Introduction to Enzyme Substrates and Their Reference Standards - Section 10.1

Solution assays designed to quantitate enzymatic activity in cell extracts or other biological fluids typically employ substrates that yield highly fluorescent or intensely absorbing water-soluble products. ELISAs also rely on these substrates for indirect quantitation of analytes. An ideal fluorogenic substrate for fluorescence-based solution assays yields a highly fluorescent, water-soluble product with optical properties significantly different from those of the substrate. If the fluorescence spectra of the substrate and product overlap significantly, analysis will likely require a separation step, especially when using excess substrate to obtain pseudo–first-order kinetics. Fortunately, many substrates have low intrinsic fluorescence or are metabolized to products that have longer-wavelength excitation or emission spectra (Figure 10.1). These fluorescent products can typically be quantitated in the presence of the unreacted substrate using a fluorometer or a fluorescence microplate reader. Microplate readers facilitate high-throughput analysis and require relatively small assay volumes, which usually reduces reagent costs. Moreover, the front-face optics in many microplate readers allows researchers to use more concentrated solutions, which may both improve the linearity of the kinetics and reduce inner-filter effects.

When the spectral characteristics of the substrate and its metabolic product are similar, techniques such as thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), capillary electrophoresis, solvent extraction or ion exchange can be used to separate the product from unconsumed substrate prior to analysis. For example, our FAST CAT Chloramphenicol Acetyltransferase Assay Kits (F2900, F6616, F6617; Substrates for Microsomal Dealkylases, Acetyltransferases, Luciferases and Other Enzymes - Section 10.6) utilize chromatography to separate the intrinsically fluorescent substrates from their fluorescent products.

Figure 10.1 Normalized emission spectra of 1) 7-hydroxy-4-methylcoumarin (H189) and 6,8-difluoro-7-hydroxy-4-methylcoumarin (DiFMU, D6566), 2) fluorescein (F1300, F36915), 3) resorufin (R363) and 4) DDAO (H6482) in aqueous solution at pH 9. These fluorophores correspond to the hydrolysis, oxidation or reduction products of several of our fluorogenic enzyme substrates.

Substrates Derived from Water-Soluble Coumarins

Hydroxy- and amino-substituted coumarins have been the most widely used fluorophores for preparing fluorogenic substrates. Coumarin-based substrates produce highly soluble, intensely blue-fluorescent products. Phenolic dyes with high pK_as, such as 7-hydroxycoumarin (often called umbelliferone) and the more common 7-hydroxy-4-methylcoumarin (β-methylumbelliferone, H189; Figure 10.2), are not fully deprotonated and therefore not fully fluorescent unless the pH of the reaction mixture is raised to above pH ~10. Thus, substrates derived from these fluorophores are seldom used for continuous measurement of enzymatic activity in solution or live cells. The similar 3-cyano-7-hydroxycoumarin (C183) and 6,8-difluoro-7-hydroxy-4-methylcoumarin (DiFMU, D6566; Figure 10.2, ) have lower pK_as (Figure 1.95), making them suitable for a broader range of applications. Ether, ester and phosphate substrates derived from these phenolic dyes may be fluorescent but invariably exhibit shorter-wavelength absorption and emission spectra that can be easily distinguished from those of their metabolic product. The phosphate ester of 6,8-difluoro-7-hydroxy-4-methylcoumarin (DiFMUP, D6567, D22065, E12020; Detecting Enzymes That Metabolize Phosphates and Polyphosphates - Section 10.3) exhibits extraordinary spectral properties, making it one of the most sensitive fluorogenic substrates for continuous high-throughput assay of alkaline phosphatase and its bioconjugates.

Figure 1.95 Comparison of the pH-dependent fluorescence changes produced by attachment of electron-withdrawing fluorine atoms to a hydroxycoumarin. 7-Hydroxy-4-methylcoumarin-3-acetic acid (, H1428) and 6,8-difluoro-7-hydroxy-4-methylcoumarin (, D6566) demonstrate the decrease of the pK_a from ~7.4 to ~6.2. Fluorescence intensities were measured for equal concentrations of the two dyes using excitation/emission at 360/450 nm.

Figure 10.2 Absorption and fluorescence emission spectra of 7-hydroxy-4-methylcoumarin (H189) in pH 9.0 buffer. The spectra of 6,8-difluoro-7-hydroxy-4-methylcoumarin (D6566) are essentially identical.

