Fluorogenic substrates that detect glycosidases (Detecting Glycosidases—Section 10.2) and phosphatases (Detecting Enzymes That Metabolize Phosphates and Polyphosphates—Section 10.3) have been by far the dominant probes for measuring enzymatic activity. Exactly the same fluorophores and chromophores—fluoresceins, resorufins and umbelliferones (7-hydroxycoumarins)—can be used to prepare substrates for other hydrolytic enzymes and ether-metabolizing microsomal dealkylase enzymes or peroxidases. In addition, we offer substrates for chloramphenicol acetyltransferase (CAT), luciferase and β-lactamase, which are usually not widely expressed in cells. These substrates are important tools for detecting cells transfected with reporter genes that encode these enzymes. We also have available several reagents that are substrates for detecting enzyme-catalyzed chemical reduction associated with cells, including the tetrazolium salts MTT and XTT (M6494, X6493; Viability and Cytotoxicity Assay Reagents—Section 15.2) and resazurin (R12204), which is useful for quantitatively measuring cell-mediated cytotoxicity, cell proliferation and mitochondrial metabolic activity in isolated neural tissue.
Metabolic oxidation of chemical compounds, including many pollutants, is the function of the cytochrome-mediated monooxygenase or mixed-function oxidase system. Several enzymes are involved, including cytochrome P448 monooxygenase (aryl hydrocarbon hydroxylase), which is induced by carcinogenic polyaromatic hydrocarbons. Cytochrome P450 (CYP) is a useful marker of endoplasmic reticulum membranes. The very low turnover rate of these enzymes can be followed using various fluorogenic alkyl ether derivatives of coumarin, resorufin and fluorescein, all of which yield cleavage products with longer-wavelength spectral properties than the parent substrates.
Resorufin ether–based substrates (R352, R441, R1147), which all yield red-fluorescent resorufin (R363, Introduction to Enzyme Substrates and Their Reference Standards—Section 10.1; excitation/emission maxima ~571/585 nm), have been extensively used to differentiate isozymes of cytochrome P450. Ethoxyresorufin O-deethylase (EROD) and total protein concentration have been simultaneously assayed in a fluorescence microplate reader using ethoxyresorufin (resorufin ethyl ether, R352) and fluorescamine (F2332, F20261; Protein Quantitation in Solution—Section 9.2).
Fluorescence detection of the deethylation of 3-cyano-7-ethoxycoumarin (C684) is reported to be 50–100 times more sensitive than that of ethoxyresorufin, primarily because of the faster turnover rate of 3-cyano-7-ethoxycoumarin; however, ethoxyresorufin exhibits lower fluorescence background due to its more favorable spectral shifts. The deethylase product of 3-cyano-7-ethoxycoumarin, 3-cyano-7-hydroxycoumarin (C183, Introduction to Enzyme Substrates and Their Reference Standards—Section 10.1), has a lower pKa than that of 7-ethoxycoumarin, allowing continuous measurements of enzyme activity at pH 7.
The cytochrome P450 substrate 7-ethoxy-4-trifluoromethylcoumarin (E2882) yields a product with a fluorescence emission that is distinct from that of the substrate and of NADPH, making this substrate useful for the direct measurement of enzymatic activity. Researchers have shown that this substrate is cleaved by at least the 1A2, 2E1 and 2B1 isozymes of cytochrome P450.
The EnzChek epoxide hydrolase substrate (E33956) is ideal for studying the epoxide hydrolase family of enzymes, including the arachidonic epoxide hydrolases (implicated in the regulation of inflammation and blood pressure) and microsomal epoxide hydrolases (reported to detoxify epoxides into diols), and their respective inhibitors. This substrate provides accurate detection of epoxide hydrolase activity in solution, with better sensitivity than colorimetric epoxide hydrolase substrates. In the presence of epoxide hydrolases, the nonfluorescent EnzChek epoxide hydrolase substrate produces a bright blue-fluorescent product with excitation and emission maxima of ~358 nm and 452 nm, respectively. Furthermore, the blue-fluorescent product of the EnzChek epoxide hydrolase substrate exhibits pH-insensitive spectra in the physiological pH range and is compatible with optics used for coumarin detection in fluorometers.
