Interference of thiol-compounds with dextrinizing activity assay of alpha-amylase by starch-iodine colour reaction: Modification of the method to eliminate this interference

Interference of Luria broth and other bacteriological media with the starch -iodine colour assay of the dextrinizing activity of alpha-amylase (Fuwa's method) was observed, complete bleaching occurring with 0.4 ml of the Luria broth. The interference was found to be due to the thiol groups present in the medium which compete with starch for iodine. Among the various metal s alts tested for counteracting the interference, ZnSO4 was found to be the b est which reverted the colour to about 73-85% of that of the blank. A combi nation of hydrogen peroxide (10 mu l of 30% solution) and CuSO4 . 5H(2)O (5 0 mu l of 0.1 M solution) completely protected the starch-iodine reaction i n the presence of even 0.5 ml of Luria broth and a modified assay was devel oped based on this finding. The colour intensity, however, was almost doubl e than that obtained for the same amount of starch and iodine in the absenc e of these protective agents. Nevertheless, with different concentrations o f starch as well as with varying amounts of enzymes, the modified method sh owed perfect linearity and could be effectively used for estimation of dext rinizing activity of alpha-amylase in the presence of thiol groups.