Interfacial enzyme kinetics at the phospholipid/water interface: Practical considerations

In dealing with interracial kinetics, numerous precautions should be observed. Care should be taken to select the appropriate aggregate form in which to present the substrate and to ensure that this does not change during the studies as other compounds are added to the assay. Any and all compounds added to the assay should be analyzed for their potential to change the 'quality of the interface' or the nature of the phospholipid aggregates. The substrate concentration should be sufficiently broad to adequately characterize the kinetic model, keeping in mind the various limitations placed on this by the nature of the aggregates. All comparisons of activity should be performed on substrates presented in identical form. Independent studies should be carried out to characterize the nature and strength of all components that can affect the binding to the surface. Whether the standard surface dilution kinetic analysis or the scooting mode analysis should be used depends on which substrate form is used and the enzyme. If vesicles or bicelles are employed, the scooting mode system should be explored. If mixed micelles are used with an enzyme that has a turnover number on the order of 1 to 10 turnovers per second, the surface dilution model can be used. This is especially true if short-chain phospholipids can be used to form the mixed micelles. By following these precautions meaningful interfacial kinetic experiments can be performed.