Determination of Calcium Ion Binding Parameter of Human Salivary Alpha-amylase by Partial Inactivation Kinetics

Aims: The aims were: i) To ascertain the applicability of a model for the determination of the effect of calcium depletion, to the effect of the presence of the calcium chloride, ii) Quantify the thermodynamic activation parameters for unfolding of the enzyme with increasing temperature at varying concentration of the salt, iii) To determine calcium binding parameters of the human salivary alpha amylase (HSaA) and cognate apparent thermodynamic parameters.

Study Design: Experimental.

Place and Duration of Study: Department of Biochemistry, Ambrose Alli University and Research Division of Ude International Concepts Limited (RC 862217) B. B. Agbor Delta, Nigeria. The research spanned between 2013 and 2016.

Methodology: Bernfeld method of enzyme assay was used. Controls were free from calcium chloride. Crude human salivary alpha amylase was assayed at different thermodynamic temperatures for duration of 5 minutes.

Results: The Gibbs free energies of activation (DG#) at 4 mM and 1 mM CaCl2 (aq) at 318.15 K were 89.62±0.00 and 89.35±0.03 kJ/mol respectively. The corresponding enthalpies of activation were 6.82±0.038 and 3.07±0.09 kJ/mol and the entropies were -260.00±1.17 and -271.21±0.29 J/mol.K respectively. The apparent unfolding rate constant, ranged from 138.50±0.07 – 154.20±0.11 × 1/104/s. The DG# of unfolding as [CaCl2(aq)]→zero at 318.15 K is 89.22±0.01 kJ/mol. The entropy of activation was -279.12±19.20 J/mol.K. Calcium ion binding constant ranged from 42.39±2.47 – 46.81±1.31 1/M. The Gibbs free energy and entropy of calcium ion binding at 318.15 K were -9.94±0.08 kJ/mol and 55.08±0.25 J/mol.K respectively. The unfolding equilibrium constant ranged from 62.83±1.35 – 135.45±5.88; the enthalpy and entropy of unfolding were 33.96±0.13 kJ/mol and 143.96±0.09 J/mol.K respectively at 318.15 K.

Conclusion: The model for the calculation of apparent inactivation rate of the enzyme is applicable to calcium treated enzyme. High Gibbs free energy of activation is due to increased “barrier” to unfolding. Large negative entropy of activation was a reflection of a more ordered transition state. Calcium ion binding and unfolding as [CaCl2(aq)]→zero were spontaneous and entropically driven.