KINETIC-STUDY OF AN ENZYMATIC CYCLING SYSTEM COUPLED TO AN ENZYMATIC STEP - DETERMINATION OF ALKALINE-PHOSPHATASE ACTIVITY

A kinetic study is made of a system consisting of a specific enzymic c ycling assay coupled to an enzymic reaction. A kinetic analysis of thi s system is presented, and the accumulation of chromophore involved in the cycle is seen to be parabolic, i.e. the rate of the reaction incr eases continuously with constant acceleration. The system is illustrat ed by the measurement of alkaline phosphatase activity using beta-NADP (+) as substrate. The enzymes alcohol dehydrogenase and diaphorase are used to cycle beta-NAD(+) in the presence of ethanol and p-Iodonitrot etrazolium Violet. During each turn of the cycle, one molecule of the tetrazolium salt is reduced to an intensely coloured formazan. A simpl e procedure for evaluating the kinetic parameters involved in the syst em and for optimizing this cycling assay is described. The method is a pplicable to the measurement of any enzyme, and its amplification capa city as well:as the simplicity of determining kinetic parameters enabl e it to be employed in enzyme immunoassays to increase the magnitude o f the measured response.