Aromatic amines, including the commonly used 7-amino-4-methylcoumarin (AMC, A191; ), are partially protonated at low pH (less than ~5) but fully deprotonated at physiological pH. Thus, their fluorescence spectra are not subject to variability due to pH-dependent protonation/deprotonation when assayed near or above physiological pH. AMC is widely used to prepare peptidase substrates in which the amide has shorter-wavelength absorption and emission spectra than the amine hydrolysis product.

Substrates Derived from Water-Soluble Green to Yellow Fluorophores

As compared with coumarin-based substrates, substrates derived from fluoresceins, rhodamines, resorufins and some other dyes often provide significantly greater sensitivity in fluorescence-based enzyme assays. In addition, most of these longer-wavelength dyes have extinction coefficients that are five to 25 times that of coumarins, nitrophenols or nitroanilines, making them additionally useful as sensitive chromogenic substrates.

Hydrolytic substrates based on the derivatives of fluorescein (fluorescein reference standard, F1300; fluorescein NIST-traceable standard, F36915; ) or rhodamine 110 (R110, R6479; ) usually incorporate two moieties, each of which serves as a substrate for the enzyme. Consequently, they are cleaved first to the monosubstituted analog and then to the free fluorophore. Because the monosubstituted analog often absorbs and emits light at the same wavelengths as the ultimate hydrolysis product, this initial hydrolysis complicates the interpretation of hydrolysis kinetics. However, when highly purified, the disubstituted fluorescein- and rhodamine 110–based substrates have virtually no visible-wavelength absorbance or background fluorescence, making them extremely sensitive detection reagents. For example, researchers have reported that the activity of as few as 1.6 molecules of β-galactosidase can be detected with fluorescein di-β-D-galactopyranoside (FDG) and capillary electrophoresis. Fluorogenic substrates based on either the AMC and R110 fluorophore are used in our EnzChek Caspase Assay Kits (Assays for Apoptosis - Section 15.5) to detect apoptotic cells.

Chemical reduction of fluorescein- and rhodamine-based dyes yields colorless and nonfluorescent dihydrofluoresceins () and dihydrorhodamines (). Although extremely useful for detection of reactive oxygen species (ROS) in phagocytic and other cells (Generating and Detecting Reactive Oxygen Species - Section 18.2), these dyes tend to be insufficiently stable for solution assays. An exception is our Patented Amplex Gold reagent, which is utilized in our Amplex Gold and DyeChrome Double Western Blot Stain Kits (Figure 9.70). These kits are described in Multiplexed Proteomics Technology for Detecting Specific Proteins in Gels and on Blots - Section 9.4.

Figure 9.70 A total-protein profile and two specific protein bands visualized on a blot. A twofold dilution series of a protein mixture containing bovine serum albumin (BSA), tubulin, ovalbumin, carbonic anhydrase and soybean trypsin inhibitor (from 1 µg to 0.24 ng each) was separated by electrophoresis through a 13% SDS-polyacrylamide gel and blotted onto a PVDF membrane. The DyeChrome Double Western Blot Stain Kit (D21887) components were used, together with two antibodies, to stain all proteins and to visualize two specific proteins. The total-protein profile was stained with the blue-fluorescent dye MDPF. Tubulin was detected using mouse monoclonal anti–α-tubulin antibody (A11126) followed by an alkaline phosphatase conjugate of goat anti-mouse IgG antibody, along with DDAO phosphate (red fluorescence). BSA was detected using an antibody against BSA followed by a horseradish peroxidase conjugate of goat anti–rabbit IgG antibody, along with the Amplex Gold reagent (yellow fluorescence). The fluorescent signals were detected separately using appropriate excitation light and emission filters on either the Fluor-S MAX MultiImager documentation system (Bio-Rad Laboratories) or the FLA3000G laser scanner (Fuji Photo Film Co.).

Substrates Derived from Water-Soluble Red Fluorophores

Long-wavelength fluorophores are often preferred because background absorbance and autofluorescence are generally lower when longer excitation wavelengths are used. Substrates derived from the red-fluorescent resorufin (R363, ) and the dimethylacridinone derivative 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one) (DDAO, H6482; Figure 10.7, ) contain only a single hydrolysis-sensitive moiety (), thereby avoiding the biphasic kinetics of both fluorescein- and rhodamine-based substrates.

