Detecting Glycosidases—Section 10.2

Page Contents

• Fluorogenic β-Galactosidase Substrates
• Modified Fluorogenic β-Galactosidase Substrates with Improved Cellular Retention
• NovaBright β-Galactosidase Chemiluminescent Detection Kits
• Amplex Red Galactose/Galactose Oxidase Assay Kit
• Fluorogenic β-Glucuronidase Substrates
• Fluorogenic β-Glucosidase Substrates
• Amplex Red Glucose/Glucose Oxidase Assay Kit
• Amplex Red Neuraminidase/Sialidase Assay Kit
• EnzChek Cellulase Substrate
• EnzChek Ultra Amylase Assay Kit
• EnzChek Ultra Xylanase Assay Kit
• EnzChek Lysozyme Assay Kit
• Chromogenic Glycosidase Substrates
• Auxiliary Products for Glycosidase Research
• Data Table
• Ordering Information

Glycosidase enzymes exhibit very high selectivity for hydrolysis of their preferred sugars. For example, β-galactosidase rapidly hydrolyzes β-D-galactopyranosides but usually does not hydrolyze either the anomeric α-D-galactopyranosides or the isomeric β-D-glucopyranosides. Endogenous glycosidase activity is frequently used to characterize strains of microorganisms and to selectively label organelles of mammalian cells; defects in glycosidase activity are characteristic of several diseases.

In addition, glycosidases are important reporter gene markers. Specifically, lacZ, which encodes β-galactosidase, is extensively used as a reporter gene in animals and yeast, whereas the β-glucuronidase (GUS) gene is a popular reporter gene in plants. Glycosidase substrates are also used in conjunction with glycosidase-conjugated secondary detection reagents in immunohistochemical techniques and enzyme-linked immunosorbent assays (ELISAs). Molecular Probes fluorogenic and chromogenic glycosidase substrates are listed in Glycosidase enzymes and their fluorogenic and chromogenic substrates—Table 10.1.

Fluorescein Digalactoside

Probably the most sensitive fluorogenic substrate for detecting β-galactosidase is fluorescein di-β-D-galactopyranoside (FDG, F1179; ) Nonfluorescent FDG is sequentially hydrolyzed by β-galactosidase, first to fluorescein monogalactoside (FMG) and then to highly fluorescent fluorescein (F1300, F36915; Introduction to Enzyme Substrates and Their Reference Standards—Section 10.1). Enzyme-mediated hydrolysis of FDG can be followed by the increase in either absorbance or fluorescence. Although the turnover rates of FDG and its analogs are considerably slower than that of the common spectrophotometric galactosidase substrate, o-nitrophenyl β-D-galactopyranoside (ONPG), the absorbance of fluorescein is about fivefold greater than that of o-nitrophenol. Moreover, fluorescence-based measurements can be several orders of magnitude more sensitive than absorption-based measurements. Fluorescence-based assays employing FDG are also reported to be 100- to 1000-fold more sensitive than radioisotope-based ELISAs.

In addition to its use in ELISAs, the FDG substrate has proven very effective for identifying lacZ-positive cells with fluorescence microscopy and flow cytometry. FDG has been employed to identify cells infected with recombinant herpesvirus, to detect unique patterns of β-galactosidase expression in live transgenic zebrafish embryos and to monitor β-galactosidase expression in bacteria. The purity of FDG and its analogs is very important because a reagent with extremely low fluorescence background is essential for most applications. Our stringent quality control ensures that the fluorescent contamination of FDG is less than 50 ppm.

The FluoReporter lacZ Flow Cytometry Kit (F1930) provides materials and protocols for quantitating β-galactosidase activity with FDG in single cells using flow cytometry. The FluoReporter lacZ Flow Cytometry Kit contains:

• FDG
• Phenylethyl β-D-thiogalactopyranoside (PETG; also available separately as a solid, P1692), a broad-spectrum β-galactosidase inhibitor for stopping the reaction
• Chloroquine diphosphate for inhibiting hydrolysis of the substrate in acidic organelles by endogenous galactosidase enzymes
• Propidium iodide for detecting dead cells
• Detailed protocol (FluoReporter lacZ Flow Cytometry Kits)

Each kit provides sufficient reagents for 50 flow cytometry assays. This assay enables researchers to detect heterogeneous expression patterns and to sort and clone individual cells expressing known quantities of β-galactosidase. Practical reviews on using FDG for flow cytometric analysis and sorting of lacZ-positive cells are available.

