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Fluorometric Protease Assays in the SpectraMax Gemini Microplate Spectrofluorometer: Example Using Caspase3 (MaxLine Application Note #35)

ZDEVD AMC. Working solutions of ZDEVDAMC stock were typically 1:100 dilutions of the stock, i.e. 5 L of the stock solution into 495 L of 1X Reaction Buffer. The stock enzyme was frozen in 2 or 4 L aliquots (to avoid repeated freeze/thaw cycles) and working enzyme solutions were prepared by diluting the stock 1:10 with 1X Reaction Buffer. The stock AMC solution was 2 mM in DMSO.


RESULTS
Background fluorescence of ZDEVDAMC
ZDEVDAMC substrate is fluorescent, with excitation/emission maxima of ~330 nm/390 nm.9 The excitation/emission maxima of the AMC product are ~342 nm/441 nm. Therefore, both the excitation spectra and emission spectra overlap. Figure 1 shows the substrate and product emission spectra, without (left) and with (right) a 435 nm emission cutoff filter. The substrate has a lower lambda maximum (~390 nm) than the product, and it is less intensely fluorescent, but its signal overlaps significantly with that of the product, even with the cutoff filter.

Because the substrate must be present in a relatively high concentration in the reaction mixture in order to maintain maximal enzyme activity (Molecular Probes recommends 100 M), it contributes a high background to the assay.


Effect of substrate on fluorescence of the product and selection of the excitation wavelength
The absorption spectrum of ZDEVDAMC substrate also affects the caspase3 assay. The substrate absorbs light at 342 nm, the excitation wavelength associated with maximal AMC signal (Figure 2.)


As a result, the substrate absorbs the excitation light and decreases (quenches) the fluorescence intensity of the product. The higher the substrate concentration, the more severe the quenching. The quenching effect of ZDEVDAMC is eliminated by employing a longer excitation wavelength at which substrate absorbance is minimized. This will cause some
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