Lipases play an essential role in the transfer of lipids in cell signaling and metabolism and generally include glycerol ester hydrolases and cholesterol esterases. Phospholipase A selectively hydrolyzes lipophilic esters of phospholipids. Because of their importance to the process of signal transduction in cells, our extensive selection of substrates and other probes for phospholipases is discussed in Probes for Lipid Metabolism and Signaling—Section 17.4.
The triacylglycerol-based EnzChek lipase substrate (E33955) offers higher throughput and better sensitivity than chromogenic (TLC or HPLC) assays, and a visible wavelength–detection alternative to pyrene-based fluorescent substrates. In the presence of lipases, the nonfluorescent EnzChek lipase substrate produces a bright, green-fluorescent product (excitation/emission maxima of ~505/515 nm) for the accurate and sensitive detection of lipase activity in solution. Furthermore, the green-fluorescent product of the EnzChek lipase substrate exhibits pH-insensitive spectra in the physiological pH range and is compatible with optics used for fluorescein detection in fluorometers.
The fluorogenic lipase substrate O-pivaloyloxymethyl umbelliferone (C-POM, P35901) was developed to deliver optimal performance in assays of lipase activity. Standard lipase substrates may exhibit high levels of undesirable nonspecific reactivity, either through spontaneous hydrolysis or direct reaction of the substrate with noncatalytic proteins such as BSA. C-POM is much less prone to these unwanted side reactions, and the resulting low level of background fluorescence yields a better signal-to-noise ratio, providing a more accurate measure of lipase catalysis. Enzymatic conversion of the essentially nonfluorescent C-POM yields a bright blue-fluorescent reaction product (excitation/emission ~360/460 nm). C-POM has been shown to serve as a substrate for a variety of lipases and displays excellent stability in solution, making it an ideal substrate for specific lipases or for general or high-throughput screening.
Unlike lipase substrates that are esters of 7-hydroxy-4-methylcoumarin (β-methylumbelliferone, H189; Introduction to Enzyme Substrates and Their Reference Standards—Section 10.1), 6,8-difluoro-4-methylumbelliferyl octanoate (DiFMU octanoate, D12200) can be used for the continuous in vitro assay of lipases at a pH greater than or equal to 6; the blue-fluorescent hydrolysis product of DiFMU octanoate, 6,8-difluoro-7-hydroxy-4-methylcoumarin (DiFMU, D6566; Introduction to Enzyme Substrates and Their Reference Standards—Section 10.1), has a pKa of 4.9.
The cholesterol produced by cholesterol esterases is readily quantitated using the Amplex Red Cholesterol Assay Kit (A12216), which is discussed in Substrates for Oxidases, Including Amplex Red Kits—Section 10.5 and Sphingolipids, Steroids, Lipopolysaccharides and Related Probes—Section 13.3.The Amplex Red cholesterol assay can be adapted to continuously measure cholesterol generated by the activity of cholesterol esterases.
Because of the close correlation between its transcript levels and enzymatic activity and the excellent sensitivity of the enzyme assay, the chloramphenicol acetyltransferase (CAT) reporter gene system has proven to be a powerful tool for investigating transcriptional elements in animal and plant cells. Most conventional CAT assays require incubation of cell extracts with radioactive substrates, typically 14C chloramphenicol or 14C acetyl CoA, followed by organic extraction and autoradiography or scintillation counting. The FAST CAT Chloramphenicol Acetyltransferase Assay Kits contain unique BODIPY chloramphenicol fluorescent substrates that take advantage of the exquisite sensitivity of fluorescence techniques, thus eliminating the need for hazardous radiochemicals, film, fluors, scintillation counters and expensive radioactive waste disposal. The original FAST CAT Kit and our FAST CAT Green and Yellow (deoxy) Kits provide detection limits similar to those achieved with conventional radioactive methods and yield results that are easily visualized using a hand-held UV lamp.
