The protocol for measuring caspase activity in cell lysates can be divided into two sections, the first involving the preparation of subcellular fractions and the second measuring the cleavage of various substrates. It is critical that the cell lysis procedure not allow inadvertent caspase activation. To avoid release of lysosomal and other granule proteases, which could conceivably activate caspases after cell lysis (16, 17), we prefer a lysis procedure that avoids detergents and thereby allows removal of sequestered proteases by differential centrifugation.
Protocol 2. Measurement of caspase activity using fluorogenic substrates
• procedures for the synthesis of potential substrates are described in detail in ref. 18
• free pNA, 7-amino-4-trifluoromethylcouarin (AFCI, or7-amino-4-methylcoumarin (AMC) to construct standard curves are available from Sigma
• chromogenic and fluorogenic caspase substrates are available from a number of suppliers, including Bachem Bioscience, Biomol, Calbiochem, and Enzyme Systems Products
Part A. Preparation ofcytosol or other subcellular fractions (18)
1. Harvest adherent cells by trypsinization followed by sedimentation at 200 g for 10 min. Wash the sample twice in ice-cold PBS. All further steps are performed at 4°C unless otherwise indicated.
2. Resuspend the pellet in a small volume of buffer A [25 mM Hepes (pH 7.5 at 4°C), 5 mM MgCI2,1 mM EGTA supplemented immediately before use with 1 mM a-phenylmethylsulfonyl fluoride (PMSF), 1 mM dithiothreitol (DTT), 10 |xg/ml pepstatin A, and 10 n,g/ml leupeptin]. Typically, 1 ml of buffer A is added to 1-3 x 108 cells. This yields a cytosolic extract with a protein concentration ranging from 4 to 8 mg/ml.
3. Incubate the sample for 20 minutes, then lyse the cells with 20-30 strokes in a tight-fitting Dounce homogenizer. Mix 3-5 (d of homo-genate with an equal volume of 0.4% (w/v) trypan blue and examine by light microscopy. Continue homogenization until >95% of the cells stain blue.
4. Remove the nuclei by sedimentation (800 g for 10 min or 16000 g for 3 min).
5. Supplement the supernatant with EDTA to a final concentration of 5 mM. Prepare cytosol from the post-nuclear supernatant by sedimentation at 280000 gfmax for 60 min in a Beckman TL-100 ultracentrifuge. Other fractions can be likewise be purified by differential sedimentation (37).
6. Freeze the cytosol (280000 g supernatant) in 50 (jlI aliquots at -70°C. Control experiments from our laboratory have indicated that activity capable of cleaving Ac-DEVD-AFC is stable for at least 3 months at -70°C.
Part B. Fluorogenic or chromogenic substrate cleavage assay
1. Determine the protein concentration of the cytosol or other subcellular fraction using the method of Bradford (20) or Smith et al. (21).
2. Thaw aliquots of cytosol or subcellular fractions containing 50 ftg of cytosolic protein at 4°C. Dilute to 50 (jlI with ice-cold buffer A containing 5 mM EDTA.
3. Dilute sample with 225 |xl of freshly prepared buffer B [25 mM Hepes (pH 7.5), 0.1% (w/v) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, 10 mM DTT, 100 U/ml aprotinin, 1 mM PMSF] containing 100 |xM substrate.
4. Incubate the reaction at 37°C. Continuous monitoring of fluorochrome release can be utilized to examine the kinetics of product release and/or the kinetics of enzyme inhibition if suitable equipment is available (38). Alternatively, the reaction can be run as an end-point assay (18). In that case, add 1.225 ml ice-cold buffer B at a fixed time point to stop the reaction.
5. Incubate negative control reactions, containing 50 jjlI of buffer A and 225 |jl1 of buffer B at 37 °C and then dilute them with 1.225 ml ice-cold buffer B in parallel with experimental samples.
6. Measure the fluorescence in a fluorometer using an excitation wavelength of 360 nm and an emission wavelength of 475 nm. The absolute amount of fluorochrome released is determined by measuring the fluorescence of a panel of standards containing various amounts of the liberated fluorophore [e.g. 0-1500 pmoles AFC].
When setting up this assay, it is important to use an appropriate substrate concentration. If the substrate concentration is below the Km for the enzyme, changes in the amount of product released might be due to either changes in the number of active enzyme molecules (altered vmax) or the affinity for the substrate (altered Km). The common interpretation that changes in the amount of product released correspond to changes in the number of active enzyme molecules is only valid when the reaction is run under conditions where the substrate is saturating.
When the assay is run as an end-point assay, it is important to confirm that product release is a linear function of the length of incubation. Important causes of non-linearity include substrate exhaustion and enzyme instability. If extracts are prepared as described above, the assay is linear for up to 4 h under the specified conditions.
Although numerous substrates can be utilized for this assay, peptide derivatives of AMC and AFC are most commonly employed because of the high quantum yield of the liberated fluorophore. Colorimetric assays utilizing chromogenic substrates (e.g. a peptide linked to pNA) have also been developed. These colorimetric assays can easily be adapted to microtitre plate readers, permitting their use in high throughput screening.
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