The fluorescent hydrolysis product of FDG is fluorescein (F1300, F36915; Introduction to Enzyme Substrates and Their Reference Standards—Section 10.1) , which rapidly leaks from cells under physiological conditions, making the use of FDG problematic for prolonged studies. Our DetectaGene Green, ImaGene Green and ImaGene Red substrates (see below) have been specifically designed to improve retention of the fluorescent hydrolysis products in cells.

Resorufin Galactoside

Unlike FDG, resorufin β-D-galactopyranoside (R1159) requires only a single-step hydrolysis reaction to attain full fluorescence. This substrate is especially useful for sensitive enzyme measurements in ELISAs. The relatively low pK_a (~6.0) of its hydrolysis product, resorufin (R363, Introduction to Enzyme Substrates and Their Reference Standards—Section 10.1), permits its use for continuous measurement of enzymatic activity at physiological pH. Resorufin galactoside has also been used to quantitate β-galactosidase activity in single yeast cells by flow cytometry and to detect immobilized β-galactosidase activity in bioreactors.

DDAO Galactoside

Although substrates based on DDAO (7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one)) are intrinsically fluorescent (excitation/emission ~460/610 nm), β-galactosidase–catalyzed hydrolysis of DDAO galactoside (D6488) liberates the DDAO fluorophore, which absorbs and emits light at much longer wavelengths (excitation/emission ~645/660 nm) (Figure 10.2.1). Not only can DDAO (H6482, Introduction to Enzyme Substrates and Their Reference Standards—Section 10.1) be excited without interference from the unhydrolyzed substrate, but its fluorescence emission is detected at wavelengths that are well beyond the autofluorescence exhibited by most biological samples. The relatively low pK_a of DDAO (~5.5) permits continuous monitoring of β-galactosidase activity at physiological pH.

Figure 10.2.1 Absorption spectra of 1) DDAO galactoside (D6488) and 2) DDAO (H6482) at equal concentrations in pH 9 aqueous buffer. These spectra show the large spectral shift accompanying enzymatic cleavage of DDAO-based substrates.

Methylumbelliferyl Galactoside

The fluorogenic β-galactosidase substrate β-methylumbelliferyl β-D-galactopyranoside (MUG, M1489MP) is commonly used to detect β-galactosidase activity in cell extracts, lysosomes and human blood serum. However, the hydrolysis product, 7-hydroxy-4-methylcoumarin (β-methylumbelliferone, H189; Introduction to Enzyme Substrates and Their Reference Standards—Section 10.1; Figure 10.2.2), has a relatively high pK_a (~7.8), precluding its use for continuous measurement of enzymatic activity.

Figure 10.2.2 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.

Carboxyumbelliferyl Galactoside and the FluoReporter lacZ/Galactosidase Quantitation Kit

Hydrolysis of 3-carboxyumbelliferyl β-D-galactopyranoside (CUG) by β-galactosidase yields 7-hydroxycoumarin-3-carboxylic acid (H185, Introduction to Enzyme Substrates and Their Reference Standards—Section 10.1). 7-Hydroxycoumarin has a pK_a below the pH at which the turnover rate is optimal, facilitating the use of CUG for continuous measurements of β-galactosidase activity. Unlike most substrates for β-galactosidase, CUG is quite water-soluble and can be used over a wide range of concentrations in enzymatic activity measurements. Our FluoReporter lacZ/Galactosidase Quantitation Kit (F2905) provides a CUG-based method for quantitating β-galactosidase activity in ELISAs or lacZ-positive cell extracts. Each kit contains:

• CUG
• 7-Hydroxycoumarin-3-carboxylic acid, a reference standard
• Detailed protocols (FluoReporter lacZ/Galactosidase Quantitation Kit) suitable for use with any fluorescence microplate reader

Sufficient reagents are provided for approximately 1000 β-galactosidase assays. We have demonstrated a practical detection limit of ~0.5 pg of β-galactosidase using this kit and a fluorescence microplate reader.