Resorufin is used to prepare several substrates for glycosidases, hydrolytic enzymes and dealkylases. In most cases, the relatively low pK_a of resorufin (~6.0) permits continuous measurement of enzymatic activity. Thiols such as DTT or 2-mercaptoethanol should be avoided in assays utilizing resorufin-based substrates. Our Amplex Red peroxidase substrate (A12222, A22177; Substrates for Oxidases, Including Amplex Red Kits - Section 10.5) is a chemically reduced, colorless form of resorufin () that is oxidized to resorufin by HRP in combination with hydrogen peroxide. Resorufin is also the product of enzyme-catalyzed reduction of resazurin (R12204; Substrates for Microsomal Dealkylases, Acetyltransferases, Luciferases and Other Enzymes - Section 10.6, Viability and Cytotoxicity Assay Reagents - Section 15.2) — also known as alamarBlue, a trademark of AccuMed International, Inc. Our Amplex UltraRed reagent (A36006, Substrates for Oxidases, Including Amplex Red Kits - Section 10.5) improves upon the performance of the Amplex Red reagent, offering brighter fluorescence and enhanced sensitivity on a per-mole basis in peroxidase or peroxidase-coupled enzyme assays.

Substrates derived from DDAO, a red He–Ne laser–excitable fluorophore, generally exhibit good water solubility, low K_ms and high turnover rates. In addition, the difference between the excitation maximum of the DDAO-based substrates and that of the phenolic DDAO product is greater than 150 nm (Figure 10.7), which allows the two species to be easily distinguished. We have utilized DDAO phosphate (D6487, Detecting Enzymes That Metabolize Phosphates and Polyphosphates - Section 10.3) in several of our Pro-Q Glycoprotein Blot Stain Kits, as well as in some of our DyeChrome and Pro-Q Western Blot Stain Kits (Multiplexed Proteomics Technology for Detecting Specific Proteins in Gels and on Blots - Section 9.4) for the sensitive detection of proteins. In our unique DyeChrome Double Western Blot Kit (D21887, Multiplexed Proteomics Technology for Detecting Specific Proteins in Gels and on Blots - Section 9.4), we have combined the DDAO phosphate substrate with both the Amplex Gold HRP substrate and MPDF, a total-protein stain, for simultaneous trichromatic detection of two specific proteins and total proteins on Western blots (Figure 9.70).

Figure 10.7 Normalized absorption and fluorescence emission spectra of DDAO, which is formed by alkaline phosphatase–mediated hydrolysis of DDAO phosphate (D6487).

Molecular Probes has developed a number of innovative strategies for investigating enzymatic activity in live cells. For example, we offer a diverse set of probes that can passively enter the cell; once inside, they are processed by intracellular enzymes to generate products with improved cellular retention. We also offer kits and reagents for detecting the expression of several common reporter genes in cells and cell extracts. These include substrates for β-galactosidase (Detecting Glycosidases - Section 10.2), β-glucuronidase (Detecting Glycosidases - Section 10.2), secreted alkaline phosphatase (SEAP, Detecting Enzymes That Metabolize Phosphates and Polyphosphates - Section 10.3), chloramphenicol acetyltransferase (CAT, Substrates for Microsomal Dealkylases, Acetyltransferases, Luciferases and Other Enzymes - Section 10.6) and luciferase (Substrates for Microsomal Dealkylases, Acetyltransferases, Luciferases and Other Enzymes - Section 10.6). Some of our EnzChek and DQ Kits are useful for study of the uptake and metabolism of proteins during phagocytosis (Probes for Following Receptor Binding, Endocytosis and Exocytosis - Section 16.1), as well as for the general screening of certain glycosidases (Detecting Glycosidases - Section 10.2) and proteases (Detecting Peptidases and Proteases - Section 10.4). Substrates for specific proteases are also useful for the detection of apoptosis (Assays for Apoptosis - Section 15.5).

