The use of starch azure for measurement of alpha-amylase activity.

Despite being widely used, there is no standard protocol for α-amylase activity measurement with starch azure substrate. Boiling pre-treatment of starch azure suspension increased the reaction rate of hydrolysis catalysed by human salivary α-amylase (HSA) or porcine pancreatic α-amylase (PPA) and the sensitivity of spectrophotometric activity measurement has been improved. Kinetic constants, KM, and vmax, obtained from parallel isothermal titration calorimetric (ITC) measurements on natural and starch azure revealed, that the blue starch derivative does not differ significantly from its natural counterpart from kinetic point of view. Interestingly, substrate inhibition was observed in starch azure hydrolysis characterised by dissociation constants 49 mg/mL and 16.4 mg/mL for HSA and PPA, respectively. In this work a new protocol has been suggested for α-amylase activity measurement using boiled insoluble starch azure as substrate at 5 mg/mL concentration.