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


Abstract

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. Study holds ProTherm entries: 9162, 9163, 9164, 9165, 9166, 9167, 9168, 9169, 9170, 9171, 9172, 9173, 9174, 9175, 9176, 9177, 9178, 9179, 9180 Extra Details: holo form alpha-lactalbumin; calcium binding; binding constant; calorimetry

Submission Details

ID: C34gPYGP

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:37 p.m.

Version: 1

Publication Details
Hendrix T;Griko YV;Privalov PL,Biophys. Chem. (2000) A calorimetric study of the influence of calcium on the stability of bovine alpha-lactalbumin. PMID:10723542
Additional Information

Number of data points 38
Proteins Alpha-lactalbumin ; Alpha-lactalbumin
Unique complexes 1
Assays/Quantities/Protocols Experimental Assay: dHcal ionic:: , details:Additives EDTA (1.2 mM), ; Experimental Assay: Tm ionic:: , details:Additives EDTA (1.2 mM), ; Experimental Assay: dHcal ionic:: , details:Additives EDTA (1.0 mM), ; Experimental Assay: Tm ionic:: , details:Additives EDTA (1.0 mM), ; Experimental Assay: dHcal ionic:: , details:Additives EDTA (0.5 mM), ; Experimental Assay: Tm ionic:: , details:Additives EDTA (0.5 mM), ; Experimental Assay: dHcal details:Additives , ionic:: ; Experimental Assay: Tm details:Additives , ionic:: ; Experimental Assay: dHcal ionic:: , details:Additives EDTA (12.4 mM), ; Experimental Assay: Tm ionic:: , details:Additives EDTA (12.4 mM), ; Experimental Assay: dHcal ionic:: , details:Additives EDTA (9.0 mM), ; Experimental Assay: Tm ionic:: , details:Additives EDTA (9.0 mM), ; Experimental Assay: dHcal ionic:: , details:Additives EDTA (7.4 mM), ; Experimental Assay: Tm ionic:: , details:Additives EDTA (7.4 mM), ; Experimental Assay: dHcal ionic:: , details:Additives EDTA (6.3 mM), ; Experimental Assay: Tm ionic:: , details:Additives EDTA (6.3 mM), ; Experimental Assay: dHcal ionic:: , details:Additives EDTA (5.6 mM), ; Experimental Assay: Tm ionic:: , details:Additives EDTA (5.6 mM), ; Experimental Assay: dHcal ionic:: , details:Additives EDTA (4.9 mM), ; Experimental Assay: Tm ionic:: , details:Additives EDTA (4.9 mM), ; Experimental Assay: dHcal ionic:: , details:Additives EDTA (3.2 mM), ; Experimental Assay: Tm ionic:: , details:Additives EDTA (3.2 mM), ; Experimental Assay: dHcal ionic:CaCl2: 10.0 mM, details:Additives ; Experimental Assay: Tm ionic:CaCl2: 10.0 mM, details:Additives ; Experimental Assay: dHcal details:Additives , ionic:CaCl2: 2.0 mM ; Experimental Assay: Tm details:Additives , ionic:CaCl2: 2.0 mM ; Experimental Assay: dHcal ionic:CaCl2: 1.0 mM, details:Additives ; Experimental Assay: Tm ionic:CaCl2: 1.0 mM, details:Additives ; Experimental Assay: dHcal details:Additives , ionic:CaCl2: 0.1 mM ; Experimental Assay: Tm details:Additives , ionic:CaCl2: 0.1 mM ; Experimental Assay: dHcal ionic:CaCl2: 0.0 mM, details:Additives ; Experimental Assay: Tm ionic:CaCl2: 0.0 mM, details:Additives
Libraries Mutations for sequence MEQLTKCEVFRELKDLKGYGGVSLPEWVCTTFHTSGYDTQAIVQNNDSTEYGLFQINNKIWCKDDQNPHSSNICNISCDKFLDDDLTDDIVCVKKILDKVGINYWLAHKALCSEKLDQWLCEKL
Sequence Assay Result Units