Fluorescent Glycosphingolipids

β-Galactosidase enzymes that act on the lipophilic sphingosyl galactosides, including galactosylceramidase (EC 3.2.1.46) and G_M1 ganglioside β-galactosidase (EC 3.2.1.23), are particularly important in neurochemistry. The preferred substrates for these enzymes are sphingolipids derived from galactose (Sphingolipids, Steroids, Lipopolysaccharides and Related Probes—Section 13.3). Galactosylceramidase converts substrates such as our BODIPY FL C₁₂-galactosylceramide (D7519) back to the ceramide. Purified G_M1 ganglioside galactosidase removes the terminal galactose residue from lactosylceramides such as our BODIPY FL C₅-lactosylceramide (D13951), yielding the corresponding glucosylceramide. BODIPY FL C₅-lactosylceramide complexed with bovine serum albumin (BSA) (B34402) may also be useful for assaying G_M1 ganglioside galactosidase. Complexing fluorescent lipids with BSA facilitates the preparation of aqueous solutions by eliminating the need for organic solvents to dissolve the lipophilic probe. The lack of a spectral shift between the substrates and the hydrolysis products in these reactions means that extraction and chromatographic separation of the products is necessary for assessment of enzyme activity.

The primary problems associated with detecting lacZ expression in live cells using fluorogenic substrates are:

• Difficulty in loading the substrates under physiological conditions
• Leakage of the fluorescent product from live cells
• High levels of endogenous β-galactosidase activity in many cells

Our DetectaGene Green, ImaGene Green and ImaGene Red Kits are designed to improve the sensitivity of β-galactosidase assays by yielding products that are better retained in viable cells and, in the case of the ImaGene Green and ImaGene Red Kits, by providing substrates that can be passively loaded into live cells. The high level of endogenous β-galactosidase activity remains an obstacle when detecting low levels of lacZ expression.

DetectaGene Green lacZ Gene Expression Kit

The substrate in our DetectaGene Green lacZ Gene Expression Kit (D2920)—5-chloromethylfluorescein di-β-D-galactopyranoside (CMFDG)—is a galactose derivative that has been chemically modified to include a mildly thiol-reactive chloromethyl group (Figure 10.2.3). Once loaded into the cell using the Influx pinocytic cell-loading reagent (I14402, included in Kit D2920; ) or by microinjection, hypotonic shock or another technique (Choosing a Tracer—Section 14.1, Techniques for loading molecules into the cytoplasm—Table 14.1), the DetectaGene substrate undergoes two reactions: 1) its two galactose moieties are cleaved by intracellular β-galactosidase and 2) either simultaneously or sequentially, its chloromethyl moiety reacts with glutathione and possibly other intracellular thiols to form a membrane-impermeant, peptide–fluorescent dye adduct (Figure 10.2.3). Because peptides do not readily cross the plasma membrane, the resulting fluorescent adduct is much better retained than is the free dye, even in cells that have been kept at 37°C. We have found that lacZ-positive cells loaded from medium containing 1 mM CMFDG are as fluorescent as cells loaded with 40-fold higher concentrations of FDG. Furthermore, unlike the free dye, the peptide–fluorescent dye adducts contain amine 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, as well as amplification of the conjugate by standard immunohistochemical techniques using our anti–fluorescein/Oregon Green dye antibody (Anti-Dye and Anti-Hapten Antibodies—Section 7.4, Anti-fluorophore antibodies and their conjugates—Table 7.8).

The CMFDG substrate in our DetectaGene Green lacZ Gene Expression Kit was used to stain lacZ-expressing floor plate cells in tissue dissected from a developing mouse embryo, to identify lacZ-enhancer–trapped Drosophila neurons in culture and to detect β-galactosidase activity in hippocampus slices. In the latter study, the fluorescence of the neurons could still be visualized 24 hours after dye loading, and the fluorescent CMFDG-loaded neurons exhibited a normal pattern and time course of axonal outgrowth and branching. CMFDG also has been microinjected into primary hepatocytes, fibroblasts and glioma cells to detect β-galactosidase activity and has been incorporated into an electrophysiological recording pipette to confirm the identity of neurons cotransfected with the lacZ gene and a second gene encoding Ca²⁺/calmodulin-dependent protein kinase II (CaM kinase II).

