Search!

ChapterSectionIndexSectionChapterPrintProduct ListData Table
Updated: May 30, 2006

Section 10.5 — Substrates for Oxidases, Including Amplex Red Kits

Printer-Friendly

Oxidases, the most useful of which is undoubtedly horseradish peroxidase (HRP), are important enzymes that are used in a wide variety of bioassays. Peroxidase activity is also present in many cells. Reagents for quantitating peroxidase and the activity of a variety of other oxidases are described in this section; reagents for detecting the activity of cellular peroxidases and the oxygen radicals produced by these peroxidases are described in Section 16.1 and Section 18.2. Antibody, protein G, avidin and streptavidin conjugates of horseradish peroxidase are listed in the product list for this section and described in Section 7.2 and Section 7.6. Tyramide signal amplification (TSA) technology (Section 6.2) makes extensive use of peroxidase-conjugated reagents and fluorescent dye– or hapten-labeled tyramides to deposit a detectable product at the site of enzymatic activity (Figure 6.5). Our exclusive Zenon technology (Section 7.3) includes the Zenon Horseradish Peroxidase Antibody Labeling Kits (Table 7.14), which permit the rapid and quantitative formation of HRP-labeled antibody complexes; the Zenon Antibody Labeling Kits are described in detail in Section 7.3.

We have used our extremely versatile Amplex Red reagent — the most stable and sensitive fluorogenic substrate known for horseradish peroxidase — to develop a variety of novel fluorogenic and chromogenic assays for enzymes that produce hydrogen peroxide. Furthermore, these coupled assays permit the ultrasensitive quantitation of a diverse assortment of analytes, including glucose, galactose, cholesterol, glutamic acid, xanthine (or hypoxanthine), uric acid, choline and acetylcholine, as well as hydrogen peroxide. Our Patented PiPer Phosphate Assay Kit and PiPer Pyrophosphate Assay Kit (P22061, P22062; Section 10.3) also utilize our exclusive Amplex Red technology for the continuous assays of enzymes that produce either inorganic phosphate or pyrophosphate.

Peroxidases

Amplex Red Reagent: Stable Substrate for Peroxidase Detection

In the presence of horseradish peroxidase (HRP), the Amplex Red reagent (10-acetyl-3,7-dihydroxyphenoxazine, A12222, A22177; structure) reacts with H2O2 with a 1:1 stoichiometry to produce highly fluorescent resorufin ref (R363, Section 10.1, Figure 10.59). The Amplex Red reagent has greater stability, yields less background and produces a red-fluorescent product that is more readily detected than the similar reduced methylene blue derivatives commonly used for colorimetric determination of lipid peroxides in plasma, sera, cell extracts and a variety of membrane systems.ref Using the Amplex Red reagent in conjunction with HRP, we have found that release of hydrogen peroxide to the medium by as few as 2000 phorbol ester–stimulated neutrophils can be detected in a fluorescence microplate reader.

The Amplex Red reagent has been used to detect the release of H2O2 from activated human leukocytes,ref to measure the activity of monoamine oxidase in cow brain tissue,ref to demonstrate the extracellular production of H2O2 produced by UV light stimulation of human keratinocytes ref and to measure L-glutamate in food samples.ref Using the Amplex Red reagent, researchers have discovered that antibodies can convert molecular oxygen to H2O2, which may be important in understanding a new chemical arm of the immune system, as well as the evolution of antibodies and the role they may play in human diseases.ref The sensitivity of the Amplex Red reagent in detecting the activity of D-amino acid oxidase has been reported to be 5- to 25-times better than that of the QuantaBlu fluorogenic peroxidase substrate,ref with a lower limit for detection of D-alanine of 2 picomoles.ref The Amplex Red reaction can be used to routinely detect as little as 10 picomoles of H2O2 in a 100 µL volume (50 nM, Figure 10.60), at least a 10-fold greater sensitivity than that attained with the commonly used scopoletin assay for H2O2.ref In the scopoletin assay, HRP catalyzes conversion of the fluorescent scopoletin to a nonfluorescent product. Unlike scopoletin, the Amplex Red reagent is a fluorogenic substrate with very low background fluorescence. Consequently, assays using Amplex Red as the substrate result in an increase in fluorescence, not a decrease — an inherently superior method for enzymatic assays. Other advantages of the Amplex Red reaction over scopoletin-based H2O2 assays include high chemical stability of the Amplex Red reagent and its fluorescent product, resorufin, and the long-wavelength spectra of resorufin. Because resorufin has excitation/emission maxima (spectra) of ~570/585 nm (versus 360/460 nm for scopoletin), there is much less interference from autofluorescence in most biological samples. The Amplex Red reagent can be purchased separately in a single 5 mg vial (A12222) or packaged as a set of 10 vials, each containing 10 mg of the substrate, for high-throughput screening applications (A22177).

