FLOW MICROCALORIMETER AUTOCALIBRATION FOR THE ANALYSIS OF IMMOBILIZEDENZYME-KINETICS

A new calibration procedure of a flow microcalorimeter (FMC) for the d irect investigation of immobilized enzyme kinetics was developed, The procedure is based on the use of a flow-through microcalorimetric colu mn packed with the immobilized enzyme adapted to the differential reac tor system with infinite flow recirculation. The true reaction rate is determined from the substrate consumption as calculated from the decr ease in thermometric response. The response is transformed to the subs trate concentration based on a microcalorimeter column calibration usi ng known substrate concentrations. The method was experimentally verif ied using the invertase immobilized by biospecific binding on concanav alin A-cellulose conjugates. From the measurement of the sucrose hydro lysis in the FMC, all kinetic parameters of immobilized invertase, V-m ax, K-m, and K-i were estimated, Results were compared to those obtain ed by the simultaneous spectrophotometric determination of the reactan t concentrations. Both techniques gave equivalent results of reactant concentrations and kinetic parameters. (C) 1997 Elsevier Science B.V.