Detecting Peptidases and Proteases—Section 10.4

Page Contents

• Peptidase Substrates
• Caspase Substrates and Assay Kits
• Substrates for HIV Protease and Renin
• EnzChek Protease Assay Kits and Fluorescein Casein
• EnzChek Gelatinase/Collagenase Assay Kit
• EnzChek Elastase Assay Kit
• DQ Substrates
• Alternative Methods for Detecting Protease Activity
• Protease Inhibitors
• Data Table
• Ordering Information

Peptidases and proteases play essential roles in protein activation, cell regulation and signaling, as well as in the generation of amino acids for protein synthesis or utilization in other metabolic pathways. In general, peptidases cleave shorter peptides, and proteases cleave longer peptides and proteins. Depending on their site of cleavage, peptidases can be classified as exopeptidases if they preferentially hydrolyze amino acid residues from the terminus of a peptide, or endopeptidases if they cleave internal peptide bonds. Exopeptidases are further divided into aminopeptidases and carboxypeptidases depending on whether they hydrolyze residues from the amine or the carboxy terminus.

Although the spectral properties of fluorogenic peptidase and protease substrates and their hydrolysis products are easily predictable, the utility of a given substrate for an enzyme depends on the kinetics of hydrolysis by the enzyme, which, in turn, depends on the substrate's concentration and amino acid sequence, as well as on the pH, temperature and presence of cofactors in the medium. For measurements in live cells, the suitability of a particular substrate also hinges on its accessibility to the enzyme and the cellular retention of the hydrolysis product. In addition to these factors, the chromophore or fluorophore conjugated to the substrate can influence its hydrolysis rate and specificity, as well as the permeability of the substrate and its hydrolysis product.

We prepare a variety of protease substrates, including selective protease substrates for caspase-3 and caspase-8—enzymes that are activated during apoptosis (Assays for Apoptosis—Section 15.5)—and for HIV protease and renin. Our EnzChek and DQ protease substrates include:

• EnzChek Protease Assay Kits (E6638, E6639, R22132), which use a heavily BODIPY dye–labeled DQ casein derivative for the assay of a wide variety of proteases (Detection limits of the EnzChek Protease Assay Kits—Table 10.4).
• EnzChek Peptidase/Protease Assay Kit (E33758), which uses a fluorescence resonance energy transfer (FRET)–based substrate to detect protease activity.
• The EnzChek Polarization Assay Kit for Proteases (E6658), which uses a green-fluorescent BODIPY FL casein conjugate with an optimal degree of labeling for fluorescence polarization-based general protease assays (Fluorescence Polarization (FP)—Note 1.4).
• EnzChek Gelatinase/Collagenase Assay Kit (E12055) and EnzChek Elastase Assay Kit (E12056), which use DQ gelatin or DQ elastin as substrates, provide the speed, high sensitivity and convenience required for measuring gelatinase (collagenase) or elastase activity and for screening protease inhibitors in a high-throughput format.
• DQ collagen (D12052, D12060), DQ BSA (D12050, D12051) and DQ ovalbumin (D12053), which are useful for screening protease activity and inhibitors (including in situ zymography ) and for studying antigen processing.

The carboxy terminus of single amino acids and short peptides can be conjugated to certain amine-containing fluorophores to create fluorogenic peptidase substrates. The dyes used to make these substrates are fluorescent at physiological pH; however, when the dyes are coupled in an amide linkage to peptides, their absorption maxima are usually shortened significantly. The resulting substrates are sometimes fluorescent but with relatively short-wavelength emission spectra. In an extreme case such as that of rhodamine 110–based substrates, detectable long-wavelength absorbance and fluorescence are completely eliminated by amide formation. Peptidase activity releases the fluorophore, restoring its free-dye fluorescence.

UV Light–Excitable Substrates Based on 7-Aminocoumarins

7-Amino-4-methylcoumarin (AMC, A191; Introduction to Enzyme Substrates and Their Reference Standards—Section 10.1) is a blue-fluorescent dye whose peptide amides are used extensively as substrates for detecting enzymatic activity in cells, homogenates and solutions. The CBZ-L-phenylalanyl-L-arginine amide of AMC (A6521) is a substrate for a variety of serine proteases, including cathepsins, kallikrein and plasmin. AMC and 7-amino-4-trifluoromethylcoumarin (AFC)—a dye with somewhat longer-wavelength spectra than AMC (excitation/emission maxima of ~380/500 nm) at pH 7—are released from the caspase-3, caspase-7 and caspase-8 substrates listed in Fluorogenic substrates for caspase activity—Table 15.5; these caspases are activated during early stages of apoptosis (Assays for Apoptosis—Section 15.5). The Z-DEVD-AMC substrate is also a component of the EnzChek Caspase-3 Assay Kit #1 (E13183, see below).

7-Amino-4-chloromethylcoumarin (CMAC, C2110; Introduction to Enzyme Substrates and Their Reference Standards—Section 10.1) is a mildly thiol-reactive analog of AMC; CMAC-based substrates yield fluorescent peptidase products with improved retention in live cells. The fluorogenic t-BOC-Leu-Met-CMAC substrate (A6520) has been used to measure calpain activity in hepatocytes following the addition of extracellular ATP.

Visible Light–Excitable Substrates Based on Rhodamine 110

Rhodamine 110 (R110, R6479; Introduction to Enzyme Substrates and Their Reference Standards—Section 10.1) is a visible light–excitable dye with much stronger absorption than AMC; R110-based substrates usually comprise two identical amino acids or peptides attached to a single fluorophore. Molecular Probes bisamide derivatives of rhodamine 110 are sensitive and selective substrates for assaying protease activity in solution or inside live cells. Originally developed by Walter Mangel and colleagues, these substrates comprise an amino acid or peptide covalently linked to each of R110's amino groups, thereby suppressing both its visible absorption and fluorescence. Upon enzymatic cleavage, the nonfluorescent bisamide substrate is converted in a two-step process first to the fluorescent monoamide and then to the even more fluorescent R110 (Figure 10.4.1). The fluorescence intensities of the monoamide and of R110 are constant from pH 3–9. Both of these hydrolysis products exhibit spectral properties similar to those of fluorescein, with peak excitation and emission wavelengths of 496 nm and 520 nm, respectively, making them compatible with flow cytometers and other instrumentation based on the argon-ion laser. Substrates based on R110 may also be useful for sensitive spectrophotometric assays because the R110 dye has intense visible absorption (extinction coefficient at 496 nm = ~80,000 cm^-1M^-1 in pH 6 solution).

