( Evelyn McGown Ph.D. and Anna Lam...)Fluorometric Protease Assays in the SpectraMax Gemini Microplate Spectrofluorometer: Example Using Caspase3 (MaxLine Application Note #35)

Evelyn McGown, Ph.D. and Anna Lam, B.S.
Molecular Devices Corporation, 8/99

INTRODUCTION
Modern assays for proteolytic activity typically use synthetic chromogenic or flu-orogenic peptide substrates. Enzyme selectivity is obtained by choosing a peptide sequence uniquely recognized by the catalytic site of a particular enzyme. The chromophore or fluorophore is attached near the peptides cleavage site and enzymatic activity is detected by the increase in color or fluorescence. Chromogenic assays are easy in that one simply monitors the rise in absorbance above a (usually) low background. Fluorometric assays are much more sensitive than chromogenic assays, but are less straightforward. The substrate itself can be fluorescent, causing a high background. If the absorption spectra of the substrate and product overlap, the substrate can quench the signal from the product by absorbing either excitation or emission light. Therefore, both the excitation and emission wavelengths should be optimized to minimize interference from the substrate, while maintaining sufficiently high product fluorescence. This application note describes how to optimize a protease assay in the SPECTRAmax GEMINI micro-plate spectrofluorometer. We chose to use the protease caspase3 and a fluorogenic peptide substrate containing a coumarin derivative.

Coumarin derivatives are among the most popular fluorophores for peptide based protease assays because they are more sensitive and more watersoluble than other fluorescent derivatives.¹³ A 7amino4methylcoumarin (AMC) derivative was first used as a substrate for chymotrypsin¹. Since then, AMC and the 7amino4trifluoromethylcoumarin (AFC) fluorophores have been used for various protease assays.¹⁴ In each case, cleavage of the fluorophore results in increased fluorescence, with a shift of excitation and emission maxima to longer wavelengths. AMC a
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