OPTIMIZING ENZYMATIC CYCLING ASSAYS - SPECTROPHOTOMETRIC DETERMINATION OF LOW-LEVELS OF PYRUVATE AND L-LACTATE

A kinetic analysis of enzymatic cycling systems covering the whole cou rse of the reaction, i.e., the transient phase and the steady state, i s presented. The cost of enzymatic cycling assays is minimalized by us ing equations to calculate the smallest amount of enzymes which should be used to obtain a given rate constant of the cycle. The model chemi cal system chosen for illustration purposes involved the determination of pyruvate and/or L-lactate via the coupling of L-lactate dehydrogen ase and L-lactate oxidase cycling and NADH mediation. The method is si mple although thorough and can be applied to any technique that uses e nzymatic cycling. (C) 1996 Academic Press, Inc.