We prepare a variety of substrates based on the rhodamine 110 fluorophore (Rhodamine 110–based bis-peptide substrates—Table 10.2). Bis-(CBZ-Arg)-R110 (BZAR, R6501) is a general substrate for serine proteases that has proven to be 50- to 300-fold more sensitive than the analogous AMC-based substrate. This enhanced sensitivity can be attributed both to the greater fluorescence of the enzymatic product and to the enhanced reactivity of the cleavage site. In addition, BZAR inhibits guanidinobenzoatase activity in tumor cells. The tripeptide derivative bis-(CBZ-Ile-Pro-Arg)-R110 (BZiPAR, R6505) allows direct and continuous monitoring of enzyme turnover, making it useful for determining individual kinetic constants of fast-acting, irreversible trypsin inhibitors. BZiPAR has been shown to enter intact cells where it is cleaved by lysosomal proteases. Simultaneous measurement of enzymatic activity with BZiPAR and Ca²⁺ transients with fura-2 (F1201, F1221, F1225, F14185; Fluorescent Ca2+ Indicators Excited with UV Light—Section 19.2) has been reported. Bis-(CBZ-Phe-Arg)-R110 (R6502) has been employed for flow cytometric analysis of the cysteine proteases cathepsin B and L in human monocytes and rat macrophages. Bis-(CBZ-Ala-Ala-Ala-Ala)-R110 (R6506), an elastase substrate, has been used in a novel DNA detection assay. Bis-(CBZ-Ala-Arg)-R110 (R6508) is a fluorogenic substrate for both elastase and trypsin. The bis-(tosyl-Gly-Pro-Arg) amide of rhodamine 110 (R22124) is a selective substrate for thrombin. Turnover of this substrate by thrombin on a membrane in the presence of thromboplastin produces both color and fluorescence that has been reported to model coagulation and blood clot formation. We also offer the human renin substrate 1 (R2931, see below) for measuring the activity of this important blood-pressure-regulating enzyme.

Figure 10.4.1 Sequential peptidase cleavage of a rhodamine 110–based substrate. The nonfluorescent bisamide substrate is first converted to the fluorescent monoamide and then to the highly fluorescent rhodamine 110.

Members of the caspase (CED-3/ICE) family of cysteine–aspartic acid specific proteases have been identified as crucial mediators of the complex biochemical events associated with apoptosis, The recognition site for caspases is marked by three to four amino acids followed by an aspartic acid residue, with the cleavage occurring after the aspartate. The caspase proteases are typically synthesized as inactive precursors. Inhibitor release or cofactor binding activates the caspase through cleavage at internal aspartates, either by autocatalysis or by the action of another protease.

CellEvent Caspase-3/7 Green Detection Reagent

Caspase-3 (CPP32/apopain) is a key effector in the apoptosis pathway, amplifying the signal from initiator caspases (such as caspase-8) and signifying full commitment to cellular disassembly. In addition to cleaving other caspases in the enzyme cascade, caspase-3 has been shown to cleave poly(ADP-ribose) polymerase (PARP), DNA-dependent protein kinase, protein kinase Cδ and actin.

CellEvent Caspase-3/7 Green detection reagent (C10423) represents a new generation of caspase substrates and is an important tool for the study of apoptosis. The cell-permeant CellEvent reagent comprises the four–amino acid peptide DEVD—which contains the recognition site for caspases 3 and 7—conjugated to a nucleic acid–binding dye. Because the DEVD peptide inhibits the ability of the dye to bind to DNA, CellEvent Caspase-3/7 Green detection reagent is intrinsically nonfluorescent . In the presence of activated caspase 3/7, the dye is cleaved from the DEVD peptide and free to bind DNA, producing a bright green-fluorescent signal (absorption/emission maxima ~502/530 nm) indicative of apoptosis. This robust assay is highly specific for caspase 3/7 activation and, as expected, we observe nearly complete inhibition of the CellEvent Caspase-3/7 Green detection reagent signal in cells pretreated with a caspase 3/7 inhibitor.

Apoptosis assays with CellEvent Caspase-3/7 Green detection reagent are extremely easy to perform. Cells are simply incubated with the CellEvent reagent in complete culture medium for 30 minutes and then imaged by traditional fluorescence microscopy (Figure 10.4.2) or high-content imaging. Apoptotic cells with activated caspase 3/7 show bright green-fluorescent nuclei, whereas cells without activated caspase 3/7 show minimal fluorescence. Because the cleaved reagent labels nuclei of caspase 3/7–positive cells, this stain can also provide information on nuclear morphology, including condensed nuclei typical of late‑stage apoptosis.

One important advantage of the CellEvent caspase-3/7 assay is that no wash steps are required, thus preserving fragile apoptotic cells that are typically lost during these rinses. The loss of apoptotic cells during wash steps may lead to an underestimation of the extent of apoptosis in the sample, resulting in poor assay accuracy. Additionally, the fluorescent signal resulting from cleavage of CellEvent Caspase-3/7 detection reagent survives formaldehyde fixation and detergent permeabilization, providing the flexibility to perform endpoint assays and to probe for other proteins using immunocytochemical techniques.

Figure 10.4.2 Multiplex imaging of apoptosis. U2OS cells were treated with 30 μM etoposide for 18 hr to induce apoptosis. The treated cells were stained first with 7.5 μM CellEvent Caspase-3/7 Green detection reagent (green fluorescence, C10423) to detect apoptosis and Hoechst 33342 nucleic acid stain (blue fluorescence, H3570) to label nuclei, and then with 150 nM MitoTracker Deep Red FM (pink fluorescence, M22426) to label mitochondria. Following fixation and permeabilization, actin was labeled with Alexa Fluor 546 phalloidin (orange fluorescence, A22283).

Other Caspase-3 Substrates

Caspase-3 (CPP32/apopain) is a key effector in the apoptosis pathway, amplifying the signal from initiator caspases (such as caspase-8) and signifying full commitment to cellular disassembly. In addition to cleaving other caspases in the enzyme cascade, caspase-3 has been shown to cleave poly(ADP-ribose) polymerase (PARP), DNA-dependent protein kinase, protein kinase C and actin. We offer a selection of fluorogenic caspase substrates (Fluorogenic substrates for caspase activity—Table 15.5). The Z-DEVD-R110 substrate, which contains containing the caspase-3 recognition site Asp-Glu-Val-Asp (DEVD), is a component of our EnzChek Caspase-3 Assay Kit #2 (E13184) and RediPlate 96 EnzChek Caspase-3 Assay Kit (R35100) described below and is available separately in a 20 mg unit size for high-throughput screening applications (R22120, Rhodamine 110–based bis-peptide substrates—Table 10.2). This nonfluorescent bisamide is first converted by caspase-3 (or a closely related protease) to the fluorescent monoamide and then to the even more fluorescent rhodamine 110 (excitation/emission maxima ~496/520 nm). In addition, the bis-L-aspartic acid amide of R110 (D₂-R110, R22122), which contains rhodamine 110 (R110) flanked by aspartic acid residues, may serve as a substrate for a variety of apoptosis-related proteases, including caspase-3 and caspase-7, and does not appear to require any invasive techniques such as osmotic shock to gain entrance into the cytoplasm ().