Thiol-Reactive Fluorogenic Substrates

Molecular Probes prepares a number of enzyme substrates for live-cell assays that incorporate a mildly thiol-reactive chloromethyl moiety. Once inside the cell, this chloromethyl group undergoes what is believed to be a glutathione S-transferase–mediated reaction to produce a membrane-impermeant, glutathione–fluorescent dye adduct, although our experiments suggest that they may also react with other intracellular components. Regardless of the mechanism, many cell types loaded with these chloromethylated substrates are both fluorescent and viable for at least 24 hours after loading and often through several cell divisions. Furthermore, unlike the free dye, the peptide–fluorescent dye adducts contain amino groups and can therefore be covalently linked to surrounding biomolecules by fixation with formaldehyde or glutaraldehyde. This property permits long-term storage of the labeled cells or tissue and, in cases where the anti-dye antibody is available (Anti-Dye and Anti-Hapten Antibodies - Section 7.4), amplification of the conjugate by standard immunochemical techniques, including the tyramide signal amplification (TSA, Tyramide Signal Amplification (TSA) Technology - Section 6.2) and Enzyme-Labeled Fluorescence (ELF, Enzyme-Labeled Fluorescence (ELF) Signal Amplification Technology - Section 6.3) technologies. Chloromethyl analogs of fluorogenic substrates for glycosidases (for example, our DetectaGene Green CMFDG Kit, (D2920); Detecting Glycosidases - Section 10.2), peptidases, dealkylases, peroxidases and esterases are available. Our CellTracker Blue CMAC and CellTracker Blue CMF₂HC dyes (C2110, ; C12881, ) are precursors to peptidase and glycosidase substrates, respectively. They are also used for long-term cell tracing (Membrane-Permeant Reactive Tracers - Section 14.2). The improved retention of the MitoTracker (Probes for Mitochondria - Section 12.2) and CellTracker (Membrane-Permeant Reactive Tracers - Section 14.2) probes in fixed cells is also based on this principle.

Lipophilic Fluorophores

Lipophilic analogs of fluorescein and resorufin exhibit many of the same properties as the water-soluble fluorophores, including relatively high extinction coefficients and good quantum yields. In most cases, however, substrates based on these lipophilic analogs load more readily into cells, permitting use of much lower substrate concentrations in the loading medium, and their fluorescent products are better retained after cleavage than their water-soluble counterparts. Lipophilic substrates and their products probably also distribute differently in cells and likely associate with lipid regions of the cell. When passive cell loading or enhanced dye retention are critical parameters of the experiment, we recommend using our lipophilic substrates for glycosidases (such as our ImaGene Green and ImaGene Red products, Detecting Glycosidases - Section 10.2) and dealkylases (Substrates for Microsomal Dealkylases, Acetyltransferases, Luciferases and Other Enzymes - Section 10.6). Like resazurin (R12204, Viability and Cytotoxicity Assay Reagents - Section 15.2), dodecylresazurin — the substrate in our LIVE/DEAD Cell Vitality Assay Kit, Vybrant Cell Metabolic Assay Kit and Vybrant Apoptosis Assay Kit #10 (V23110, L34951, V35114; Viability and Cytotoxicity Assay Kits for Diverse Cell Types - Section 15.3, Assays for Apoptosis - Section 15.5) — is reduced to dodecylresorufin by metabolically active cells; however, this lipophilic substrate is more useful than resazurin for microplate assays of all metabolic activity and permits single-cell analysis of cell metabolism by flow cytometry and cell counting (Figure 15.33, Figure 15.34, Figure 15.35, Figure 15.95). Dodecylresorufin is also the product produced by hydrolysis of the β-galactosidase substrate () used in our ImaGene Red C₁₂RG lacZ Gene Expression Kit (I2906, Detecting Glycosidases - Section 10.2).

Figure 15.33 Flow cytometric analysis of Jurkat cells using the LIVE/DEAD Cell Vitality Assay Kit (L34951). Jurkat human T-cell leukemia cells were first exposed to 10 µM camptothecin for 4 hours at 37°C, 5% CO₂. The cells were then treated with the reagents in the LIVE/DEAD Cell Vitality Assay Kit as specified in the kit protocol (Product Information Sheet) and analyzed by flow cytometry. This dot plot of SYTOX Green fluorescence versus resorufin fluorescence shows resolution of live-, injured- and dead-cell populations.