The DetectaGene Green CMFDG lacZ Gene Expression Kit (D2920) contains:

• DetectaGene Green CMFDG substrate (Figure 10.2.3)
• Phenylethyl β-D-thiogalactopyranoside (PETG; also available separately as a solid, P1692), a broad-spectrum β-galactosidase inhibitor for stopping the reaction
• Verapamil for inhibiting product efflux
• Chloroquine diphosphate for inhibiting acidic hydrolysis of the substrate
• Propidium iodide for detecting dead cells
• Influx pinocytic cell-loading reagent for introducing CMFDG into cells
• Detailed protocols for detecting β-galactosidase activity (DetectaGene Green CMFDG lacZ Gene Expression Kit)

When used at the recommended dilutions, sufficient reagents are provided for approximately 200 flow cytometry tests with the DetectaGene Green CMFDG Kit. Verapamil has been added to the DetectaGene Green CMFDG lacZ Gene Expression Kit because we have observed that cell retention of the fluorescent dye–peptide adduct can be considerably improved in many cell types by adding verapamil to the medium.

Figure 10.2.3 Sequential β-galactosidase hydrolysis and peptide conjugate formation of CMFDG, a component of the DetectaGene Green CMFDG lacZ Gene Expression Kit (D2920).

PFB Aminofluorescein Digalactoside

5-(Pentafluorobenzoylamino)fluorescein di-β-D-galactopyranoside (PFB-FDG, P11948; ) yields the green-fluorescent 5-(pentafluorobenzoylamino)fluorescein (PFB-F), which appears to localize to endosomal and lysosomal compartments when loaded into cells by pinocytosis (), similar to our PFB aminofluorescein diglucoside (PFB-FDGlu, P11947; see below). Thus, PFB-FDG is potentially useful for studying lysosomal storage diseases, including Krabbe disease, G_M1 gangliosidosis, galactosialidosis and Morquio syndrome, which are all associated with deficient lysosomal β-galactosidase activity.

ImaGene Green and ImaGene Red lacZ Reagents and Gene Expression Kits

The fluorescein- and resorufin-based galactosidase substrates in our ImaGene Green and ImaGene Red lacZ Gene Expression Kits (I2904, I2906) have been covalently modified to include a 12-carbon lipophilic moiety. Unlike FDG or CMFDG, these lipophilic fluorescein- and resorufin-based substrates—abbreviated C₁₂FDG () and C₁₂RG () for the ImaGene Green and ImaGene Red substrates, respectively—can be loaded simply by adding the substrate to the aqueous medium in which the cells or organisms are growing, either at ambient temperatures or at 37°C. Once inside the cell, the substrates are cleaved by β-galactosidase, producing fluorescent products that are well retained by the cells, probably by incorporation of their lipophilic tails within the cellular membranes. Mammalian NIH 3T3 lacZ-positive cells grown for several days in medium containing 60 µM C₁₂FDG appear morphologically normal, continue to undergo cell division and remain fluorescent for up to three cell divisions after replacement with substrate-free medium.

The C₁₂FDG substrate in our ImaGene Green lacZ Expression Kit (I2904) is superior to FDG for flow cytometric detection of β-galactosidase activity in live mammalian cells. Using C₁₂FDG with flow cytometric methods, researchers have:

• Analyzed senescence-associated β-galactosidase activity
• Identified endocrine cell precursors in dissociated fetal pancreatic tissue based on their high levels of endogenous acid β-galactosidase
• Measured β-galactosidase activity in single recombinant Escherichia coli bacteria
• Detected the activity of β-galactosidase fusion proteins in yeast
• Sorted β-galactosidase–expressing mouse sperm cells and insect cells that harbor recombinant baculovirus

The C₁₂FDG substrate was also useful in a fluorescence microscopy study of zebrafish expressing a lacZ reporter gene that was under the control of a mammalian homeobox gene promoter. In some cell types, C₁₂FDG produces high levels of background fluorescence that may prohibit its use in assaying low β-galactosidase expression.

The ImaGene Green C₁₂FDG or ImaGene Red C₁₂RG lacZ Gene Expression Kits contain:

• ImaGene Green C₁₂FDG (in Kit I2904) or ImaGene Red C₁₂RG (in Kit I2906)
• Phenylethyl β-D-thiogalactopyranoside (PETG; also available separately as a solid, P1692), a broad-spectrum β-galactosidase inhibitor for stopping the reaction
• Chloroquine diphosphate for inhibiting acidic hydrolysis of the substrate
• Detailed protocols for detecting β-galactosidase activity (ImaGene Green C12FDG lacZ Gene Expression Kit, ImaGene Red C12RG lacZ Gene Expression Kit)

Each kit provides sufficient reagents for 100–200 assays, depending on the volume used for each experiment. 5-Dodecanoylaminofluorescein di-β-D-galactopyranoside (C₁₂FDG) is available separately (D2893). We also offer 5-acetylaminofluorescein di-β-D-galactopyranoside (C₂FDG, A22010), which has been shown to be useful for detecting lacZ reporter gene expression in slow-growing mycobacteria, including Mycobacterium tuberculosis, using a fluorescence microplate reader or a flow cytometer.