Caspase-8 Substrates

Caspase-8 plays a critical role in the early cascade of apoptosis, acting as an initiator of the caspase activation cascade. Activation of the enzyme itself is accomplished through direct interaction with the death domains of cell-surface receptors for apoptosis-inducing ligands. The activated protease has been shown to be involved in a pathway that mediates the release of cytochrome c from the mitochondria and is also known to activate downstream caspases, such as caspase-3. The fluorogenic substrate Z-IETD-R110 (R22125, R22126; green fluorescent after cleavage) contains the caspase-8 recognition sequence Ile-Glu-Thr-Asp (IETD) (Fluorogenic substrates for caspase activity—Table 15.5).

Other Caspase and Granzyme B Substrates

In addition to our R110-derived caspase-3 and -8 substrates, we offer R110-based substrates for caspase-1, -2, -6, -9 and -13, as well as substrates for granzyme B (Fluorogenic substrates for caspase activity—Table 15.5). Granzyme B, a serine protease contained within cytotoxic T lymphocytes and natural killer cells, is thought to induce apoptosis in target cells by activating caspases and causing mitochondrial cytochrome c release.

EnzChek Caspase-3 Assay Kits

The EnzChek Caspase-3 Assay Kits permit the detection of apoptosis by assaying for increases in caspase-3 and caspase-3–like protease activities (Figure 10.4.3, Figure 10.4.4). Our EnzChek Caspase-3 Assay Kit #1 (E13183) contains the 7-amino-4-methylcoumarin (AMC)–derived substrate Z-DEVD-AMC (where Z represents a benzyloxycarbonyl group). This substrate, which is weakly fluorescent in the UV spectral range (excitation/emission maxima ~330/390 nm), yields the blue–fluorescent product AMC (A191, Introduction to Enzyme Substrates and Their Reference Standards—Section 10.1), which has excitation/emission maxima of 342/441 nm upon proteolytic cleavage.

The EnzChek Caspase-3 Assay Kit #2 (E13184) contains the R110-derived substrate, Z-DEVD-R110. This substrate is a bisamide derivative of R110, containing DEVD peptides covalently linked to each of R110's amino groups, thereby suppressing both the dye's visible absorption and fluorescence. Upon enzymatic cleavage by caspase-3 (or a closely related protease), the nonfluorescent bisamide substrate is converted in a two-step process first to the fluorescent monoamide and then to the even more fluorescent R110 (R6479, Introduction to Enzyme Substrates and Their Reference Standards—Section 10.1, Figure 10.4.1). Both of these hydrolysis products exhibit spectral properties similar to those of fluorescein, with excitation/emission maxima of 496/520 nm. The Z-DEVD-R110 substrate (R22120) is also available separately in a 20 mg unit size for high-throughput screening applications.

Either kit can be used to continuously measure the activity of caspase-3 and closely related proteases in cell extracts and purified enzyme preparations using a fluorescence microplate reader or fluorometer. AMC-based DEVD substrates, which yield blue fluorescence upon proteolytic cleavage, are widely used to monitor caspase-3 activity. The longer-wavelength spectra and higher extinction coefficient of the green-fluorescent products of the R110-based substrate in Kit #2 (E13184) should provide even greater sensitivity. The reversible aldehyde-based inhibitor Ac-DEVD-CHO can be used to confirm that the observed fluorescence signal in both induced and control cell populations is due to the activity of caspase-3–like proteases.

The EnzChek Caspase-3 Assay Kits contain:

• Z-DEVD-AMC (in Kit #1, E13183) or Z-DEVD-R110 (in Kit #2, E13184)
• Dimethylsulfoxide (DMSO)
• Concentrated cell-lysis buffer
• Concentrated reaction buffer
• Dithiothreitol (DTT)
• Ac-DEVD-CHO, a reversible aldehyde-based inhibitor
• 7-Amino-4-methylcoumarin (AMC) (in Kit E13183) or rhodamine 110 (in Kit E13184) reference standard to quantitate the amount of fluorophore released in the reaction
• Detailed protocols (EnzChek Caspase-3 Assay Kit #1 *Z-DEVD-AMC Substrate*, EnzChek Caspase-3 Assay Kit #2 *Z-DEVD-R110 Substrate*)

Each kit provides sufficient reagents for performing ~500 assays using a volume of 100 µL per assay. For information about additional kits and reagents for studying apoptosis, see Assays for Apoptosis—Section 15.5.

Figure 10.4.3 Detection of caspase-3 activity using the EnzChek Caspase-3 Assay Kit #1 (E13183). Increasing amounts of purified active human (recombinant) caspase-3 (PharMingen) were allowed to react with 100 µM Z-DEVD–AMC in 1X reaction buffer for ~45 minutes at room temperature. Fluorescence was measured in a fluorescence microplate reader using excitation at 360 ± 17.5 nm and emission detection at 465 ± 17.5 nm. Background fluorescence (386 arbitrary units), determined for a no-enzyme control, was subtracted from each value.

Figure 10.4.4 Detection of protease activity in Jurkat cells using the EnzChek Caspase-3 Assay Kit #1 with Z-DEVD-AMC substrate (E13183). Cells were either treated with 10 µM camptothecin for four hours at 37°C to induce apoptosis (induced) or left untreated (control). Both induced and control cells were then harvested, lysed and assayed. Reactions were carried out at room temperature, and fluorescence was measured in a fluorescence microplate reader using excitation at 360 ± 20 nm with emission detection at 460 ± 20 nm after the indicated amount of time.

