A calorimetric study of the influence of calcium on the stability of bovine alpha-lactalbumin.
Bovine alpha-lactalbumin has been studied by differential scanning calorimetry 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 significant increase of heat capacity. Analysis of the observed heat effect shows that this temperature-induced process closely approximates a two-state transition. The transition temperature increases in proportion with the logarithm of the calcium concentration, which results in an increase in the transition enthalpy as expected from the observed heat capacity increment of denaturation. As the total concentration of free calcium in solution is decreased below that of the proteins, there are two temperature-induced heat 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 the holo-protein, while the low-temperature peak is within the temperature range associated with the unfolding of the apo-protein. Statistical thermodynamic modeling of this process shows that the bimodal character of the thermal denaturation of bovine alpha-lactalbumin at non-saturated calcium concentrations 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.
Hendrix
T
T
Department of Biology, Johns Hopkins University, Baltimore, MD 21218, USA.
Griko
Y V
YV
Privalov
P L
PL
D000818:Animals; D001059:Apoproteins / Q000737:chemistry / Q000378:metabolism; D002118:Calcium / Q000737:chemistry* / Q000378:metabolism; D002152:Calorimetry, Differential Scanning / Q000379:methods; D002417:Cattle; D004305:Dose-Response Relationship, Drug; D006358:Hot Temperature; D007700:Kinetics; D007768:Lactalbumin / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding; D013816:Thermodynamics; D014325:Tromethamine
D001059:Apoproteins; C041966:apo-alpha-lactalbumin; D014325:Tromethamine; D007768:Lactalbumin; D002118:Calcium