The green-fluorescent BODIPY FL chloramphenicol substrate in our original FAST CAT Chloramphenicol Acetyltransferase Assay Kit (F2900) has a KM for purified CAT of 7.4 µM and a Vmax of 375 picomoles/unit/minute, values that are similar to those of 14C-labeled chloramphenicol (KM = 12 µM and Vmax = 120 picomole/unit/minute). To perform the assay, cell extracts are simply incubated with BODIPY FL chloramphenicol and acetyl coenzyme A. After a suitable incubation period, the products and remaining substrate are extracted and separated by thin-layer chromatography (TLC). The brightly fluorescent, well-resolved spots can be immediately visualized with a hand-held UV lamp or quantitated with a laser scanner or CCD camera (). Alternatively, quantitation can be accomplished using a fluorometer or spectrophotometer after a simple extraction. HPLC analysis of the fluorescent products has also been used to further enhance the assay's sensitivity.
These attributes have enabled researchers to use this FAST CAT substrate to measure CAT activity in crude cellular extracts of transfected ovarian granulosa cells. The FAST CAT Chloramphenicol Acetyltransferase Assay Kit has also been employed to study hormonal regulation of prodynorphin gene expression and to measure the rate of hair growth in single follicles of transgenic mice.
Each FAST CAT Chloramphenicol Acetyltransferase Assay Kit (F2900) is available in a 100-test size and includes:
- BODIPY FL chloramphenicol substrate
- Mixture of the 1- and 3-acetyl and 1,3-diacetyl BODIPY FL derivatives, which serve as a reference standard for the fluorescent products
- Detailed protocols (FAST CAT Chloramphenicol Acetyltransferase Assay Kit)
The FAST CAT (deoxy) Chloramphenicol Acetyltransferase Assay Kits (F6616, F6617) contain substrates that greatly simplify the quantitation of chloramphenicol acetyltransferase (CAT) activity and extend the linear detection range of the original Molecular Probes FAST CAT assay. The BODIPY FL chloramphenicol substrate in our original FAST CAT kit contains two acetylation sites, only one of which is acetylated by the CAT enzyme. Once the CAT enzyme adds an acetyl group to this position, the acetyl group can be nonenzymatically transferred to the second site, leaving the original position open for another enzymatic acetylation. Therefore, enzyme acetylation of our original BODIPY FL FAST CAT substrate produces three products—one diacetylated and two monoacetylated chloramphenicols—thus complicating the quantitative analysis of CAT gene activity. More importantly, because the nonenzymatic transacetylation is the rate-limiting step, the rate of product accumulation may not accurately reflect CAT activity.
To overcome this limitation, we have modified the original FAST CAT substrate, producing reagents that undergo a single acetylation reaction (Figure 10.6.1). The green-fluorescent BODIPY FL deoxychloramphenicol and yellow-fluorescent BODIPY 543/569 deoxychloramphenicol substrates in the FAST CAT Green and FAST CAT Yellow (deoxy) Chloramphenicol Acetyltransferase Assay Kits (F6616, F6617) are acetylated at a single position, yielding only one fluorescent product (Figure 10.6.1). This simplified reaction scheme provides a straightforward and reliable measure of CAT activity and extends the linear detection range of our original FAST CAT assay.
The FAST CAT Green and FAST CAT Yellow (deoxy) Chloramphenicol Acetyltransferase Assay Kits are available in a 100-test size and include:
- BODIPY FL 1-deoxychloramphenicol substrate (in Kit F6616) or BODIPY TMR 1-deoxychloramphenicol substrate (in Kit F6617)
- 3-Acetyl BODIPY FL derivative (in Kit F6616) or BODIPY TMR derivative (in Kit F6617), which serves as a reference standard for the fluorescent product
- Detailed protocols (FAST CAT (deoxy) Chloramphenicol Acetyltransferase Assay Kits)
The CAT substrate in our FAST CAT Green (deoxy) Chloramphenicol Acetyltransferase Assay Kit (F6616) is spectrally identical to the green-fluorescent BODIPY FL chloramphenicol substrate in our original FAST CAT Kit. The FAST CAT Yellow (deoxy) Chloramphenicol Acetyltransferase Assay Kit (F6617) contains a red-orange–fluorescent BODIPY TMR derivative. The availability of two spectrally distinct CAT substrates allows researchers to choose the optimal fluorophore for a particular excitation source or multicolor labeling experiment.