RediPlate 96 EnzChek Caspase-3 Assay Kit

Our EnzChek Caspase-3 Assay Kit #2 is also available as a convenient RediPlate 96 EnzChek Caspase-3 Assay Kit (R35100, RediPlate 96 EnzChek Caspase-3 Assay Kit), which includes one 96-well microplate, contained in a resealable foil packet to ensure the integrity of the fluorogenic components, plus all necessary buffers and reagents for performing the assay (Figure 10.4.5). The enzyme sample to be assayed is added to the microplate in a suitable buffer, along with any compounds to be tested. Then, after incubation, the resultant fluorescence is quantitated on a fluorescence microplate reader equipped with filters appropriate for the green-fluorescent R110, with excitation/emission maxima of 496/520 nm. The microplate consists of twelve removable strips, each with eight wells, allowing researchers to perform only as many assay as required for the experiment (Figure 10.4.6). Eleven of the strips (88 wells) are preloaded with the Z-DEVD-R110 substrate. The remaining strip, marked with blackened tabs, contains a dilution series of free R110 that may be used as a fluorescence reference standard. The reversible aldehyde-based inhibitor Ac-DEVD-CHO, which is supplied in a separate vial, can be used to confirm that the observed fluorescence signal in both induced and control cell populations is due to the activity of caspase-3–like proteases.RediPlate Assay Kits—Table 10.3 summarizes our other RediPlate 96 Assay Kits for protease activity, phosphatase activity (Detecting Enzymes That Metabolize Phosphates and Polyphosphates—Section 10.3) and RNA quantitation (Nucleic Acid Quantitation in Solution—Section 8.3).

Figure 10.4.5 Detection of protease activity in Jurkat cells using the RediPlate 96 EnzChek Caspase-3 Assay Kit (R35100). Jurkat human T-cell leukemia cells were first exposed to 10 µM camptothecin at 37°C to induce apoptosis, and then harvested and lysed according to the kit protocol. The cell lysate was separated into two samples, one of which was treated with the Ac-DEVD-CHO inhibitor (provided in the RediPlate 96 EnzChek Caspase-3 Assay Kit). Assay reactions on both the inhibited and the uninhibited samples were carried out at 37°C, and fluorescence was measured in a fluorescence microplate reader (excitation/emission = 485/535 nm).

Figure 10.4.6 A RediPlate 96 microplate.

Image-iT LIVE Green Caspase Detection Kits for Fluorescence Microscopy

The Image-iT LIVE Green Caspase-3 and -7 Detection Kit, Image-iT LIVE Green Caspase-8 Detection Kit and Image-iT LIVE Green Poly Caspases Detection Kit (I35106, I35105, I35104) employ a novel approach to detect active caspases that is based on a fluorescent inhibitor of caspases (FLICA methodology). The FLICA inhibitor comprises a fluoromethyl ketone (FMK) moiety, which can react covalently with a cysteine, a caspase-selective amino acid sequence and a fluorescent carboxyfluorescein (FAM) reporter group. Essentially an affinity label, the FLICA inhibitor is thought to interact with the enzymatic reactive center of an activated caspase via the recognition sequence, and then to attach covalently to a cysteine through the reactive FMK moiety. The FLICA inhibitor's recognition sequence is aspartic acid–glutamic acid–valine–aspartic acid (DEVD) for caspase-3 and-7 detection, leucine–glutamic acid–threonine–aspartic acid (LETD) for caspase-8 detection and valine–alanine–aspartic acid (VAD) for detection of most caspases (including caspase-1, -3, -4, -5, -6, -7, -8 and -9). Importantly, the FLICA inhibitor is cell permeant and not cytotoxic; unbound FLICA molecules diffuse out of the cell and are washed away. The remaining green-fluorescent signal (excitation/emission maxima ~488/530 nm) can be used as a direct measure of the amount of active caspase that was present at the time the inhibitor was added. FLICA reagents have been used widely to study apoptosis with flow cytometry and microscopy. Recent work indicates that cellular fluorescence from the bound FLICA reagent is strongly linked to caspase activity in apoptotic cells; however, the interaction of the FLICA reagent with other cellular sites may contribute to signal intensity in nonapoptotic cells. Appropriate controls should be included in any experimental design.

The Image-iT LIVE Green Caspase Detection Kit includes:

• FAM-DEVD-FMK caspase-3 and -7 reagent (in Kit I35106), FAM-LETD-FMK caspase-8 reagent (in Kit I35105) or FAM-VAD-FMK poly caspases reagent (in Kit I35104)
• Hoechst 33342
• Propidium iodide
• Dimethylsulfoxide (DMSO)
• Apoptosis fixative solution
• Concentrated apoptosis wash buffer
• Detailed protocols for fluorescence microscopy assays (Image-iT LIVE Green Caspase Detection Kits)

In addition to a specific FLICA reagent, each kit provides Hoechst 33342 and propidium iodide stains, which allow the simultaneous evaluation of caspase activation, nuclear morphology and plasma membrane integrity. Sufficient reagents are provided for 25 assays, based on labeling volumes of 300 µL. These Image-iT LIVE Green Caspase Detection Kits can also be used in combination with other reagents for multiparametric study of apoptosis.

Image-iT LIVE Red Caspase Detection Kits for Fluorescence Microscopy

The Image-iT LIVE Red Caspase-3 and -7 Detection Kit and Image-iT LIVE Red Poly Caspases Detection Kit (I35102, I35101) are analogous to the Image-iT LIVE Green Caspase Detection Kits except that the FLICA reagent contains a red-fluorescent sulforhodamine (SR) reporter group instead of a green-fluorescent carboxyfluorescein (FAM) reporter group. This assay's red-fluorescent signal (excitation/emission maxima ~550/595 nm) can be used as a direct measure of the amount of active caspase that was present at the time the inhibitor was added.

The Image-iT LIVE Red Caspase Detection Kit includes:

• SR-DEVD-FMK caspase-3 and -7 reagent (in Kit I35102) or SR-VAD-FMK poly caspases reagent (in Kit I35101)
• Hoechst 33342
• SYTOX Green nucleic acid stain
• Dimethylsulfoxide (DMSO)
• Apoptosis fixative solution
• Concentrated apoptosis wash buffer
• Detailed protocols for fluorescence microscopy assays (Image-iT LIVE Red Caspase Detection Kits)

In addition to a specific FLICA reagent, each kit provides Hoechst 33342 and SYTOX Green nucleic acid stains, which allow the simultaneous evaluation of caspase activation, nuclear morphology and plasma membrane integrity. Sufficient reagents are provided for 25 assays, based on labeling volumes of 300 µL.

Vybrant FAM Caspase Assay Kits for Flow Cytometry

Like the Image-iT Kits described above, the Vybrant FAM Caspase Assay Kits for flow cytometry are based on a fluorescent caspase inhibitor (FLICA methodology). We offer three different Vybrant FAM Caspase Assay Kits designed to target different caspases. The Vybrant FAM Caspase-3 and -7 Assay Kit (V35118) provides a FLICA inhibitor containing the caspase-3 and -7 recognition sequence DEVD; the Vybrant FAM Caspase-8 Assay Kit (V35119) provides a FLICA inhibitor containing the caspase-8 recognition sequence Leu-Glu-Thr-Asp (LETD); and the Vybrant FAM Poly Caspases Assay Kit (V35117) provides a FLICA inhibitor containing the caspase recognition sequence Val-Ala-Asp (VAD), which is recognized by caspase-1, -3, -4, -5, -6, -7, -8 and -9. In addition to the selective FLICA reagent, these kits contain the Hoechst 33342 and propidium iodide nucleic acid stains to permit simultaneous evaluation of caspase activation, membrane permeability and cell cycle.