The NovaBright β-Galactosidase Enzyme Reporter Gene Chemiluminescent Detection Kits for mammalian cells (N10563, N10564) and for yeast cells (N10565, N10566) are designed for the rapid, simple and sensitive detection of the β-galactosidase reporter enzyme directly in microwell cultures of mammalian and yeast cells, respectively. This chemiluminescence-based assay uses the Galacton-Star 1,2-dioxetane substrate (Figure 10.2.4) with a luminescence enhancer to generate glow light emission kinetics. A single reagent, which provides cell lysis components, luminescent substrate and enhancer, is added directly to cells in culture medium. Light emission typically reaches maximum intensity within 60–90 minutes and remains constant for 45–90 minutes or longer, depending upon assay temperature. Light emission is measured in a variety of luminometers without the need for solution injection.

The simple assay format and glow light emission achieved with the NovaBright β-Galactosidase Enzyme Reporter Gene Chemiluminescent Detection Kit provide an ideal assay system for automated high-throughput screening applications, enabling simple processing and measurement of multiple microplates. The dynamic range of the assay spans five orders of magnitude, from picogram to nanogram levels, enabling detection of a wide range of reporter enzyme concentration in cells.

Each NovaBright β-Galactosidase Enzyme Reporter Gene Chemiluminescent Detection Kit provides:

• Galacton-Star substrate concentrate
• Assay buffer for mammalian cells (200 assays, N10563; 1000 assays, N10564) or for yeast cells (200 assays, N10565; 1000 assays, N10566)
• Detailed protocols (NovaBright β-Galactosidase Enzyme Reporter Gene Chemiluminescent Detection System for Yeast or Mammalian Cells)

Chemiluminescent 1,2-dioxetane substrates for β-galactosidase, including the Galacton-Star substrate described here and the Galacton-Plus substrate provided in the NovaBright β-Galactosidase and Firefly Luciferase Dual Enzyme Reporter Gene Chemiluminescent Detection Kits (N10561, N10562; Substrates for Microsomal Dealkylases, Acetyltransferases, Luciferases and Other Enzymes—Section 10.6) provide highly sensitive enzyme detection and have been utilized extensively in reporter assays in mammalian cell and tissue extracts.

Figure 10.2.4 Galacton-Star chemiluminescent β-galactosidase substrate.

Molecular Probes Amplex Red reagent (10-acetyl-3,7-dihydroxyphenoxazine; A12222, A22177) is an unusually stable peroxidase substrate that we have used in coupled reactions to detect a wide variety of analytes, including both enzymes and their substrates (see Substrates for Oxidases, Including Amplex Red Kits—Section 10.5 for a list of all of our Amplex Red Kits and reagents). Most of the assays can be performed as continuous assays at neutral or slightly acidic pH and are particularly suitable for automation and high-throughput screening using either an absorption- or fluorescence-based microplate reader.

Rather than requiring an unnatural fluorogenic or chromogenic substrate for β-galactosidase (or α-galactosidase), our Amplex Red reagent–based technology permits the direct quantitation of free galactose, which is produced by a wide variety of enzymes. Even enzymes that act on polysaccharides and glycolipids but are difficult to assay using known chromogenic substrates can, in some cases, be detected and their activity quantitated using the Amplex Red reagent in combination with galactose oxidase and horseradish peroxidase. Unlike glucose oxidase, galactose oxidase produces H₂O₂ from either free galactose or from polysaccharides—including glycoproteins in solution and on cell surfaces—and from certain glycolipids in which galactose is the terminal residue (Figure 10.2.5). Because the galactose oxidase–catalyzed reaction does not require prior cleavage of the glycoside to free galactose by a galactosidase, appropriate control reactions must be used to ascertain whether the rate-limiting step is the galactosidase- or galactose oxidase–mediated reaction.

