active-enzyme centrifugation

a technique for determining the sedimentation and diffusion coefficients of an enzyme-substrate complex. A thin lamella of an enzyme solution is layered onto a substrate solution in an ultracentrifuge cell and, on rotation, as the enzyme molecules sediment in a band through the substrate solution they catalyse the enzymic reaction, the progress of which is observed optically (e.g. by spectrophotometry). The required coefficients are then calculated from either the rate of appearance of the product of the reaction or the rate of disappearance of the substrate. The centrifugation may be performed on impure enzyme preparations and at the very low concentrations of enzyme used in kinetic studies.

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