Firefly luciferase (Photinus-luciferin:oxygen 4-oxidoreductase or luciferin 4-monooxygenase, EC 188.8.131.52) produces light by the ATP-dependent oxidation of luciferin (Figure 10.6.2). The 560 nm chemiluminescence from this reaction peaks within seconds, with light output that is proportional to luciferase concentration when substrates are present in excess. The luc gene, which encodes the 62,000-dalton firefly luciferase, is a popular reporter gene for plants, bacteria and mammalian cells and for monitoring baculovirus gene expression in insects. Chemiluminescent techniques are virtually background-free, making the luc reporter gene ideal for detecting low-level gene expression.
The substrate for firefly luciferase, D-(–)-2-(6'-hydroxy-2'-benzothiazolyl)thiazoline-4-carboxylic acid, commonly known as luciferin, was first isolated by Bitler and McElroy (9 mg from approximately 15,000 fireflies!). In the firefly, spent luciferin (oxyluciferin) is recycled back to luciferin. We prepare synthetic luciferin (L2911) and its water-soluble sodium (L2912) and potassium (L2916) salts. The physical properties of these derivatives are identical to those of the natural compound. Typically, luciferase expression is measured by adding the substrates ATP and luciferin to cell lysates and then analyzing light production with a luminometer. As little as 0.02 pg (250,000 molecules) of luciferase can be reliably measured using a standard scintillation counter. Moreover, a CCD-based imaging method of detecting luc gene expression in single cells has been developed.
The NovaBright β-Galactosidase and Firefly Luciferase Dual Enzyme Reporter Gene Chemiluminescent Detection Kit (200 assays, N10561; 600 assays, N10562) allows rapid and sensitive sequential detection of firefly luciferase and β-galactosidase, enabling experimental and control reporter gene enzymes to be measured in the same cell extract sample.
This kit employs the chemiluminescent luciferase substrate luciferin and the chemiluminescent β-galactosidase substrate Galacton-Plus for the detection of 1 fg to 20 ng and 10 fg to 20 ng of purified luciferase and β-galactosidase, respectively. Cell lysate is mixed with assay buffer for the luciferase reaction, and the luciferase signal is measured immediately after the injection of substrate dilution buffer, which contains both luciferin and Galacton-Plus substrates. The enhanced luciferase reaction produces a light signal that decays with a half-life of approximately 1 minute. Light signal from the β-galactosidase reaction is negligible due to lack of enzyme turnover time, low pH (7.8) and absence of enhancer. After a 30–60 minute incubation, light signal from the accumulated product of the β-galactosidase/Galacton-Plus reaction is initiated by adding the accelerator, which raises the pH and provides the Sapphire-II luminescence enhancer to increase light intensity. Light emission from the β-galactosidase reaction exhibits glow kinetics with a half-life of 180 minutes. Residual light from the luciferase reaction is minimal, due to rapid kinetic signal decay and quenching by accelerator. Generally, only very high luciferase concentrations (ng levels of enzyme) interfere with detection of β-galactosidase. A longer delay after the addition of accelerator prior to measurement results in decreased residual luciferase signal when extremely high levels are present. It is important, however, to maintain consistent timing for the addition of substrate dilution buffer and the measurement of the β-galactosidase signal after adding accelerator.
Each NovaBright β-Galactosidase and Firefly Luciferase Dual Enzyme Reporter Gene Chemiluminescent Detection Kit provides:
- Lysis buffer
- Assay buffer
- Substrate dilution buffer
- Galacton-Plus substrate
- Detailed protocols (NovaBright β-Galactosidase and Firefly Luciferase Dual Enzyme Reporter Gene Chemiluminescent Detection System)
Chemiluminescent 1,2-dioxetane substrates for β-galactosidase, including the Galacton-Plus substrate described here and the Galacton-Star substrate provided in the NovaBright β-Galactosidase Enzyme Reporter Gene Chemiluminescent Detection Kits (N10563, N10564, N10565, N10566; Detecting Glycosidases—Section 10.2) provide highly sensitive enzyme detection and have been utilized extensively in reporter assays in mammalian cell and tissue extracts.
