Calcium Ion Binding Characteristics of Porcine Pancreatic Alpha Amylase outside Active Site Domain and Implications: Theory and Experimentation
Issue: 2016 - Volume 7 [Issue 4]
Ikechukwu Iloh Udema *
Department of Biochemistry, Faculty of Natural Science, Ambrose Alli University, Ekpoma, Nigeria and Research Division of Ude International Concepts Limited (RC 862217) B.B. Agbor, Delta State, Nigeria and Owa Alizomor Mixed Sec. Sch. Owa Alizomor, Delta State, Nigeria
*Author to whom correspondence should be addressed.
Aims: To: i) show that unfolding and folding of an enzyme can be investigated or interpreted using different aspects of the same model ii) determine the folding rate constant; iii) determine the equilibrium constant for folding; iv) determine activation parameters for unfolding and folding and apparent thermodynamic parameters for calcium ion binding and folding of the enzyme.
Study Design: Experimental.
Place and Duration: 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. Porcine alpha amylase was assayed at different thermodynamic temperatures for duration of 5 minutes.
Results: The enthalpy of activation (e.g., 401.32±7.07 J/mol) for unfolding, was higher at lower temperature and higher salt concentration. The magnitude of entropy of activation (e.g., - 279±0.06 J/mol.K) and free energy of activation (89.09±0.01 kJ/mol) for unfolding was higher at higher temperature and lower salt concentration. Activation energy (2.98±0.01 kJ/mol) was higher at higher concentration of calcium chloride. Free energy of activation, exothermic enthalpy and negative entropy for folding were higher at higher temperatures except at 333.15 K with respect to entropy change. Calcium ion binding constant decreased with increase in temperature and binding was spontaneous, exothermic and positive in entropic term. Folding equilibrium constant values were lower at higher temperatures. Free energy of folding though spontaneous showed irregular trend while the positive entropy increased but decreased at 333.15 K. The exothermic enthalpy for folding was low.
Conclusion: The data generated fitted well to the first order equation for calcium ion treated enzyme. The presence of calcium salt stabilized the enzyme against unfolding hence the high free energy and negative entropy of activation (an expression of increased order of the complex) of unfolding. Calcium ion binding and folding of porcine pancreatic alpha amylase, though exothermic, are entropically driven (entropic term > enthalpic term) and spontaneous. The presence of calcium ion resulted in significant change in the velocity of amylolysis (P < 0.05).
Keywords: Porcine pancreatic alpha amylase, activation and apparent thermodynamic parameters, calcium binding constant, folding first order rate constant, folding equilibrium constant, conformational stability