Each Vybrant FAM Caspase Assay Kit includes:

• FAM-DEVD-FMK caspase-3 and -7 reagent (in Kit V35118), FAM-LETD-FMK caspase-8 reagent (in Kit V35119) or FAM-VAD-FMK poly caspases reagent (in Kit V35117)
• Hoechst 33342
• Propidium iodide
• Dimethylsulfoxide (DMSO)
• Apoptosis fixative solution
• Concentrated apoptosis wash buffer
• Detailed protocols for flow cytometry assays (Vybrant FAM Caspase-3 and -7 Assay Kit, Vybrant FAM Caspase-8 Assay Kit, Vybrant FAM Poly Caspases Assay Kit)

Sufficient reagents are provided for 25 assays, based on labeling volumes of 300 µL. These Vybrant FAM Caspase Assay Kits can be used in combination with other fluorescent probes, such as the far-red–fluorescent allophycocyanin annexin V (A35110), for a multiparameter study of apoptosis.

Alternative strategies have been employed to create substrates specifically for some endopeptidases. Our HIV protease and renin substrates (H2930, R2931) utilize fluorescence resonance energy transfer (FRET) to generate a spectroscopic response to protease cleavage (Fluorescence Resonance Energy Transfer (FRET)—Note 1.2). In this type of substrate, both an acceptor molecule and a fluorescent molecule are attached to the peptide or protein. The acceptor molecule is carefully chosen so that its absorbance overlaps with the fluorophore's excited-state fluorescence (Figure 10.4.7), thus ensuring that the fluorescence is quenched through resonance energy transfer. Enzyme hydrolysis of the substrate results in spatial separation of the fluorophore and the acceptor molecule, thereby restoring the fluorophore's fluorescence (Figure 10.4.8). See Peptide Analysis, Sequencing and Synthesis—Section 9.5 for a discussion of our reagents for synthesizing labeled peptides and peptidase substrates, including our QSY series of nonfluorescent dyes, which have broad visible or near-infrared absorption spectra and serve as almost universal quenchers of most fluorescent donors that emit in the visible, with unusually high efficiency (Molecular Probes amine-reactive dyes—Table 1.1).

Figure 10.4.7 Spectral overlap between EDANS fluorescence and dabcyl absorption, which is required for efficient quenching of EDANS fluorescence by resonance energy transfer to the nonfluorescent dabcyl chromophore. Spectra are normalized to the same intensities.

Figure 10.4.8 Principle of the fluorogenic response to protease cleavage exhibited by HIV protease substrate 1 (H2930). Quenching of the EDANS fluorophore (F) by distance-dependent resonance energy transfer to the dabcyl quencher (Q) is eliminated upon cleavage of the intervening peptide linker.

Substrate for Detecting HIV Protease Activity

HIV protease substrate 1 (H2930) is a peptide that includes the HIV protease cleavage site, along with two covalently modified amino acid residues—one that has been linked to EDANS and the other to dabcyl. Proteolytic cleavage releases a fragment containing only the EDANS fluorophore, thus liberating it from the quenching effect of the nearby dabcyl chromophore (Figure 10.4.8). HIV protease activity can be measured by exciting the sample at ~340 nm and measuring the resulting fluorescence at 490 nm. HIV protease substrate 1 has been used to analyze the effects of solvent composition, incubation time and enzyme concentration on HIV-1 protease activity and to investigate a newly designed inhibitor of the enzyme. HIV protease substrate 1 has also been employed to follow the inhibition of HIV-1 protease activity after the enzyme's two cysteine residues are reversibly modified by nitric oxide. One milligram of HIV protease substrate 1 is sufficient for approximately 120 enzyme assays using 2 mL assay volumes and standard fluorescence cuvettes or ~1600 assays using 150 µL assay volumes and microcuvettes.

Human Renin Substrate 1

Assaying renin activity with human renin substrate 1 (R2931) is analogous to assaying HIV protease activity with the HIV protease substrate described above. Renin, an aspartic protease, plays an important role in blood-pressure regulation and is therefore a target for anti-hypertension therapeutics. Using renin substrate 1, researchers have discovered a stable, partially active conformational variant of recombinant human prorenin. This substrate has also been used to investigate the kinetics and pH stability of recombinant human renin. A fluorogenic substrate similar to our renin substrate 1 was used to develop a microplate assay for screening renin inhibitors. One milligram of the renin substrate 1 is sufficient for approximately 100 enzyme assays using 2 mL volumes and standard fluorescence cuvettes or ~1400 assays using 150 µL assay volumes and microcuvettes. The short-wavelength excitation maximum (335 nm) of the EDANS fluorophore precludes use of this substrate in most fluorescence microplate readers. We also has a fluorogenic substrate for thrombin (rhodamine 110, bis-(tosyl-Gly-Pro-Arg) amide, R22124; see above) that is useful for measuring the activity of this enzyme, which is important for coagulation and blood clot formation.

Often it is necessary to have fluorogenic substrates for the assay of purified enzymes with unknown specificity or for which there are no known useful substrates. Assay for contamination of biological preparations by unknown proteases requires substrates that can detect a variety of enzymes. We have developed a method of relieving the fluorescence quenching of BODIPY dye–labeled biopolymers by enzymatic hydrolysis (Figure 10.4.9), which we have applied to several of the general or selective protease assay kits and DQ reagents described in this section. We have also described the method's use in an assay for dextranase, and others have applied it to the assay of O-sialoglycoprotein endopeptidase and enzymes that process vesicular stomatitis virus (VSV). The DQ and EnzChek protease assay reagents may have significant potential for detecting matrix metalloproteinase (MMP) activity in living tissues by simple incubation of the tissue with the protein-based fluorogenic substrates. Fluorescent products have been shown to accumulate on the cell's surface where proteases are active, including in living human breast cancer cells. These quenched protease substrates are particularly useful for following cell migration through matrices ().

The EnzChek Protease Assay Kits provide exceptionally fast, simple and direct fluorescence assays for detecting metallo-, serine, acid and thiol proteases. The EnzChek Protease and EnzChek Peptidase/Protease Assay Kits (E6638, E6639, E33758) measure the increase in fluorescence intensity that results from protease hydrolysis of a quenched substrate, whereas our EnzChek Polarization Assay Kit for Proteases (E6658) monitors fluorescence polarization changes that occur during protease hydrolysis of a lightly labeled fluorescent casein derivative. Although the detection principles of these protease assays are quite different, no separation steps are required for either, and both types of assays are rapid, sensitive and versatile.