Amplex UltraRed Reagent: Brighter and More Sensitive than the Amplex Red Reagent

Our Amplex UltraRed reagent (A36006) improves upon the performance of the Amplex Red reagent, offering brighter fluorescence and enhanced sensitivity on a per-mole basis in horseradish peroxidase or horseradish peroxidase-coupled enzyme assays (Figure 10.62). Fluorescence of the oxidized Amplex UltraRed reagent is also less sensitive to pH (Figure 10.63), and the substrate and its oxidation product exhibit greater stability that the Amplex Red reagent in the presence of hydrogen peroxide (H2O2) or thiols such as dithiothreitol (DTT). Like the Amplex Red reagent, the nonfluorescent Amplex UltraRed reagent reacts with H2O2 in a 1:1 stoichiometric ratio to produce a brightly fluorescent and strongly absorbing reaction product (excitation/emission maxima ~568/581 nm) (spectra). Because the reaction product has long-wavelength spectra, there is little interference from the blue or green autofluorescence found in most biological samples.

The Amplex UltraRed reagent can provide increased sensitivity in peroxidase-based enzyme-linked immunosorbent assays (ELISAs). With a high extinction coefficient and good quantum efficiency, the fluorescence-based Amplex UltraRed reagent is more sensitive than standard colorimetric reagents and provides a broader measurement range for ELISAs. In contrast to commonly used ELISA reagents such as ABTS and TMB, the Amplex UltraRed reagent is exceptionally resistant to autooxidation, making it a superior alternative for peroxidase detection (Table 10.5). Like the Amplex Red reagent, the versatile Amplex UltraRed reagent can be detected using either fluorescence- or absorption-based instrumentation. The Amplex UltraRed reagent, which should be suitable for any of the applications described for the Amplex Red reagent (Section 10.5), is available as a set of five vials, each containing 1 mg of the substrate (A36006).

Coupled Enzymatic Reactions with the Amplex Red and Amplex UltraRed Reagents

Because H2O2 is produced in many different enzymatic reactions, the Amplex Red and Amplex UltraRed reagents can be used in coupled enzymatic reactions to detect the activity of many different enzymes such as NADPH oxidase ref and lysyl oxidase,ref or, when the substrate concentration is limited, to assay solutions for metabolically active constituents such as glucose, acetylcholine and cholesterol (Figure 10.65). Advantages of Amplex Red reagent–and Amplex UltraRed reagent–based assays include the following:

  • The Amplex Red and Amplex UltraRed reagents are fluorogenic substrates with extremely low background color or fluorescence.
  • Stock solutions of the Amplex Red and Amplex UltraRed reagents are chemically stable.
  • The fluorescent peroxidase reaction products are also stable.
  • The long-wavelength spectra of the peroxidase reaction products (spectra, spectra) result in little interference from blue or green autofluorescence in biological samples.
  • Detection can be by either absorption or fluorescence.
  • The peroxidase reaction products can be detected by either fluorescence- or absorption-based methods.
  • In most cases, Amplex Red and Amplex UltraRed assays can detect either unlabeled natural biomolecules, including amino acids, sugars or lipids, or the activity of enzymes that metabolize these substrates.

The Amplex Red reagent is also utilized as the detection reagent in our many Amplex Red assay kits. Substituting the Amplex UltraRed reagent in these kits should offer even greater sensitivity. The Amplex Red assay kits include:

The Amplex UltraRed assay kits include:

Most of these Amplex Red and Amplex UltraRed kits are further discussed in this section; however, some are only presented in the sections listed above. The Amplex Red and Amplex UltraRed kits and reagents are sold for noncommercial use and for high-throughput screening applications only.

Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit

The Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit (A22188) provides a simple, sensitive, one-step assay for detecting H2O2 or the activity of horseradish peroxidase either by measuring fluorescence with a fluorescence-based microplate reader or a fluorometer (Figure 10.66) or by measuring absorption with an absorption-based microplate reader or a spectrophotometer. The Amplex Red peroxidase substrate can detect the presence of active peroxidases and the release of H2O2 from biological samples, including cells and cell extracts ref and is also useful for detecting H2O2 that is produced as a product of enzyme-coupled reactions.ref The Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit contains:

Each kit provides sufficient reagents for approximately 500 assays using a fluorescence- or absorption-based microplate reader and a reaction volume of 100 µL per assay.

Amplex Red ELISA Kits

Molecular Probes' Amplex Red ELISA Kits offer an extremely sensitive fluorometric or colorimetric detection method for horseradish peroxidase (HRP)–amplified enzyme-linked immunosorbent assays (ELISAs). The Amplex Red ELISA Kit #1 (A22170) contains an HRP goat anti–mouse IgG antibody conjugate, which can be used for the ELISA detection of any mouse IgG antibody. The Amplex Red ELISA Kit #2 (A22171) contains the versatile protein G conjugate of HRP, which can be used for the ELISA detection of IgGs from most commonly used species, including human, mouse, rabbit, goat, sheep, bovine and horse. The Amplex Red reagent (10-acetyl-3,7-dihydroxyphenoxazine, structure) provided in these kits is a highly sensitive and stable probe for the detection of HRP activity. In the presence of HRP, the Amplex Red reagent reacts with hydrogen peroxide with a 1:1 stoichiometry to form the fluorescent product resorufin ref (R363, Section 10.1; absorption/emission maxima ~571/ 585 nm, Figure 10.59). Because resorufin also has strong absorption, the assay can be performed either fluorometrically or spectrophotometrically. The Amplex Red ELISA Kit #1 with the HRP–goat anti–mouse IgG antibody conjugate has detection limits of as little as 10 pg/microplate well of a mouse IgG by fluorometry or 50 pg/microplate well by colorimetry (Figure 7.50). The Amplex Red ELISA Kit #2 with HRP–protein G has detection limits of as little as 1 ng/microplate well of a mouse IgG by fluorometry or 3 ng/microplate well by colorimetry (Figure 7.51) in 96-well plates.

Each Amplex Red ELISA Kit contains:

Each kit provides sufficient reagents for approximately 1000 ELISA assays using either a fluorescence- or absorption-based microplate reader and a reaction volume of 100 µL per assay. Our HRP conjugates of the goat anti–mouse IgG antibody (G21040), goat anti–rabbit IgG antibody (G21234) and protein G (P21041) are available separately. HRP conjugates of additional antibodies that can be used with the Amplex Red reagent (A12222, A22177; Section 10.5) are listed in Table 7.4.

Amplex ELISA Development Kits for Rabbit and Mouse IgG

The Amplex ELISA Development Kits for Mouse IgG (A33851) and for Rabbit IgG (A33852) provide a comprehensive set of components for creating a fluorescence-based ELISA using a mouse or rabbit primary antibody, respectively. This assay is based on the Amplex UltraRed reagent, a fluorogenic substrate for horseradish peroxidase (HRP) that reacts with H2O2 in a 1:1 stoichiometric ratio to produce a brightly fluorescent and strongly absorbing reaction product (excitation/emission maxima ~568/581 nm) (spectra). Because the Amplex UltraRed peroxidation product has long-wavelength spectra, there is little interference from the blue or green autofluorescence found in most biological samples. With a high extinction coefficient, good quantum efficiency and resistance to autooxidation, the fluorescence-based Amplex UltraRed reagent delivers better sensitivity and a broader assay range than colorimetric reagents. In a sandwich ELISA format using C-reactive protein, Molecular Probes scientists are routinely able to detect 75 pg of antigen using goat anti–mouse IgG antibody and 1 pg using goat anti–rabbit IgG antibody); these detection limits are 25-fold lower than those obtained from the same sandwich ELISA format using the common colorimetric reagent TMB. Each Amplex ELISA Development Kit contains:

Sufficient reagents are provided in each kit for 500 microplate assays in a 96-well fluorescence microplate format (100 µL per assay).