Luciferin has been used in an exquisitely sensitive and specific ATP assay, which allows the detection of femtomolar quantities of ATP. This bioluminescent ATP assay has been employed to determine cell proliferation and cytotoxicity in both bacteria and mammalian cells. We provide all the reagents needed for this important assay in the ATP Determination Kit (A22066, Detecting Enzymes That Metabolize Phosphates and Polyphosphates—Section 10.3). Researchers have also adapted the luciferin–luciferase ATP assay system for detecting single base changes in a solid-phase DNA sequencing method. In addition, amphipathic and hydrophobic substances, including certain anesthetics and hormones, compete with luciferin for the hydrophobic site on the luciferase molecule, providing a convenient method to assay subnanomolar concentrations of these substances. A protein A–luciferase fusion protein has been developed that can be used in bioluminescence-based immunoassays.
Although luciferase activity is sometimes measured in live cells,in vivo quantitation appears to be limited by the difficulty in delivering luciferin into intact cells. Molecular Probes DMNPE-caged luciferin (L7085) readily crosses cell membranes. Once the caged luciferin is inside the cell, active luciferin can be released either instantaneously by a flash of UV light or continuously by the action of endogenous intracellular esterases, which are found in many cell types. This probe facilitates in vivo luciferase assays in two important ways. First, caged luciferin improves the sensitivity and quantitative analysis of these assays by allowing more efficient delivery of luciferin into intact cells. Second, hydrolysis by intracellular esterases provides a continuous supply of active luciferin, permitting long-term measurements and reducing the need for rapid mixing protocols and costly injection devices. Moreover, DMNPE-caged luciferin may make it easier to follow dynamic changes in gene expression in live cells. We also offer DMNPE-caged ATP (A1049, Photoactivatable Reagents, Including Photoreactive Crosslinkers and Caged Probes—Section 5.3), which can be used in conjunction with DMNPE-caged luciferin for in vivo luciferase assays.
Coelenterazine and its analogs are substrates for the bioluminescent Renilla luciferase. We offer coelenterazine (C2944) and several synthetic coelenterazine analogs, including coelenterazine cp, f and h (C14260, C6779, C6780; Protein-Based Ca2+ Indicators—Section 19.5; Coelenterazines and their properties—Table 19.4). Luciferin and coelenterazine have been used together for dual detection of firefly and Renilla luciferases in live mice. Coelenterazine analogs have been characterized for their effectiveness in measuring Renilla luciferase in both live cells and live animals.
Fluorocillin Green 495/525 substrate (F33952) is a robust reporter for ELISA protocols that employ TEM-1 β-lactamase conjugates. Upon cleavage, Fluorocillin Green 495/525 reagent is converted to a green-fluorescent soluble product (excitation/emission maxima ~495/525 nm). Fluorocillin Green 495/525 reagent has a broad dynamic range of fluorescence signal, is more sensitive than common colorimetric substrates such as nitrocefin and displays only modest hydrolysis when incubated for extended periods from pH 5.5 to 8.0. Additionally, Fluorocillin Green 495/525 reagent consistently reports β-lactamase activity in the presence of EDTA, many detergents, salts and sodium azide. Fluorocillin Green 495/525 reagent is available as a dry powder, packaged in five vials each containing 100 µg (F33952), and as a component of the SensiFlex ELISA Development Kits (S33853, S33854; Secondary Immunoreagents—Section 7.2).
Fluorocillin Green 345/530 β-lactamase substrate (F33951) was developed as a precipitating dye generally compatible with a variety of β-lactamase enzymes and corresponding antibody conjugates. We have demonstrated the utility of this reagent in immunohistochemistry applications, and it may also prove useful as a marker for endogenous β-lactamase activity in prokaryotes, possibly detected by microscopy or even flow cytometry. It is important to note, however, that in its precipitated form, Fluorocillin Green 345/530 β-lactamase substrate produces a crystal size incompatible with immunocytochemical analysis. This form of β-lactamase substrate is also incompatible with TEM-1 β-lactamase and its conjugates, but can, for example, be cleaved by P99 β-lactamase.