EnzChek Protease Assay Kits for Fluorescence Intensity Measurements

The EnzChek Protease Assay Kits contain a casein derivative that is heavily labeled with either the green-fluorescent BODIPY FL or red-fluorescent BODIPY TR-X dye, resulting in almost total quenching of the conjugate's fluorescence; they typically exhibit <3% of the fluorescence of the corresponding free dyes. Protease-catalyzed hydrolysis relieves this quenching, yielding brightly fluorescent BODIPY FL dye– or BODIPY TR-X dye–labeled peptides (Figure 10.4.9). The increase in fluorescence, which can be measured with a spectrofluorometer, minifluorometer or fluorescence microplate reader, is directly proportional to protease activity.

In contrast to the conventional fluorescein thiocarbamoyl (FTC)–casein protease assay, these EnzChek assays do not involve any separation steps and, consequently, can be used to continuously measure the kinetics of a variety of exopeptidases and endopeptidases over a wide pH range. They can also be used to measure the total substrate turnover at a fixed time following addition of the enzyme. We have found that these protease assays are over 100-times more sensitive and much easier to perform than the labor-intensive FTC–casein assay. Detection limits for fluorescence intensity measurements with these kits are given in Detection limits of the EnzChek Protease Assay Kits—Table 10.4.

Hydrolysis of the fluorogenic substrates by proteases provides a sensitive assay of cell proliferation and a means of detecting the sterility of a sample. In addition to their utility for detecting protease contamination in culture medium and other experimental samples, BODIPY FL casein and BODIPY TR-X casein have significant potential as general, nontoxic, pH-insensitive markers for phagocytic cells in culture (Probes for Following Receptor Binding and Phagocytosis—Section 16.1). We have shown that uptake of these quenched conjugates by neutrophils is accompanied by hydrolysis of the labeled proteins by intracellular proteases and generation of fluorescent products that are well retained in cells. This phagocytosis assay is readily performed in a fluorescence microplate reader or a flow cytometer; localization of the fluorescent products can be determined by fluorescence microscopy. The same substrates can readily detect secretion of proteases from live cells.

BODIPY FL casein and BODIPY TR-X casein can be used interchangeably, depending on whether green or red fluorescence is desired. The peptide hydrolysis products of BODIPY FL casein exhibit green fluorescence that is optimally excited by the argon-ion laser, permitting flow sorting of the cells. The red-fluorescent BODIPY TR-X–labeled peptides, with excitation and emission spectra similar to those of the Texas Red fluorophore, should be useful for multilabeling experiments or measurements in the presence of green autofluorescence.

The EnzChek Protease Assay Kit includes:

• BODIPY FL casein (in Kit E6638) or BODIPY TR-X casein (in Kit E6639)
• Concentrated digestion buffer
• Detailed protocols (EnzChek Protease Assay Kit)

Each kit provides sufficient reagents for ~100 assays using 2 mL assay volumes and standard fluorescence cuvettes or ~1000 assays using 200 µL assay volumes and 96-well microplates.

The EnzChek Protease Assay Kit containing red-fluorescent BODIPY TR-X casein is also available as a convenient RediPlate 96 Protease Assay Kit (R22132). Each RediPlate 96 EnzChek Protease Assay Kit includes one 96-well microplate, with all of the necessary reagents predispensed into the wells, where 88 wells (11 lanes) are intended for assays and 8 wells (1 lane) include a dilution series of an appropriate reference standard for generation of standard curves. The enzyme sample to be assayed is added to the microplate in a suitable buffer, along with any compounds to be tested. Then, after incubation, the resultant fluorescence is quantitated on a fluorescence microplate reader equipped with filters appropriate for the green- or red-fluorescent dye. Each RediPlate 96 microplate has removable lanes that allow researchers to perform only as many assays as required for the experiment (Figure 10.4.6). Resealable packaging ensures plate and well integrity between experiments. RediPlate Assay Kits—Table 10.3 summarizes our other RediPlate Assay Kits for caspase-3 activity (see above), phosphatase activity (Detecting Enzymes That Metabolize Phosphates and Polyphosphates—Section 10.3) and RNA quantitation (Nucleic Acid Quantitation in Solution—Section 8.3).

Figure 10.4.9 Principle of enzyme detection via the disruption of intramolecular self-quenching. Enzyme-catalyzed hydrolysis of the heavily labeled and almost totally quenched substrates provided in the EnzChek Protease Assay Kits (E6638, E6639), EnzChek Ultra Amylase Assay Kit (E33651), EnzChek Gelatinase/Collagenase Assay Kit (E12055), EnzChek Elastase Kit (E12056), EnzChek Lysozyme Assay Kit (E22013)—as well as the stand-alone quenched substrates DQ BSA (D12050, D12051), DQ collagen (D12052, D12060), DQ ovalbumin (D12053) and DQ gelatin (D12054)—relieves the intramolecular self-quenching, yielding brightly fluorescent reaction products.

EnzChek Peptidase/Protease Assay Kit

The EnzChek Peptidase/Protease Assay Kit (E33758) provides a FRET (fluorescence resonance energy transfer)–based method for the simple and accurate quantitation of a wide range of protease activities. The EnzChek peptidase/protease substrate comprises a fluorophore and a quencher moiety separated by an amino acid sequence. Upon protease cleavage, the fluorophore separates from the quencher and is free to emit a detectable fluorescent signal (excitation/emission maxima ~502/528 nm). The magnitude of the resultant signal is proportional to the degree of substrate cleavage and can therefore be used to quantitate the enzyme activity present (Figure 10.4.10). The assay is performed in a simple mix–incubate–read format and can be completed in 60 minutes or less.

The EnzChek Peptidase/Protease Assay Kit contains:

• EnzChek® peptidase/protease substrate
• Concentrated digestion buffer (200 mM Tris-HCl, pH 7.8)
• Substrate solvent (50% DMSO in 10 mM Tris-HCl, pH 7.8)
• Detailed protocols (EnzChek Peptidase/Protease Assay Kit)

Sufficient materials are supplied for 100 assays, based on a 100 µL assay volume in a 96-well microplate format. The EnzChek peptidase/protease assay can be adapted for use in cuvettes or 384-well microplates.

Figure 10.4.10 Sample standard curves obtained with the EnzChek Peptidase/Protease Assay Kit. Trypsin (EC 3.4.21.4) was assayed in 10 mM Tris-HCl (pH 7.8) digestion buffer from 10 to 1200 mU/mL using the EnzChek peptidase/protease substrate. The inset shows a separate experiment using the same enzyme, but at activities from 2 to 100 mU/mL. Samples were incubated for 60 minutes at room temperature. Fluorescence was measured at 490/520 nm; background fluorescence was subtracted from the inset data.