Figure 15.34 Flow cytometric analysis of Jurkat cells stained with C₁₂-resazurin. Cells were loaded with 0.1 µM C₁₂-resazurin, a component of the Vybrant Cell Metabolic Assay Kit (V23110), and 1 mM SYTOX Green (S7020). After a 15-minute incubation, the cells were analyzed. Healthy (live) cells reduce C₁₂-resazurin into red-fluorescent C₁₂-resorufin and exclude the cell impermeant green-fluorescent SYTOX Green. Dead cells show little reduction of the C₁₂-resazurin, but strong staining by SYTOX Green. Cells indicated in the Figure .s dying are of indeterminate viability, showing both reduction of C₁₂-resazurin and compromised membrane integrity.

Figure 15.35 Detection limit of C₁₂-resazurin and linear response with increasing cell number using our Vybrant Cell Metabolic Assay Kit (V23110). Jurkat cells were loaded with 5 µM C₁₂-resazurin for 15 minutes. The resulting signal was measured in a fluorescence microplate reader with excitation/emission at 530/590 nm. For comparison, the detection limit for alamarBlue (resazurin) in a similar experiment was ~8000 cells/well (data not shown).

Figure 15.95 Flow cytometric analysis of Jurkat cells using the Vybrant Apoptosis Assay Kit #10 (V35114). Jurkat human T-cell leukemia cells were first exposed to either 10 µM camptothecin or 2 mM hydrogen peroxide for 4 hours at 37°C, 5% CO₂. The cells were then combined, treated with the reagents in the Vybrant Apoptosis Assay Kit #10 and analyzed by flow cytometry. A) The SYTOX Green fluorescence versus allophycocyanin (APC) annexin fluorescence dot plot shows resolution of live, apoptotic and dead cell populations. The cell populations can be evaluated for metabolic activity using B) the dodecylresorufin fluorescence versus SYTOX Green fluorescence dot plot and C) the dodecylresorufin fluorescence versus allophycocyanin fluorescence dot plot.

Pentafluorobenzoyl Fluorogenic Enzyme Substrates

Detecting enzyme activity in live cells with fluorogenic substrates has been difficult both because the cell membrane is often a barrier to substrate penetration and because, once formed, the fluorescent product tends to leak from viable cells. We have found that our pentafluorobenzoyl (PFB) fluorogenic substrates address both of these difficulties. First, when compared with conventional fluorescein-based substrates, several of our PFB substrates exhibit improved penetration through the cell membrane, permitting cell loading directly from culture medium. Second, the green-fluorescent PFB aminofluorescein (PFB-F, P12925; ) released upon hydrolysis of the PFB-F substrates exhibits better cell retention than does fluorescein, the hydrolysis product of the fluorescein-based substrates. The hydrolysis products of the PFB substrates appear to be retained in viable cells by two mechanisms: 1) retention of the relatively lipophilic PFB group of the hydrolysis products in the cell membrane, and 2) glutathione S-transferase–catalyzed reaction of the nonfluorescent substrate and its fluorescent hydrolysis products with intracellular glutathione.

Alkaline phosphatase, β-galactosidase and horseradish peroxidase (HRP) conjugates are widely used as secondary detection reagents for immunohistochemical analysis and in situ hybridization, as well as for protein and nucleic acid detection by Western, Southern and Northern blots. Also, various methods such as chromatography, isoelectric focusing and gel electrophoresis are commonly employed to separate enzymes preceding their detection. A review by Weder and Kaiser discusses the use of a wide variety of fluorogenic substrates for the detection of electrophoretically separated hydrolases.

In order to precisely localize enzymatic activity in a tissue or cell, on a blot or in a gel, the substrate must yield a product that immediately precipitates or reacts at the site of enzymatic activity. In addition to the commonly used chromogenic substrates, including X-Gal, BCIP and NBT, Molecular Probes has developed fluorogenic ELF substrates for alkaline phosphatase and several other hydrolytic enzymes (Enzyme-Labeled Fluorescence (ELF) Signal Amplification Technology - Section 6.3). Our ELF substrates fluoresce only weakly in the blue range. However, upon enzymatic cleavage, these substrates form the intensely yellow-green–fluorescent ELF 97 alcohol (E6578), which precipitates immediately at the site of enzymatic activity (, , ). The fluorescent ELF alcohol precipitate is exceptionally photostable (Figure 6.16) and has a high Stokes shift (Figure 6.17). We offer several ELF kits based on our ELF 97 phosphatase substrate; see Enzyme-Labeled Fluorescence (ELF) Signal Amplification Technology - Section 6.3 for a complete discussion of our ELF technology. The similar ELF 39 phosphate (Figure 9.65) is used for detection of specific proteins in some of our DyeChrome Western Blot Stain Kits (Multiplexed Proteomics Technology for Detecting Specific Proteins in Gels and on Blots - Section 9.4, ). DDAO phosphate is very useful for solution assays but we have also been able to adapt it to yield a fluorescent precipitate that can detect proteins in Western blots; several kits containing DDAO phosphate are described in Multiplexed Proteomics Technology for Detecting Specific Proteins in Gels and on Blots - Section 9.4.