The Amplex Red Galactose/Galactose Oxidase Assay Kit (A22179) provides an ultrasensitive fluorescence-based method for detecting galactose (Figure 10.2.6) and galactose oxidase (Figure 10.2.7) activity. This assay utilizes the Amplex Red reagent to detect H₂O₂ generated by galactose oxidase–mediated oxidation of desialated galactose moieties. In the presence of horseradish peroxidase (HRP), the H₂O₂ thus produced reacts with the Amplex Red reagent in a 1:1 stoichiometry to generate the red-fluorescent oxidation product resorufin. Resorufin has absorption and fluorescence emission maxima of approximately 571 nm and 585 nm, respectively, and because its extinction coefficient is high (54,000 cm^-1M^-1), the assay can be performed either fluorometrically or spectrophotometrically. The Amplex Red Galactose/Galactose Oxidase Assay Kit provides all the reagents and a general protocol for the assay of galactose-producing enzymes or for the assay of galactose oxidase, including:

• Amplex Red reagent
• Dimethylsulfoxide (DMSO)
• Horseradish peroxidase (HRP)
• Hydrogen peroxide (H₂O₂)
• Concentrated reaction buffer
• Galactose oxidase from Dactylium dendroides
• D-Galactose
• Detailed protocols (Amplex Red Galactose/Galactose Oxidase Kit)

Sufficient reagents are provided for approximately 400 assays using either a fluorescence- or absorption-based microplate reader and reaction volumes of 100 µL per assay. The Amplex Red galactose/galactose oxidase assay accurately measures as low as 4 µM galactose and 2 mU/mL galactose oxidase activity (Figure 10.2.6, Figure 10.2.7). Because of the high absorbance of resorufin, the spectrophotometric assay has only slightly lower sensitivity than the fluorometric assay.

Figure 10.2.5 Detection scheme utilized in the Amplex Red Galactose/Galactose Oxidase Assay Kit (A22179). Oxidation of the terminal galactose residue of a glycoprotein, glycolipid or polysaccharide results in the generation of H₂O₂, which, in the presence of horseradish peroxidase (HRP), reacts stoichiometrically with the Amplex Red reagent to generate the red-fluorescent oxidation product, resorufin.

Figure 10.2.6 Detection of galactose using the Amplex Red Galactose/Galactose Oxidase Assay Kit (A22179). Each reaction contained 50 µM Amplex Red reagent, 0.1 U/mL HRP, 2 U/mL of galactose oxidase and the indicated amount of galactose in 1X reaction buffer. Reactions were incubated at 37°C. After 30 minutes, fluorescence was measured in a fluorescence microplate reader using excitation at 530 ± 12.5 nm and fluorescence detection at 590 ± 17.5 nm. A background fluorescence of 93 units was subtracted from each data point.

Figure 10.2.7 Detection of galactose oxidase activity using the Amplex Red Galactose/Galactose Oxidase Assay Kit (A22179). Each reaction contained 50 µM Amplex Red reagent, 0.1 U/mL HRP, 100 µM galactose and the indicated amount of galactose oxidase in 1X reaction buffer. Reactions were incubated at 37°C. After 20 minutes, fluorescence was measured in a fluorescence microplate reader using excitation at 530 ± 12.5 nm with fluorescence detection at 590 ± 17.5 nm.

The substrate 4-methylumbelliferyl β-D-glucuronide (MUGlcU, M1490) is probably the most commonly used fluorogenic reagent for identifying E. coli contamination and for detecting GUS reporter gene expression in plants and plant extracts. However, β-glucuronidase substrates based on fluorescein may be much more sensitive and yield products that are fluorescent at physiological pH, making them useful for continuous monitoring of enzymatic activity.

Fluorescein Diglucuronide

Fluorescein di-β-D-glucuronide (FDGlcU, F2915) is colorless and nonfluorescent until it is hydrolyzed to the monoglucuronide and then to highly fluorescent fluorescein (F1300, F36915; Introduction to Enzyme Substrates and Their Reference Standards—Section 10.1). FDGlcU has been used to detect β-glucuronidase activity in plant and animals containing the GUS reporter gene and may also be useful for assaying lysosomal enzyme release from neutrophils. FDGlcU has also been used in the flow cytometry assay of individual mammalian cells expressing the E. coli β-glucuronidase gene.

PFB Aminofluorescein Diglucuronide

5-(Pentafluorobenzoylamino)fluorescein di-β-D-glucuronide (PFB-FDGlcU, P11949) yields the green-fluorescent 5-(pentafluorobenzoylamino)fluorescein (PFB-F), which appears to localize in endosomal and lysosomal compartments when PFB-FDGlcU is loaded into cells by pinocytosis, similar to PFB aminofluorescein diglucoside (PFB-FDGlu, P11947) described below. PFB-FDGlcU has been used for the quantitative analysis of β-glucuronidase activity in viable cells and for sorting high-expressing cells by flow cytometry.

