Merck UK: Apoptosis Resource - Using Fluorometric Substrates for Measuring Caspase Activity

		Technical Resources

		Technical Information

		Calbiochem Information

		Apoptosis Resource

		Appendix

Apoptosis Resource: Technical Protocols

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III. General Guidelines for Using Fluorometric Substrates for Measuring Caspase Activity
Substrate Principle
A synthetic peptide substrate is labeled with AFC (7-amino-4- trifluoromethylcoumarin), a fluorescent molecule, to form a fluorogenic compound that can be used for measuring caspase activity. Alternatively, the substrate may be conjugated to other fluorescent molecules, such as AMC (7-amino-4-methylcoumarin).

When AFC is attached to the substrate, it produces a blue fluorescence upon exposure to light (excitation max.: ~400 nm). Caspase enzymatically cleaves the AFC-substrate and releases free AFC. Free AFC produces a yellow fluorescence (emission max.: ~505 nm).

AFC has several advantages over other fluorogenic labels. The larger Stoke’s shift between bound and free AFC enables the substrate to be both chromogenic (yellow-green color is visible to the naked eye) and fluorogenic (detection of emission at ~505 nm with a fluorimeter). With a larger Stoke’s shift, greater sensitivity can be achieved.

Assay Principle
A fluorimeter is first calibrated with known amounts of free AFC or AMC. The release of AFC or AMC in the reaction mixture is monitored with a fluorimeter. Caspase activity in the sample is proportional to the amount of free AFC or AMC produced. A unit is defined as the amount of caspase required to produce 1 pmol of AFC (or AMC)/min at 25�C at saturating substrate concentrations.

Example
Purified or partially purified caspase preparations (~15 ng enzyme). If the sample is not purified, a negative control containing a specific caspase inhibitor should be assayed.

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General Fluorometric Assay Procedure

1.	Prepare a 500 mM stock solution of caspase substrate in DMSO.
2.	Prepare a 500 mM stock solution of caspase inhibitor in DMSO.
3.	Buffer: 100 mM HEPES, 10% sucrose, 10 mM DTT, 500 M EDTA. Adjust pH to 7.5 using 0.1 N NaOH or HCl.
4.	Prepare several dilutions of sample using the caspase buffer (see 3 above).
5.	Ideally, each sample dilution should be tested in three different reaction mixtures: • Substrate only (blank) • Sample + inhibitor + substrate (negative control) • Sample + substrate (sample)
6.	Prepare a calibration curve by measuring known amounts of AFC (excitation max: ~400 nm; emission max: ~505 nm) or AMC (excitation max: ~380 nm; emission max: ~460 nm) in a fluorometer.
7.	Reaction with inhibitor should be started first because of the time required for the inhibitor to react with the sample before substrate addition. A preliminary time course for maximum effect should be determined. Example: Mix 440 ml of caspase buffer with 20 ml of inhibitor in a tube, add 20 ml sample. Mix gently. Incubate at 30�C for 30 min to 12 h.
8.	To blank tubes add 480 ml of buffer, 20 ml of substrate.
9.	Add 20 ml substrate to negative control tubes.
10.	To sample tubes add 460 ml of caspase buffer, 20 ml of substrate. Mix well then add 20 ml of sample.
11.	Incubate all tubes at 30�C for 60 minutes and measure fluorescence for time zero.
12.	Measure fluorescence after another 60 minutes (t1).
13.	Calculate change in fluorescence (∆FU) for each sample at t1 as follows:
14.	∆FU = (sample FU at t1 - blank FU at t1) - (Sample FU at time zero - Blank FU at time zero)
15.	Calculate enzyme activity for t1. If the activity is low, assay should be allowed to proceed for a longer time (up to 24 h).
16.	For final results, use sample dilution that gives highest sample reading and lowest negative control reading.

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