An evaluation of active enzyme centrifugation as a zonal and boundary technique by the analysis of simulated data

Abstract

An evaluation is presented of the zonal technique of active enzyme centrifugation which involved analyzing computer-simulated data generated from known centrifugal and kinetic models. The relative merits of the commonly used methods of measurement are discussed as are potential artifacts in data interpretation arising from the technique itself. In addition, an extension of the technique is proposed to make it applicable to interacting systems. This involves converting it from a zonal to a boundary technique and is achieved by layering sufficient enzyme on to the substrate solution to ensure a plateau region in which the enzyme concentration remains constant. A procedure for analyzing difference curves generated from the product distribution curves is presented and its suitability illustrated by application to a simulated system involving a monomeric and dimeric enzyme in a rapidly established equilibrium.