The influence of Al3+ ion on porcine pepsin activity in vitro

The in vitro effect of Al³⁺ ions in the concentration range 1.7·10^{? 6} M–8.7·10^{? 3} M on pepsin activity at pH 2, via kinetic parameters and its electrophoretic mobility was evaluated. Kinetic study demonstrated the existence of an activation effect of Al³⁺ at pH 2 on pepsin molecule. Kinetic analysis with respect to concentrations of haemoglobin showed that Al³⁺ ions increase the maximal velocity (V_max) and k_cat values rather than apparent affinity for substrate (K_S) implying the non-competitive nature of activation which indicated that aluminium was a non-essential activator of partial non-competitive type. The values of the equilibrium constants K_S and K_mA for dissociation of corresponding complexes were evaluated as 0.904 ± 0.083 mM and 8.56 ± 0.51 μM, respectively. Dissociation constant K_A, of activator from enzyme-activator complex calculated via kinetic and direct measurement of Al³⁺ binding data, as well as activation constant A₅₀, the activator concentration that gives a rate equal to half at a saturating concentration of activator, were found to be 8.82 ± 0.90 μM, 8.39 ± 0.76 μM, and 8.05 ± 0.48 μM respectively. Native PAGE electrophoresis shows the decrease in electrophoretic mobility of pepsin and confirms modification of the electric charge and conformational changes of pepsin caused by bound Al³⁺ on the pepsin molecule. Al³⁺ induced conformational changes of pepsin were verified by UV-VIS and IR spectra. Moreover, the absence of conformational changes in the haemoglobin molecule in the presence of Al³⁺ ions confirms that the obtained activation is a consequence of conformational changes caused only in the pepsin molecule.