. As the total concentration of free calcium in solution is d ecreased below that of the proteins, there are two temperature-induced heal absorption peaks whose relative area depends on the calcium concentration, such that further decrease of calcium concentration results in a increase of the low-temperature peak and a decrease of the high-temperature one. The high-temperature peak occurs at the same temperature as the unfolding of t he hole-protein, while the low-temperature peak is within the temperature r ange associated with the unfolding of the apo-protein. Statistical thermody namic modeling of this process shows that the bimodal character of the ther mal denaturation of bovine alpha-lactalbumin at non-saturated calcium conce ntrations is due to a high affinity of Ca2+ for alpha-lactalbumin and a low rate of calcium exchange between the holo- and apo-forms of this protein. Using calorimetric data, the calcium-binding constant for alpha-lactalbumin has been determined to be 2.9 x 10(8) M-1. (C) 2000 Elsevier Science B.V. All rights reserved.