New approaches for verification of kinetic parameters of immobilized concanavalin A: invertase preparations investigated by flow microcalorimetry.

ABSTRACT

In our preceding article, we demonstrated a procedure based upon enzymic flow microcalorimetry using an enzyme thermistor (ET) to characterize the microkinetic properties of an immobilized enzyme (IME) and its further application in the screening of IMEs. To consider the ET method (single ET unit, ET system 1) as standard, it was necessary to show that the estimated relative kinetic parameter (DeltaT(max)) calorimetrically corresponds with the absolute value for the reaction rate within the whole measurement range. This article presents three experimental verification procedures. Two procedures are based on adaptation of the flow-through ET column to a mini-differential-reactor (DR) system with substrate recirculation and post-ET-column methods for determination of the concentration change of the product (spectrophotometrically in ET system 2) or the substrate (calorimetrically in ET system 3) with the IME-catalyzed enzymic hydrolysis. The third procedure is an independently operating DR system which spectrophotometrically estimates the concentration change of the product. The results obtained exhibited good correlation (r = 0.921) between the relative kinetic parameter DeltaT(max), as determined calorimetrically by ET system 1, and the absolute value for the reaction rate (r(max)) as determined by ET systems 2 and 3. These data proved that, within the whole range of experimental conditions applied in this study, the parameter DeltaT(max) instead of the true reaction rate could be employed for the IME screening. Moreover, the generality of the detection principle and the standardized configuration of the ET favor ET systems 2 and 3 for normal screening of IMEs and as miniaturized DR systems allowing dual measurements of kinetic parameters.