Kinetic readout

The fluorimetric imaging plate reader (FLIPR , Molecular Devices) system is a highly specialized, advanced plate reader that is able to produce a kinetic readout for fluorimetric, cell-based assays in a homogeneous format. Examples of such assays are measurements of intracellular concentration, membrane potential,... 

Other readouts, such as high content imaging or enzyme kinetic rate determinations, require extended detection intervals in the plate reader and are best automated on a dedicated workcell or semiautomated workstation platform. 

Figures 3 and 4 illustrate how G( r) changes with Keq in the fast reaction regime (tc -C to). For both figures, k and the equilibrium constant A e( = l

The complexation process is characterized by its thermodynamic and kinetic stability and selectivity, i.e. by the amount of energy and the amount of information brought into operation. Thus, conceptually, energy (interaction) and information are at the bottom of the recognition process of one chemical entity by another, and the design of molecular systems capable of forming stable and selective complexes becomes a problem in information storage and readout at the molecular level. 

In the second section of this chapter, strategies to identify substrates for biochemical protease assays are discussed. Section 2.3 focuses on theoretical and practical aspects of various fluorescence-based readouts for biochemical protease assays. Finally concrete experiments for the determination of enzyme kinetics relevant for the development of robust and sensitive biochemical protease assays are summarized in Section 2.4. Altogether this chapter offers guidelines for the development of biochemical protease assays for the purpose of protease inhibitor-directed drug discovery. 

Most biochemical assays used to determine the potency of inhibitors against (1) aminopeptidases and (2) endopeptidases with minor contributions to the substrate binding efficiency by the S site, are based on the measurement of FI as readout. AMC and RhllO are the most common fluorophores in these cases. For studies of enzyme kinetics, the application of AMC is sufficient. If an assay is intended to determine the inhibitory potencies of small chemical compounds, it is recommended to use red-shifted dyes like RhllO to minimize interference of compound fluorescence with the fluorescence originating from the assay dye. 

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