Coumarin Glucuronides

4-Methylumbelliferyl β-D-glucuronide (MUGlcU, M1490) has been used extensively to detect E. coli in food, water, urine and environmental samples. MUGlcU is stable to the conditions required for sterilization of media. A fluorogenic bioassay using MUGlcU has been developed to assess the detrimental effects of Li⁺, Al³⁺, Cr⁶⁺ and Hg²⁺ on the proliferation of E. coli. MUGlcU is also commonly used to identify plant tissue expressing the GUS reporter gene, including nondestructive assays that allow propagation of the transformed plant lines. In addition, MUGlcU has served as a sensitive substrate for lysosomal enzyme release from neutrophils.

ImaGene Green β-D-Glucuronidase Substrate

We also offer a lipophilic analog of fluorescein di-β-D-glucuronide in the ImaGene Green C₁₂FDGlcU GUS Gene Expression Kit (I2908). As with our similar ImaGene Green and ImaGene Red substrates for β-galactosidase (see above), we have shown that this lipophilic β-glucuronidase substrate freely diffuses across the membranes of viable cultured tobacco leaf cells or protoplasts under physiological conditions. Furthermore, the fluorescent cleavage product is retained in the plant cell for hours to days, facilitating long-term measurements of GUS gene expression. In thin sections of transgenic tomato leaf, the ImaGene Green C₁₂FDGlcU GUS Gene Expression Kit provided a simple and reliable GUS assay that, coupled with confocal laser-scanning microscopy, yielded good cellular resolution. The substrate has also been used to detect β-glucuronidase activity in an Acremonium transformant containing the GUS reporter gene.

The ImaGene Green C₁₂FDGlcU GUS Gene Expression Kit contains:

• ImaGene Green C₁₂FDGlcU
• D-Glucaric acid-1,4-lactone, a β-glucuronidase inhibitor for stopping the reaction
• Detailed protocols for detecting β-glucuronidase activity (ImaGene Green C12FDGlcU GUS Gene Expression Kit)

Each kit provides sufficient reagents for approximately 100 tests, depending on the volume used for each experiment.

β-Glucosidase, which is a marker for the endoplasmic reticulum (Probes for the Endoplasmic Reticulum and Golgi Apparatus—Section 12.4), is present in nearly all species. Its natural substrate is a glucosylceramide (Sphingolipids, Steroids, Lipopolysaccharides and Related Probes—Section 13.3). People with Gaucher disease have mutations in the acid β-glucosidase gene that result in abnormal lysosomal storage. Enzyme replacement therapy in Gaucher disease patients requires sensitive and selective methods for measuring β-glucosidase activity (Glycosidase enzymes and their fluorogenic and chromogenic substrates—Table 10.1).

Fluorescein Diglucoside

As with the other fluorescein diglycosides, our fluorogenic fluorescein di-β-D-glucopyranoside (FDGlu, F2881) is likely to yield the greatest sensitivity for detecting β-glucosidase activity in both cells and emulsion droplets. This substrate has been used to demonstrate the utility of Saccharomyces cerevisiae and Candida albicans exo-1,3-β-glucanase genes as reporter genes. Because these reporter genes encode secreted proteins, assays for reporter gene expression do not require cell permeabilization. FDGlu has been reported to be a selective substrate for the flow cytometric assay of lysosomal glucocerebrosidase activity in a variety of cells. The assay demonstrated the inordinately low glucocerebrosidase activity present in fibroblasts of Gaucher disease patients.

PFB Aminofluorescein Diglucoside

Through a collaboration with Matthew Lorincz and Leonard A. Herzenberg at Stanford University Medical School, our PFB aminofluorescein diglucoside (PFB-FDGlu, P11947) has proven to be an excellent substrate for the flow cytometric discrimination of normal peripheral blood mononuclear cells (PBMC) from the PBMC of patients with Gaucher disease, a genetic deficiency in lysosomal β-glucocerebrosidase activity.