Figure 6.16 Photostability comparison for ELF 97 alcohol– and fluorescein-labeled tubulin preparations. Tubulin in acetone-fixed CRE BAG 2 mouse fibroblasts was labeled with an anti–β-tubulin monoclonal antibody and then detected using biotin-XX goat anti–mouse IgG antibody (B2763) in conjunction with either our ELF 97 Cytological Labeling Kit (E6603, ) or fluorescein streptavidin (S869, ). Alternatively, anti-tubulin labeling was detected directly using fluorescein goat anti–mouse IgG antibody (F2761, ). The photostability of labeling produced by the three methods was compared by continuously illuminating stained samples on a fluorescence microscope using Omega Optical longpass optical filter sets. Images were acquired every 5 seconds using a Star 1 CCD camera (Photometrics); the average fluorescence intensity in the field of view was calculated with Image-1 software (Universal Imaging Corp.) and expressed as a fraction of the initial intensity. Three data sets, representing different fields of view, were averaged for each conjugate to obtain the plotted time courses.

Figure 6.17 The normalized fluorescence excitation and emission spectra of the ELF 97 alcohol precipitate (E6578), which is generated by enzymatic cleavage of the soluble ELF 97 phosphatase substrate (E6588, E6589).

Figure 9.65 Structure of ELF 39 phosphate, a component of DyeChrome Western Blot Stain Kits #4, #5 and #6 (D21884, D21885, D21886).

Tyramide signal amplification (TSA) technology (Tyramide Signal Amplification (TSA) Technology - Section 6.2) utilizes a unique concept in fluorescent substrates. Tyramide derivatives labeled with detectable moieties such as biotin or fluorophores are activated by HRP to a phenoxyl radical that is trapped near the site of its formation by reaction with nearby tyrosine residues (Figure 6.5). The covalent bond formed results in detection of HRP-labeled targets with high spatial resolution.

The principle of excited-state energy transfer can also be used to generate fluorogenic substrates (Fluorescence Resonance Energy Transfer (FRET) - Note 1.2 ). For example, the EDANS fluorophore in our HIV protease and renin substrates is effectively quenched by a nearby dabcyl acceptor chromophore (Figure 10.10). This chromophore has been carefully chosen for maximal overlap of its absorbance with the fluorophore's fluorescence, thus ensuring that the fluorescence is quenched through excited-state energy transfer. Proteolytic cleavage of the substrate results in spatial separation of the fluorophore and the acceptor chromophore, thereby restoring the fluorophore's fluorescence. Many of the dyes described in Fluorophores and Their Amine-Reactive Derivatives - Chapter 1 have been used to form energy-transfer pairs, some of which can be introduced during automated synthesis of peptides using modified amino acids described in Reagents for Peptide Analysis, Sequencing and Synthesis - Section 9.5. Molecular Probes' nonfluorescent quenchers and photosensitizers - Table 1.10 lists our nonfluorescent quenching dyes and their spectral properties. Our QSY dyes (Long-Wavelength Rhodamines, Texas Red Dyes and QSY Quenchers - Section 1.6, Reagents for Analysis of Low Molecular Weight Amines - Section 1.8, Thiol-Reactive Probes Excited with Visible Light - Section 2.2) have spectral properties that are superior to those of the dabcyl chromophore (Molecular Probes' amine-reactive dyes - Table 1.1, Figure 1.70), making the QSY dyes useful as nonfluorescent quenchers for a broad range of fluorescent donor dyes (Figure 8.51).

Figure 10.10 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.

Figure 8.51 Fluorescence quenching of 5'-tetramethylrhodamine–labeled M13 primers by nonfluorescent dyes attached at the 3'-end. The comparison represents equal concentrations of oligonucleotides with (1) no 3'-quencher (control), (2) 3'-dabcyl quencher, (3) 3'-QSY 7 quencher.

