Intrinsic Fluorescence Studies of Metal Ion Binding Effects on the Thermodynamic Stability of Human α-Lactalbumin

The stability of the protein human α-lactalbumin (HLA) after binding a series of metal cations (Mg2+, Zn2+, Cd2+, Co2+, Mn2+, Sr2+, Ca2+, Na+ and K+) has been examined and compared with that of α-lactalbumin from other species by monitoring the fluorescence of tryptophan residues upon thermal-induced denaturation. The melting temperature (Tm) was determined from the wavelength shift in λmax data as well as the fluorescence intensity data as the protein unfolds. Mathematical expressions for determining thermodynamic parameters (ΔH, ΔG and ΔS) were introduced based on the assumption that the thermal denaturing process was a simple two-state model between the folded state and the unfolded state. These newly developed expressions are especially useful because they allow one to easily calculate the thermodynamic parameters at every temperature as long as the fitting parameters are known. The thermal-induced unfolding experiments revealed that the binding of metal ions to apo-α-lactalbumin increased its stability, but the degree of stabilization varied significantly for each metal ion. From the λmax data and the fluorescence intensity data, the melting temperatures ranged from 28.15℃ to 63.59℃ and from 26.12℃ to 64.42℃, respectively. At Tm, ΔH and ΔS were determined to range from 129.19 kJ/mol to 273.69 kJ/mol and from 0.40 kJ/mol·K to 0.81 kJ/mol·K, respectively. At physiological temperature (37℃), ΔH and ΔS were determined to range from 121.84 kJ/mol to 238.65 kJ/mol and from 0.38 kJ/mol·K to 0.68 kJ/mol·K, respectively. At Tm, ΔG was 0 as expected, but it ranged from -5.29 kJ/mol to 19.91 kJ/mol at 37℃. Overall, the monovalent cations Na+ and K+ were found to destabilize HLA whereas the divalent cations Mg2+, Zn2+, Cd2+, Co2+, Mn2+, Sr2+ and Ca2+ were found to stabilize HLA. As α-lactalbumin is a Ca2+-binding protein, Ca2+ was found to have the greatest effect on the protein stability with the Tm value of 63.59℃.