Molecular Probes

Section 10.1 — Introduction to Enzyme Substrates and Their Reference Standards

Molecular Probes offers a large assortment of common and uncommon fluorogenic and chromogenic enzyme substrates. We prepare substrates for enzyme-linked immunosorbent assays (ELISAs), as well as substrates for detecting very low levels of enzymatic activity in fixed cells, tissues, cell extracts and purified preparations. Our RediPlate product line includes enzyme substrates predispensed in 96-well or 384-well plates for high-throughput applications, along with the appropriate reference standards and other reaction components. We have also developed effective methods for detecting some enzymes in live cells. In this section, we describe the characteristics of our enzyme substrates and the fluorophores and chromophores from which they are derived, focusing primarily on the suitability of these substrates for different types of enzyme assays. The fluorophores that are available as reference standards — including a NIST-traceable fluorescein standard — can be found in the data table and product list associated with this section. Substrates for specific enzymes are described in subsequent sections of this chapter.

Substrates Yielding Soluble Fluorescent Products

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; Section 10.6) utilize chromatography to separate the intrinsically fluorescent substrates from their fluorescent products.

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; 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.

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 (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 (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 Section 9.4.

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; 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; Section 10.6, Section 15.2) — also known as alamarBlue, a trademark of AccuMed International, Inc. Our Amplex UltraRed reagent (A36006, 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, 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 (Section 9.4) for the sensitive detection of proteins. In our unique DyeChrome Double Western Blot Kit (D21887, 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).

Substrates for Live-Cell Enzyme Assays

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 (Section 10.2), β-glucuronidase (Section 10.2), secreted alkaline phosphatase (SEAP, Section 10.3), chloramphenicol acetyltransferase (CAT, Section 10.6) and luciferase (Section 10.6). Some of our EnzChek and DQ Kits are useful for study of the uptake and metabolism of proteins during phagocytosis (Section 16.1), as well as for the general screening of certain glycosidases (Section 10.2) and proteases (Section 10.4). Substrates for specific proteases are also useful for the detection of 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 (Section 7.4), amplification of the conjugate by standard immunochemical techniques, including the tyramide signal amplification (TSA, Section 6.2) and Enzyme-Labeled Fluorescence (ELF, Section 6.3) technologies. Chloromethyl analogs of fluorogenic substrates for glycosidases (for example, our DetectaGene Green CMFDG Kit, (D2920); 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 (Section 14.2). The improved retention of the MitoTracker (Section 12.2) and CellTracker (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, Section 10.2) and dealkylases (Section 10.6). Like resazurin (R12204, 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; Section 15.3, 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, Section 10.2).

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.

Substrates Yielding Insoluble Fluorescent Products

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 (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 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 (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 Section 9.4.

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.

Substrates Based on Excited-State Energy Transfer

The principle of excited-state energy transfer can also be used to generate fluorogenic substrates (Technical Focus: Fluorescence Resonance Energy Transfer (FRET)). 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 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 Section 9.5. Table 1.10 lists our nonfluorescent quenching dyes and their spectral properties. Our QSY dyes (Section 1.6, Section 1.8, Section 2.2) have spectral properties that are superior to those of the dabcyl chromophore (Table 1.11, Figure 1.70), making the QSY dyes useful as nonfluorescent quenchers for a broad range of fluorescent donor dyes (Figure 8.51).

The protease substrates in three of our EnzChek Protease Assay Kits and their RediPlate 96 and RediPlate 384 versions (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; 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; Section 17.4; Figure 17.24) is useful as a specific phospholipase-A₂ substrate.

Fluorescent Derivatization Reagents for Discontinuous Enzyme Assays

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 Chapter 1, Chapter 2 and Chapter 3, usually followed by a separation step in order to generate a product-specific fluorescent signal. For example, fluorescamine (F2332, F20261; Section 1.8) or o-phthaldialdehyde (OPA, P2331MP; 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; Section 5.2) or 7-fluorobenz-2-oxa-1,3-diazole-4-sulfonamide (ABD-F, F6053; 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 (Section 17.4).

Substrates that Yield Insoluble Chromophoric Products

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; 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; Section 10.2) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP, B6492, 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.