The protease substrates in three of our EnzChek Protease Assay Kits and their RediPlate 96 and RediPlate 384 versions (Detecting Peptidases and Proteases - Section 10.4) are heavily labeled casein conjugates; the close proximity of dye molecules results in considerable self-quenching. Hydrolysis of the protein to smaller fragments is accompanied by a dramatic increase in fluorescence, which forms the basis of a simple and sensitive continuous assay for a variety of proteases. In addition, we offer a phospholipase A substrate (bis-BODIPY FL C₁₁-PC, B7701; Probes for Lipid Metabolism and Signaling - Section 17.4) that contains a BODIPY FL fluorophore on each phospholipid acyl chain. Proximity of the BODIPY FL fluorophores on adjacent phospholipid acyl chains causes fluorescence self-quenching that is relieved only when the fluorophores are separated by phospholipase A–mediated cleavage. PED6, a phospholipid with a green-fluorescent BODIPY fatty acid on the lipid portion of the molecule and a 2,4-dinitrophenyl quencher on the polar head group (PED6, D23739; Probes for Lipid Metabolism and Signaling - Section 17.4; Figure 17.24) is useful as a specific phospholipase-A₂ substrate.

Figure 17.24 Mechanism of phospholipase activity–linked fluorescence enhancement responses of bis-BODIPY FL C₁₁-PC (B7701) and PED6 (D23739). Note that enzymatic cleavage of bis-BODIPY FL C₁₁-PC yields two fluorescent products, whereas cleavage of PED6 yields only one.

The mechanism of some enzymes makes it difficult to obtain a continuous optical change during reaction with an enzyme substrate. However, a discontinuous assay can often be developed by derivatizing the reaction products with one of the reagents described in Fluorophores and Their Amine-Reactive Derivatives - Chapter 1, Thiol-Reactive Probes - Chapter 2 and Reagents for Modifying Groups Other Than Thiols or Amines - Chapter 3, usually followed by a separation step in order to generate a product-specific fluorescent signal. For example, fluorescamine (F2332, F20261; Reagents for Analysis of Low Molecular Weight Amines - Section 1.8) or o-phthaldialdehyde (OPA, P2331MP; Reagents for Analysis of Low Molecular Weight Amines - Section 1.8) can detect the rate of any peptidase reaction by measuring the increase in the concentration of free amines in solution. The activity of enzymes that produce free coenzyme A from its esters can be detected using thiol-reactive reagents such as 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB, D8451; Chemical Crosslinking Reagents - Section 5.2) or 7-fluorobenz-2-oxa-1,3-diazole-4-sulfonamide (ABD-F, F6053; Thiol-Reactive Probes Excited with Visible Light - Section 2.2). The products of enzymes that metabolize low molecular weight substrates can frequently be detected by chromatographic or electrophoretic analysis. HPLC or capillary zone electrophoresis can also be used to enhance the sensitivity of reactions that yield fluorescent products. Measuring the activity of phospholipases, in particular, often requires chromatographic means to separate the detectable hydrolysis products (Probes for Lipid Metabolism and Signaling - Section 17.4).

A number of chromogenic substrates for hydrolytic enzymes are derived from indolyl chromophores. These initially form a colorless — and sometimes blue-fluorescent — 3-hydroxyindole ("indoxyl"), which spontaneously, or through mediation of an oxidizing agent such as nitro blue tetrazolium (NBT, N6495; Detecting Enzymes That Metabolize Phosphates and Polyphosphates - Section 10.3) or potassium ferricyanide, is converted to an intensely colored indigo dye that typically precipitates from the medium (Figure 9.69). Halogenated indolyl derivatives, including 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-Gal, B1690, B22015; Detecting Glycosidases - Section 10.2) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP, B6492, Detecting Enzymes That Metabolize Phosphates and Polyphosphates - Section 10.3) are generally preferred because they produce finer precipitates that are less likely to diffuse from the site of formation, making them especially useful for detecting enzymatic activity in cells and tissues, on blots and in gels.

Figure 9.69 Principle of enzyme-linked detection using the reagents in our NBT/BCIP Reagent Kit (N6547). Phosphatase hydrolysis of BCIP is coupled to reduction of NBT, yielding a formazan and an indigo dye that together form a black-purple–colored precipitate.