Other Substrates for Peroxidase Assays

Although HRP is an important enzyme for both histochemistry and ELISAs, fluorogenic peroxidase substrates have not been extensively used for its detection. Fluorogenic peroxidase substrates such as the dihydrofluoresceins (also known as fluorescins) (D399, C400, C13293), dihydrocalcein AM (D23805, structure), dihydrorhodamines (D632, D633, D23806; Section 18.3) and dihydroethidium (hydroethidine; D1168, D11347, D23107; Section 18.2) are converted to highly fluorescent products in the presence of the enzyme and hydrogen peroxide. Because these substrates are insufficiently stable for routine use in ELISA assays, Molecular Probes has converted the dihydrofluoresceins to diacetates. When used in intracellular applications, the acetates are cleaved by endogenous esterases, releasing the intact substrate. However, when used for in vitro assays, an esterase or a mild base must first be added to cleave the acetates, releasing the substrate. The dihydrofluoresceins have been used to measure peroxidase activity ref and to detect hydroperoxide formation.ref

In addition to being a reagent for derivatization of aldehydes and ketones (Section 3.2) and detection of nitric oxide (Section 18.3), NBD methylhydrazine (N-methyl-4-hydrazino-7-nitrobenzofurazan, M20490) has been reported to be useful as a fluorogenic peroxidase substrate, with a sensitivity limit for detection of H2O2 of about 75 nM.ref

Luminol and MCLA: Chemiluminescent Peroxidase Substrates

Nonisotopic immunoassays utilizing peroxidase conjugates and the chemiluminescent horseradish peroxidase substrate luminol (L8455) have provided a rapid and sensitive method for quantitating a wide variety of analytes, including cholesterol,ref digoxin ref and acetylcholine.ref Addition of trace amounts of luciferin (L2911, L2912, L2916; Section 10.6) has been shown to considerably enhance the sensitivity in the assay of thyroxine, digoxin, α-fetoprotein and other analytes.ref A method that employs luminol has been developed for the quantitation of very limiting samples of human DNA from single hairs, saliva, small blood stains and paraffin-embedded and fixed tissue sections. Using a biotinylated oligodeoxynucleotide probe to membrane-immobilized DNA, horseradish peroxidase streptavidin and luminol, researchers have detected 150 pg of human DNA.ref

MCLA (M23800) is principally utilized as a superoxide-sensitive chemiluminescent probe (Section 18.2). MCLA has also been utilized for the determination of both horseradish peroxidase ref and myeloperoxidase.ref

DAB Histochemistry Kits

The use of horseradish peroxidase (HRP) for enzyme-amplified immunodetection — commonly referred to as immunoperoxidase labeling — is a well-established standard histochemical technique.ref The most widely used HRP substrate for these applications is diaminobenzidine (DAB), which generates a brown-colored polymeric oxidation product localized at HRP-labeled sites. The DAB reaction product can be visualized directly by bright-field light microscopy or, following osmication, by electron microscopy. We offer DAB Histochemistry Kits for detecting mouse IgG primary antibodies (D22185) and biotinylated antibodies and tracers (D22187). Each kit contains:

  • Diaminobenzidine (DAB)
  • HRP-labeled goat anti–mouse IgG antibody (in Kit D22185) or streptavidin (in Kit D22187) conjugate
  • Hydrogen peroxide (H2O2) reaction additive
  • Blocking reagent
  • Staining buffer
  • Detailed staining protocols (Diaminobenzidine Histochemistry Kits)

Each kit provides sufficient materials to stain approximately 200 slides.

