Temporal resolution of individual steps in an enzymic reaction at low temperature

Media are described which make it possible to study mechanisms of enzyme action at low temperature. The applicability of techniques developed are illustrated by results obtained on formation kinetics of complexes produced by the interaction of horseradish peroxidase with hydrogen peroxide. It is shown that the various steps in the time course of the reaction can be readily resolved with isolation of each intermediate in concentrations sufficient to permit rate studies between consecutive steps. The potential of the method for elaboration of enzyme mechanisms is discussed.