8] Measurement of hydrodynamic properties of active enzyme by sedimentation

Abstract

This chapter discusses measurement of hydrodynamic properties of active enzyme by sedimentation. Active enzyme centrifugation (AEC) is a simple and precise method to obtain both sedimentation and diffusion coefficients for the active species. As with any technique, there are a series of assumptions, limitations, and possible artifacts which should be taken into consideration in the experimental design. However, AEC provides an excellent means to directly observe the hydrodynamic properties of the fully active enzyme substrate complex in either a purified or crude state. The technique also allows for the investigation of enzyme interactions with other cellular components such as filaments, or nucleic acids. The ability to simulate AEC data is important not only because it allows the detection of possible artifacts, but it allows one, having completed an experiment, to subsequently test the interpretation of the data to ensure that it is consistent with the experimentally observed results. The potential of AEC for analyzing crude enzyme mixtures in a cellular environment with sensitivity and specificity is unmatched, and in combination with theoretical simulation will provide a valuable tool for future investigations.