Myeloperoxidase

Myeloperoxidase (MPO, EC 1.11.1.7) is a lysosomal hemeoprotein located in the azurophilic granules of polymorphonuclear (PMN) leukocytes and monocytes. It is a dimeric protein composed of two 59 kD and two 13.5 kD subunits. MPO is a unique peroxidase that catalyzes the conversion of hydrogen peroxide (H2O2) and chloride to hypochlorous acid, the major strong oxidant with powerful antimicrobial activity and broad-spectrum reactivity with biomolecules. MPO has been considered as an important marker for inflammatory and autoimmune diseases and cancer. MPO is also experimentally and clinically important for distinguishing myeloid from lymphoid leukemia and, due to its role in the pathology of atherogenesis, has been advocated as a prognostic marker of cardiovascular disease.

The ferric, or native, MPO reacts with hydrogen peroxide (H2O2) to form the active redox and enzyme intermediate compound MPO-I, which oxidizes chloride (Cl–) to HOCl; these reactions make up the chlorination cycle. MPO also oxidizes a variety of substrates, including phenols and anilines, via the classic peroxidation cycle. The relative concentrations of chloride and the reducing substrate (AH) determine whether MPO uses hydrogen peroxide for chlorination or peroxidation. Assays based on measurement of chlorination activity are more specific for MPO than those based on peroxidase substrates such as tetramethylbenzidine (TMB).

EnzChek Myeloperoxidase (MPO) Activity Assay Kit

The EnzChek Myeloperoxidase (MPO) Activity Assay Kit (E33856) provides assays for rapid and sensitive determination of both chlorination and peroxidation activities of MPO in solution and in cell lysates. For detection of chlorination, the kit provides nonfluorescent 3'-(p-aminophenyl) fluorescein (APF), which is selectively cleaved by hypochlorite (–OCl) to yield fluorescein. Peroxidation is detected using nonfluorescent Amplex UltraRed reagent, which is oxidized by the H2O2-generated redox intermediates MPO-I and MPO-II to form a fluorescent product with excitation/ emission maxima of ~568/581 nm (spectra). The EnzChek Myeloperoxidase Activity Assay Kit can be used to continuously detect these activities at room temperature over a broad dynamic range (1.5 to 200 ng/mL). The speed (30 minutes), sensitivity, and mix-and-read convenience make this kit ideal for measuring MPO activities and for high-throughput screening for MPO-specific inhibitors. Each EnzChek Myeloperoxidase (MPO) Activity Assay Kit contains:

  • 3'-(p-aminophenyl) fluorescein (APF)
  • Amplex UltraRed reagent
  • Human myeloperoxidase (MPO) standard
  • Chlorination inhibitor
  • Peroxidation inhibitor
  • Hydrogen peroxide (H2O2)
  • Phosphate-buffered saline (PBS)
  • Detailed protocols (EnzChek(R) Myeloperoxidase (MPO) Activity Assay Kit)

Sufficient reagents are provided to perform 200 assays for chlorination and 200 assays for peroxidation activity in a 96-well fluorescence microplate format (100 µL per assay).

Zen Myeloperoxidase (MPO) ELISA Kit

The Zen Myeloperoxidase (MPO) ELISA Kit (Z33857) provides a comprehensive set of components for accurate and sensitive quantitation of MPO in a variety of biological samples, including human serum. This assay is based on the Amplex UltraRed reagent, a fluorogenic substrate for horseradish peroxidase (HRP) that reacts with H2O2 in a 1:1 stoichiometric ratio to produce the Amplex UltraRed product, a brightly fluorescent and strongly absorbing product with excitation/ emission maxima of ~568/581 nm (spectra). Because the Amplex UltraRed product has long-wavelength emission, there is little interference from the blue or green autofluorescence found in most biological samples. With a high extinction coefficient, good quantum efficiency, and resistance to autooxidation, the fluorescence-based Amplex UltraRed reagent delivers better sensitivity and a broader assay range than colorimetric reagents. Each kit contains:

  • Amplex UltraRed reagent
  • Dimethylsulfoxide (DMSO)
  • Concentrated phosphate-buffered saline (PBS)
  • Horseradish peroxidase (HRP)–labeled goat anti–rabbit IgG antibody
  • Amplex stop reagent
  • Hydrogen peroxide (H2O2)
  • MPO standard
  • Bovine serum albumin (BSA)
  • Tween 20
  • Mouse anti-MPO antibody (primary capture antibody)
  • Rabbit anti-MPO antibody (secondary capture antibody)
  • Zen microplates
  • Detailed protocols (Zen Myeloperoxidase (MPO) ELISA Kit)

Sufficient reagents are provided for ~200 assays in a microplate format, using a 100 µL per well reaction volume. The microplate-based formulation provides speed and convenience yet is highly sensitive, and it can be used to detect MPO over a broad dynamic range (0.2 to 100 ng/mL) at room temperature.

