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

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The stability of the protein human ГҺВұ-lactalbumin (HLA) after binding a series of metal cations (Mg<SUP>2+</SUP>, Zn<SUP>2+</SUP>, Cd<SUP>2+</SUP>, Co<SUP>2+</SUP>, Mn<SUP>2+</SUP>, Sr<SUP>2+</SUP>, Ca<SUP>2+</SUP>, Na<SUP>+</SUP> and K<SUP>+</SUP>) has been examined and compared with that of ГҺВұ-lactalbumin from other species by monitoring the fluorescence of tryptophan residues upon thermal-induced denaturation. The melting temperature (T<SUB>m</SUB>) was determined from the wavelength shift in ГҺВ»<SUB>max</SUB> data as well as the fluorescence intensity data as the protein unfolds. Mathematical expressions for determining thermodynamic parameters (ГҺВ”H, ГҺВ”G and ГҺВ”S) 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-ГҺВұ-lactalbumin increased its stability, but the degree of stabilization varied significantly for each metal ion. From the ГҺВ»<SUB>max</SUB> data and the fluorescence intensity data, the melting temperatures ranged from 28.15ГўВ„Вғ to 63.59ГўВ„Вғ and from 26.12ГўВ„Вғ to 64.42ГўВ„Вғ, respectively. At T<SUB>m,</SUB> ГҺВ”H and ГҺВ”S were determined to range from 129.19 kJ/mol to 273.69 kJ/mol and from 0.40 kJ/molГӮВ·K to 0.81 kJ/molГӮВ·K, respectively. At physiological temperature (37ГўВ„Вғ), ГҺВ”H and ГҺВ”S were determined to range from 121.84 kJ/mol to 238.65 kJ/mol and from 0.38 kJ/molГӮВ·K to 0.68 kJ/molГӮВ·K, respectively. At T<SUB>m</SUB>,<SUB> </SUB>ГҺВ”G was 0 as expected, but it ranged from -5.29 kJ/mol to 19.91 kJ/mol at 37ГўВ„Вғ. Overall, the monovalent cations Na<SUP>+</SUP> and K<SUP>+</SUP> were found to destabilize HLA whereas the divalent cations Mg<SUP>2+</SUP>, Zn<SUP>2+</SUP>, Cd<SUP>2+</SUP>, Co<SUP>2+</SUP>, Mn<SUP>2+</SUP>, Sr<SUP>2+</SUP> and Ca<SUP>2+</SUP> were found to stabilize HLA. As ГҺВұ-lactalbumin is a Ca<SUP>2+</SUP>-binding protein, Ca<SUP>2+</SUP> was found to have the greatest effect on the protein stability with the T<SUB>m</SUB> value of 63.59ГўВ„Вғ.