EnzChek Protease Assay Kit for Fluorescence Polarization Measurements

When a fluorescent molecule tethered to a protein is excited by polarized fluorescent light, the polarization of fluorescence emission is dependent on the rate of molecular tumbling. Upon proteolytic cleavage of the fluorescently labeled protein, the resultant smaller peptides tumble faster, and the emitted light is depolarized relative to the light measured from the intact conjugate. The EnzChek Polarization Assay Kit for Proteases (E6658) contains green-fluorescent BODIPY FL casein with an optimal degree of labeling for fluorescence polarization–based protease assays. Fluorescence polarization technology is more sensitive than many nonradioactive protease assays and allows measurements to be taken in real time, permitting the collection of kinetics data (Fluorescence Polarization (FP)—Note 1.4). Our BODIPY FL dye has an adequate fluorescence lifetime and pH-insensitive fluorescence—two prerequisites for successful measurement of protease activity by fluorescence polarization.

The EnzChek Polarization Assay Kit for Proteases contains:

• BODIPY FL casein
• Concentrated digestion buffer
• Detailed protocols

Each kit provides sufficient reagents for ~100 assays using 2 mL assay volumes and standard fluorescence cuvettes or ~1000 assays using 200 µL assay volumes and 96-well microplates. With the advent of high-capacity automated instrumentation, this kit provides an important tool for high-throughput screening of proteases and their inhibitors in research laboratories.

Fluorescein Casein

We also offer fluorescein casein (C2990) for assaying protease activity. In this assay, unhydrolyzed fluorescein casein must be precipitated with trichloroacetic acid, separated by centrifugation, transferred for measurement and then pH-adjusted for fluorescein signal enhancement. Fluorescein casein may be useful for a continuous assay if monitored by fluorescence polarization. Fluorescein casein is rapidly degraded by Bacteroides gingivalis but only slowly by streptococci.

Collagen is a major component of the extracellular matrix, which not only serves as scaffolding to stabilize tissue structure, but also influences the development, migration, proliferation and metabolism of cells that contact it. Gelatinases and collagenases—matrix metalloproteinases (MMPs) that digest collagen or gelatin (denatured collagen)—are increasingly important to our understanding of both normal development and carcinogenesis. For example, gelatinase A (20,000-dalton MMP-2) is primarily responsible for degrading the helical domains of type IV collagen, the principal collagen of basement membranes. Thus, gelatinase A likely plays a major role in the turnover of basement membrane during fetal tissue development, wound healing, angiogenesis and tumor invasion.

The EnzChek Gelatinase/Collagenase Assay Kit (E12055) provides the speed, high sensitivity and convenience required for measuring gelatinase or collagenase activity or for screening inhibitors in a high-throughput format. This kit contains:

• DQ gelatin, a gelatin conjugate so heavily labeled with fluorescein that its fluorescence is quenched; this substrate is also available separately (D12054)
• 1,10-Phenanthroline, a general metalloproteinase inhibitor
• Type IV collagenase from Clostridium histolyticum for use as a positive control
• Concentrated reaction buffer
• Detailed protocols (EnzChek Gelatinase/Collagenase Assay Kit)

DQ gelatin, the highly quenched gelatinase/collagenase substrate provided, is efficiently digested by most, if not all, gelatinases and collagenases, releasing brightly fluorescent peptides (Figure 10.4.9). The increase in fluorescence upon digestion is proportional to proteolytic activity and can be monitored with a fluorescence microplate reader, minifluorometer or spectrofluorometer. Depending on the substrate concentration used in each reaction, sufficient reagents are supplied for 250–1000 assays using 200 µL assay volumes and 96-well microplates. Using 100 µg/mL DQ gelatin and a two-hour incubation period, we have detected as little as 2 × 10^-3 U/mL (7 ng protein/mL) of C. histolyticum collagenase, where one unit is defined as the amount of enzyme required to liberate 1 µmole L-leucine equivalents from collagen in 5 hours at 37°C, pH 7.5. Longer incubation times increase the sensitivity, whereas higher enzyme concentrations decrease the incubation times. Using human gelatinase A (not provided), 100 µg/mL DQ gelatin and a 24-hour incubation period, we have detected concentrations of gelatinase as low as 3 × 10^-4 U/mL, where one unit is defined as the amount of enzyme that can hydrolyze 1 mg of type IV collagen in one hour at 37°C, pH 7.5. DQ gelatin and some of our other highly quenched protease substrates have been utilized for in situ detection of matrix metalloproteinase and other protease activity in cell preparations, tissue sections and SDS gels.

The EnzChek Elastase Assay Kit (E12056) provides the speed, high sensitivity and convenience required for measuring elastase activity or for screening inhibitors in a high-throughput format. This kit contains DQ elastin—soluble bovine neck ligament elastin that has been labeled with our BODIPY FL dye such that the conjugate's fluorescence is quenched.Upon digestion by elastase or other proteases, the fluorescence is revealed (Figure 10.4.9). The resulting increase in fluorescence can be monitored with a fluorescence microplate reader, minifluorometer or spectrofluorometer. Digestion products from the DQ elastin substrate have absorption maxima at ~505 nm and fluorescence emission maxima at ~515 nm. Because the assay is continuous, kinetics data can be obtained easily. Furthermore, because fluorescence of the BODIPY FL dye is pH insensitive between pH 3 and 9, the assay can be performed under a variety of buffer conditions. Please note that DQ elastin is also digested by proteases other than elastase.

The EnzChek Elastase Assay Kit (A10256) contains:

• DQ elastin substrate
• Concentrated reaction buffer
• Elastase from pig pancreas for use as a positive control
• N-Methoxysuccinyl-Ala-Ala-Pro-Val-chloromethyl ketone, a selective elastase inhibitor
• Detailed protocols (EnzChek Elastase Assay Kit)

Each kit provides sufficient reagents for approximately 600 assays using a fluorescence microplate reader and reaction volumes of 200 µL. The N-methoxysuccinyl-Ala-Ala-Pro-Val-chloromethyl ketone inhibitor can be used to confirm the identity of the protease responsible for substrate digestion or, alternatively, as a control inhibitor for use when screening for elastase inhibitors.