Catalase

The Amplex Red Catalase Assay Kit (A22180) provides an ultrasensitive, yet simple, assay for measuring catalase activity. Catalase is a heme-containing redox protein found in nearly all animal and plant cells, as well as in aerobic microorganisms. In eukaryotic cells, it is concentrated in the peroxisomes. Catalase is an important enzyme because H2O2 is a powerful oxidizing agent that is potentially damaging to cells. By preventing excessive buildup of H2O2, catalase allows important cellular processes that produce H2O2 as a by-product to take place safely.

In the assay, catalase first reacts with H2O2 to produce water and oxygen (O2). Next, the Amplex Red reagent reacts with a 1:1 stoichiometry with any unreacted H2O2 in the presence of horseradish peroxidase to produce the highly fluorescent oxidation product, resorufin. Therefore, as catalase activity increases, the signal from resorufin decreases (Figure 10.67). The results are typically plotted by subtracting the observed fluorescence from that of a no-catalase control. Using this kit, it is possible to detect catalase activity in a purified system at levels as low as 50 mU/mL.

Resorufin has absorption and fluorescence emission maxima of approximately 571 nm and 585 nm, respectively (spectra). Because the absorption is strong, the assay can be performed either fluorometrically or spectrophotometrically. The Amplex Red Catalase Assay Kit contains:

  • Amplex Red reagent
  • Dimethylsulfoxide (DMSO)
  • Horseradish peroxidase (HRP)
  • Hydrogen peroxide (H2O2)
  • Concentrated reaction buffer
  • Catalase
  • Detailed protocols (Amplex(R) Red Catalase Assay Kit)

Each kit provides sufficient reagents for approximately 400 assays using either a fluorescence- or absorption-based microplate reader and a reaction volume of 100 µL per assay.

Glucose and Glucose Oxidase

Glucose oxidase is widely used for glucose determination and, when conjugated to antibodies, for use in enzyme immunoassays (EIAs). Molecular Probes has found that the Amplex Red reagent can be utilized for the ultrasensitive detection of both glucose and glucose oxidase. In this enzyme-coupled assay, glucose oxidase reacts with glucose to form gluconolactone and H2O2. The H2O2 is then detected using the Amplex Red reagent peroxidase substrate (Figure 10.59). The Amplex Red Glucose/Glucose Oxidase Assay Kit (A22189) can be used to detect glucose levels as low as 3 µM or 0.5 µg/mL (Figure 10.25) and is at least 10-fold more sensitive than assays using o-dianisidine as the peroxidase substrate. This kit can also be used to detect glucose oxidase levels as low as 0.05 mU/mL (Figure 10.68). We have even shown that the Amplex Red reagent can detect glucose liberated from native dextrans by dextranase ref and from carboxymethylcellulose. The Amplex Red Glucose/Glucose Oxidase Assay Kit contains:

The kit provides sufficient reagents for approximately 500 assays using either a fluorescence- or absorption-based microplate reader and a reaction volume of 100 µL per assay.

Galactose and Galactose Oxidase

The Amplex Red Galactose/Galactose Oxidase Assay Kit (A22179) provides the reagents and a general protocol (Amplex(R) Red Galactose/Galactose Oxidase Kit) for the assay of terminal galactosylated proteins, galactose-producing enzymes and for the assay of galactose oxidase. Unlike glucose oxidase, galactose oxidase can produce H2O2 from either free galactose or from polysaccharides — including glycoproteins in solution or on cell surfaces — in which galactose is the terminal residue, producing an aldehyde moiety on the 6-position of the galactose (Figure 10.14). We have used our Amplex Red galactose oxidase assay for the quantitative assay of mucin-type glycoproteins by using a method similar to one described by Kinosita and collaborators.