Enzymatic Kinetic Determinations

Abstract

Kinetic methods for analytical applications can be divided into noncatalytic and catalytic methods. Enzyme kinetic determinations are a special type of catalytic methods. Enzymatic reactions are used analytically to determine enzyme activities – for instance in the diagnosis of diseases, substrate concentrations in food industry or medicine and concentrations of effectors – for example in trace analysis. Due to the high selectivity of enzymatic analysis the importance of this method in various fields is growing rapidly. The widest use is observed in clinical chemistry and in food chemistry. This article can only give a general survey of the great potential of enzymatic analysis, with references to more detailed literature. Enzymatic reactions are characterized by some special features, explained after the introduction. The theoretical background for kinetic determination is the comprehension of the principles on enzymatic kinetics. In section 3 the simplest mechanism, outlined by Michaelis and Menten and several transformations of this equation will be described. In this section two kinetic parameters, enzyme activity and the Michaelis constant, will be introduced. Section 4 deals with substances, so called effectors, influencing the rate of an enzymatic reaction. Simple mechanisms of these effectors will be shown. The different methods for determination of substrates, enzymes and effectors, illustrated by many examples, are represented in section 5. While substrate concentrations can be determined using both ways, equilibrium or kinetic methods, the determination of enzymes and effectors can only be performed by kinetic approaches. In the last two sections the application of immobilized enzymes in flow systems and biosensor development will be shown.