蛋白酶的荧光分析

Modern assays for proteolytic activity typically use synthetic chromogenic or fluorogenic 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 peptide’s cleavage site and enzymatic activity is detected by the increase in color or fluorescence. Chromogenicassays 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 microplate spectrofluorometer. We chose to use the protease caspase–3 and a fluorogenic peptide substrate containing a coumarin derivative.