Physico-Chemical Studies of Molybednum (VI) -Amylase InteractionAuthor(s): R. P. Singh and R. Chaudhary
The binding of molybdate ions has been studied with b-amylase using polarography and equilibirum dialysis techniques. The extent of binding has been determined by means of Tanford's method and the value of average association constant (log K) and maximal number of binding site (n) have been calculated from Scatchard plot at pH 5.57. These have been found to be 3.3600 and 17, respectively. The non linear plots from polarographic and equilibrium dialysis at pH 5.57 are analysed for primary and secondary sites as well as well their apparent association constants. Their log K1 and log K2 have been found to be 3.9884 and 2.9348 from polarographic and 4.0355 and 3.2907 from equilibrium dialysis, respectively. The number of primary (n1) and secondary (n2) sites have been found to be 4 and 13, respectively. The involvement of single primary sites has also been confirmed from absorption spectra of Mo (VI) -;-amylase mixture, which revealed absorption peak at pH 5.57 and not at other higher pH values. In higher pH range, where polarographic is not applicable, so Mo (VI) binding to b-amylase is determined by dialysis equilibrium method at higher pH-range. These have been found to be 3.4070, 2.2788, 2.7980, 2.7568 and 17, 16, 14, 7 at pH values 5.57, 7.50, 9.50 and 11.50, respectively. A good correspondence among the data obtained from the two techniques supported that electrostatic effects are negligible and the alterations of binding constants is solely due to molybdate ion- -amylase combination. The number of sites available for binding with molybdate ions is much less than the actual number of cationic groups on -amylase molecule. The qualitative binding results of pHmetry and spectroscopy are also strongly support the results of diffusion current and dialysis methods. The pH dependence of Mo (VI) - ; - amylase interaction could be explained by assuming an interaction with the positively charged groups such as -ammonium, imidazolium and guanidinium groups, which are ionized at different pH levels. The involvement of fewer such groups in binding processing may be due to the in availability of all cationic groups due to folded structure of ;-amylase molecule. However, owing to the complexity of the macromolecular structure, it was difficult to predict the nature of exact groups that were involved in the molybdate ;- amylase interaction. The free energies of molybdenum (VI) - ; - amylase complexes were also determined for the support of binding of Mo (VI) with -amylase.