Acid phosphatase assay with alpha-naphthyl phosphate as substrate and the use of diazonium salt (Fast Red TR) for chromophore formation was kinetically analysed as a system of two chemical reactions coupled to an enzymic reaction. This system follows a mechanism defined as enzymic-chemical-chemical (EzCC). The accumulation of chromophore with reaction time presented a marked lag period, which was only dependent on the rate constants of the chemical reactions and was independent of the enzymic step. The specific rate constants of each chemical step were determined in 3.8-5.0 pH and 10-35 degrees C temperature ranges. Thermodynamic parameters of the chemical steps were also obtained. Measurement of acid phosphatase activity can be carried out in the pH range 3.8-5.0 (4.8 was optimal pH) without the need to eliminate the lag period.
Kinetic analysis of chemical reactions coupled to an enzymic step. Application to acid phosphatase assay with Fast Red