Resazurin (R12204), which under the name alamarBlue (a trademark of AccuMed International, Inc.) has been reported to be useful for quantitatively measuring cell-mediated cytotoxicity, cell proliferation and mitochondrial metabolic activity in isolated neural tissue, is also a useful substrate for measuring the dehydrogenase activity or a wide variety of dehydrogenase enzymes in vitro. Among the assays reported are the use of resazurin to detect:
- Argininosuccinate lyase and NAPDH by a coupled diaphorase–resazurin reaction sequence
- Bile acids in human urine, feces and serum using NAD+ 3α-hydroxysteroid dehydrogenase
- Glucose 6-phosphate dehydrogenase (G6PD) activity
- NADH and bile acids with NADH oxidoreductase
- Serum formate using formate dehydrogenase and NAD+
- Triacylglycerols with glycerol dehydrogenase
- Urinary acylcarnitines in an immobilized enzyme reactor
Our Vybrant Cytotoxicity Assay Kit (V23111, Viability and Cytotoxicity Assay Kits for Diverse Cell Types—Section 15.3) employs the resazurin dehydrogenase substrate to monitor the release of G6PD from the cytosol of damaged cells into the surrounding medium (Figure 10.6.3); this method, however, also provides an extremely sensitive and specific assay for G6PD in cell-free extracts. The dehydrogenase substrate in our Vybrant Cell Metabolic Assay Kit and LIVE/DEAD Cell Vitality Assay Kit (V23110, L34951; Viability and Cytotoxicity Assay Kits for Diverse Cell Types—Section 15.3) is dodecylresazurin, a more lipophilic version of resazurin. Because this substrate readily penetrates the membranes of live cells and its fluorescent reduction product (dodecylresorufin) is better retained in cells, it is preferred for both microscopy and flow cytometry assays.
Figure 10.6.3 Detection of dead and dying cells using the Vybrant Cytotoxicity Assay Kit (V23111). Jurkat cells were treated with 10 µM camptothecin for six hours, then assayed for glucose 6-phosphate dehydrogenase release. An untreated control sample is shown for comparison. The fluorescence was measured in a microplate reader (excitation/emission ~530/590 nm). A background of 55 fluorescence units was subtracted from each value.
We have developed a unique fluorogenic substrate that can detect the enzymatic activity of certain enzymes that reduce nitro compounds to amines or inorganic nitrate to nitrite. 6-Chloro-9-nitro-5-oxo-5H-benzo[a]phenoxazine (CNOB, C22220) is reduced to an aminophenoxazine dye that absorbs maximally at ~620 nm and has an emission maximum near 630 nm. We have shown that CNOB is a good substrate for at least some bacterial nitroreductases but apparently is not a good substrate for a mammalian nitroreductase. The utility of CNOB for detection of nitroreductase activity or detection of hypoxia in tumor cells has not yet been tested; however, it is known that some nitroimidazoles and other nitroaromatic compounds are reduced to amines under highly reducing conditions.
|C684||215.21||L||DMSO||356||20,000||411||pH 7||3-cyano-7-hydroxycoumarin (C183)|
|C2944||423.47||FF,D,LL,AA||MeOH||429||7500||see Notes||pH 7|
|F2900||583.44||F,D,L||MeOH||504||80,000||511||MeOH||see Notes||2, 3|
|F6616||567.44||F,D,L||MeOH||504||81,000||510||MeOH||see Notes||3, 4|
|F6617||673.57||F,D,L||MeOH||545||60,000||570||MeOH||see Notes||3, 4|
|L2911||280.32||F,D,L,A||pH >6, DMSO||328||18,000||532||pH 7||see Notes||5|
|L2912||302.30||F,D,L,A||pH >6||328||17,000||533||pH 7||see Notes||5|
|L2916||318.41||F,D,L,A||pH >6||328||18,000||533||pH 7||see Notes||5|
|L7085||489.52||FF,D,LL||DMSO, DMF||334||22,000||none||MeOH||see Notes||6, 7|
|R12204||251.17||L||H2O, MeOH||604||60,000||none||MeOH||Resorufin (R363)|
For Research Use Only. Not for use in diagnostic procedures.