Flow microcalorimeter auto-calibration for the analysis of immobilized enzyme kinetics

A new calibration procedure of a flow microcalorimeter (FMC) for the direct investigation of immobilized enzyme kinetics was developed. The procedure is based on the use of a flow-through microcalorimetric column packed with the immobilized enzyme adapted to the differential reactor system with infinite flow recirculation. The true reaction rate is determined from the substrate consumption as calculated from the decrease in thermometric response. The response is transformed to the substrate concentration based on a microcalorimeter column calibration using known substrate concentrations. The method was experimentally verified using the invertase immobilized by biospecific binding on concanavalin A - cellulose conjugates. From the measurement of the sucrose hydrolysis in the FMC, all kinetic parameters of immobilized invertase, Vmax, Km, and Ki were estimated. Results were compared to those obtained by the simultaneous spectrophotometric determination of the reactant concentrations. Both techniques gave equivalent results of reactant concentrations and kinetic parameters.