DQ Collagens

Our DQ collagens, type I (D12060) and type IV (D12052), are complementary reagents to the DQ gelatin provided in the EnzChek Gelatinase/Collagenase Assay Kit. Like DQ gelatin, these highly quenched substrates are heavily labeled with fluorescein and release fluorescent peptides when enzymatically cleaved. DQ collagen, type I should be useful in assays detecting matrix metalloproteases (MMP-1) activity. DQ collagen, type IV may prove particularly useful in the development of assays for gelatinase A (MMP-2) and gelatinase A inhibitors, as well as for other gelatinases and collagenases that specifically degrade type IV collagen. DQ collagens may be used with the EnzChek Gelatinase/Collagenase Assay Kit, and a sample protocol is included. Because of its more complex structure, DQ collagens generally require either greater amounts of the enzyme or longer incubation periods than does DQ gelatin. Please note that both the DQ collagens and DQ gelatin can be digested by proteases other than gelatinases and collagenases. The fluorescence generated by hydrolysis of DQ collagen (D12052) by cellular collagenase has been used to visualize the migratory pathway followed by tumor cells during invasion of a gelatin matrix and to image proteolysis by living breast cancer cells.

In addition to the DQ substrates, we have prepared gelatin and collagen conjugates that have been labeled to maximize probe fluorescence and minimize dye quenching. We offer two green-fluorescent conjugates of gelatin, one in which gelatin is coupled to our photostable Oregon Green 488 dye (G13186, Probes for Cell Adhesion, Chemotaxis, Multidrug Resistance and Glutathione—Section 15.6) and the other to fluorescein (G13187, Probes for Cell Adhesion, Chemotaxis, Multidrug Resistance and Glutathione—Section 15.6).

DQ BSA

DQ Green BSA (D12050, excitation/emission maxima ~500/506 nm) and DQ Red BSA (D12051, excitation/emission maxima ~589/617 nm) are bovine serum albumin (BSA) conjugates that have been labeled to such a high degree that the BODIPY dyes used to label them are strongly self-quenched. Proteolysis of the DQ BSA can easily be monitored as proteolytic fragments containing the fluorophores are released from the larger conjugate and become brightly fluorescent (Figure 10.4.9). An unlabeled neutrophil making its way through a gelatin matrix containing a DQ Green BSA prototype, as well as dihydrotetramethylrosamine, a nonfluorescent probe that fluoresces bright orange upon oxidation, leaves behind a fluorescent trail (). The alternating green- and orange-fluorescent bands dramatically demonstrate that the proteolytic and oxidative activities of the migrating neutrophil are oscillatory and are 180° out of phase with each other. Intracellular processing of a similar BODIPY BSA conjugate by J774 macrophages can be completely inhibited by protease inhibitors. DQ Green BSA has been embedded in a gelatin matrix and used to image both extracellular and intracellular proteolysis in living cells. DQ BSA can be complexed with our rabbit IgG fraction anti-BSA (A11133, Anti–Epitope Tag and Anti-Reporter Antibodies—Section 7.5) to form an immune complex that is internalized through the Fc receptor and processed in the phagovacuole to highly fluorescent peptides (Probes for Following Receptor Binding and Phagocytosis—Section 16.1). DQ Green BSA has been used for imaging proteolysis in living breast cancer cells.

DQ Ovalbumin

DQ ovalbumin (D12053) is a self-quenched ovalbumin conjugate designed specifically for the study of antigen processing. Ovalbumin is efficiently processed through mannose receptor–mediated endocytosis by antigen-presenting cells and is widely used for studying antigen processing. DQ ovalbumin is labeled with our pH-insensitive, green-fluorescent BODIPY FL dye such that the fluorescence is almost completely quenched. Upon endocytosis and proteolysis, highly fluorescent peptides are released. DQ ovalbumin appears to be an excellent indicator of macrophage-mediated antigen processing in flow cytometry and microscopy assays.

Peptidases typically liberate a free amine for each hydrolysis step. Thus, fluorogenic amine detection reagents such as fluorescamine (F2332, F20261; Reagents for Analysis of Low Molecular Weight Amines—Section 1.8) and o-phthaldialdehyde (P2331MP, Reagents for Analysis of Low Molecular Weight Amines—Section 1.8) have been used to detect the rate of amine production by peptidases.

Peptidases that liberate single free amino acids for which specific oxidases exist can be analyzed by coupling the hydrolytic reaction to oxidation of our Amplex Red reagent (Substrates for Oxidases, Including Amplex Red Kits—Section 10.5) to the red-fluorescent dye resorufin. For example, glutamic acid production can be monitored using glutamate oxidase and D-amino acid liberation monitored using a D-amino acid oxidase.

Protease assays conducted in highly autofluorescent or strongly light-scattering solutions (such as crude cell and tissue extracts) often can be improved by extracting the fluorescent hydrolysis product from the assay mixture with an organic solvent such as toluene, chloroform or ethyl acetate. Most unhydrolyzed peptidase substrates will remain in the aqueous layer.

Endopeptidase substrates that are singly labeled at the amine terminus with a fluorophore usually do not undergo a fluorescence change upon hydrolysis of internal peptide bonds; however, fluorescence (or absorbance) of the fluorophore that remains attached to the cleaved peptide can be used to detect the hydrolysis product following separation by TLC, HPLC or capillary electrophoresis.

Alexa Fluor 488 Soybean Trypsin Inhibitor

Trypsin inhibitor from soybean (SBTI) is a 21,000-dalton protein that inhibits the catalytic activity of serine proteases. SBTI binds to acrosin, an acrosomal serine protease that is associated with binding of spermatozoa and penetration of the zona pellucida, and SBTI binding patterns in non-fixed human sperm have demonstrated its usefulness for detecting acrosome-reacted sperm. In particular, an Alexa Fluor 488 conjugate of the protein has been used to measure the acrosomal status of macaque sperm and to determine the localization of acrosin during the reaction. We provide a fluorescent SBTI conjugate with one of our best fluorophores, the Alexa Fluor 488 dye (T23011). Alexa Fluor 488 dye (Alexa Fluor Dyes Spanning the Visible and Infrared Spectrum—Section 1.3) has spectral characteristics similar to fluorescein (excitation at 495 nm and emission at 519 nm) but produces conjugates that are brighter and more photostable. Furthermore, the fluorescence of Alexa Fluor 488 conjugates is insensitive to pH from 4 to 10.

BODIPY FL Pepstatin

Pepstatin A is an inhibitor of carboxyl (acid) proteases that contain aspartate residues at their active sites, including cathepsin D, pepsin and renin. We have prepared the green-fluorescent BODIPY FL pepstatin A analog (P12271), which we have shown binds to cathepsin D in live cells (Assays for Apoptosis—Section 15.5).

Leupeptin Hemisulfate

Leupeptin hemisulfate (L6543) can be used to inhibit serine and cysteine proteases. It does not inhibit chymotrypsin and thrombin.