The Amplex Red reagent (10-acetyl-3,7-dihydroxyphenoxazine, A12222, A22177; Substrates for Oxidases, Including Amplex Red Kits—Section 10.5) is a colorless, stable and extremely versatile peroxidase substrate. In an application similar to our use of the Amplex Red reagent to detect galactose-producing enzymes (see above), we have shown that it is practical to detect free glucose with high specificity at levels as low as 0.5 µg/mL using the Amplex Red reagent in combination with excess glucose oxidase (Figure 10.2.8). Because the peroxidase- and glucose oxidase–mediated reactions can be coupled, it is potentially possible to measure the release of glucose by any glucosidase enzyme—for instance, α-glucosidase, β-glucosidase and glucocerebrosidase—in either a continuous or discontinuous assay (Figure 10.2.9). This assay should also be very useful for quantitation of glucose levels in foods, fermentation media and bodily fluids. The long-wavelength spectral properties of resorufin and high sensitivity of the assay result in little interference from colored components in the samples.

Our Amplex Red Glucose/Glucose Oxidase Assay Kit (A22189) provides all the reagents required for the assay of glucose and enzymes that produce glucose. This kit is also useful for the assay of glucose oxidase activity from cell extracts. We have even shown that the Amplex Red reagent can detect glucose liberated from native dextrans by dextranase and from carboxymethylcellulose by cellulase. The Amplex Red Glucose/Glucose Oxidase Assay Kit contains:

• Amplex Red reagent
• DMSO
• Horseradish peroxidase (HRP)
• Hydrogen peroxide (H₂O₂) for use as a positive control
• Concentrated reaction buffer
• Glucose oxidase
• D-glucose
• Detailed protocols (Amplex Red Glucose/Glucose Oxidase Assay Kit)

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.

Figure 10.2.8 Detection of glucose using the Amplex Red Glucose/Glucose Oxidase Assay Kit (A22189). Reactions containing 50 µM Amplex Red reagent, 0.1 U/mL HRP, 1 U/mL glucose oxidase and the indicated amount of glucose in 50 mM sodium phosphate buffer, pH 7.4, were incubated for one hour at room temperature. Fluorescence was then measured with a fluorescence microplate reader using excitation at 530 ± 12.5 nm and fluorescence detection at 590 ± 17.5 nm. Background fluorescence (arbitrary units), determined for a no-glucose control reaction, has been subtracted from each value. The inset shows the sensitivity and linearity of the assay at low levels of glucose (0–15 µM).

Figure 10.2.9 Principle of coupled enzymatic assays using our Amplex Red reagent. Oxidation of glucose by glucose oxidase results in generation of H₂O₂, which is coupled to conversion of the Amplex Red reagent to fluorescent resorufin by HRP. The detection scheme shown here is used in our Amplex Red Glucose/Glucose Oxidase Assay Kit (A22189).

Neuraminidase (NA, also known as sialidase) is a very common enzyme that hydrolyzes terminal sialic acid residues on polysaccharide chains, most often exposing a galactose residue. Although NA is found in mammals, it is predominantly expressed in microorganisms such as bacteria and viruses, including the negative-stranded RNA influenza virus. NA located on the surface of the influenza virus is thought to play a key role in its invasion of target cells and subsequent replication through cleavage of target cell receptor sialic acid moieties. Anti-influenza drug design has therefore focused on the inhibition of NA. Various methods using chemiluminescence, absorption and fluorescence have been developed to quantitate NA in biological fluids for detecting influenza virus and for screening inhibitors of NA activity in drug development. The ultimate goal has been to develop a rapid, single-step assay that is sensitive and adaptable for a high-throughput screening format.

The Amplex Red Neuraminidase (Sialidase) Assay Kit (A22178) provides an ultrasensitive fluorescence-based method for detecting NA activity. This assay utilizes the Amplex Red reagent to detect H₂O₂ generated by galactose oxidase–mediated oxidation of desialated galactose, the end result of NA action. In the presence of horseradish peroxidase (HRP), the H₂O₂ thus produced reacts with a 1:1 stoichiometry with the Amplex Red reagent to generate the red-fluorescent oxidation product, resorufin. Resorufin has absorption and fluorescence emission maxima of approximately 571 nm and 585 nm, respectively, and because the extinction coefficient is high (54,000 cm^-1M^-1), the assay can be performed either fluorometrically or spectrophotometrically. In a purified system with fetuin as the substrate, NA levels as low as 0.2 mU/mL have been detected with the Amplex Red Neuraminidase (Sialidase) Assay Kit (Figure 10.2.10). NA activity can also be detected in biological samples such as serum (Figure 10.2.11). Kit contents include:

• Amplex Red reagent
• Dimethylsulfoxide (DMSO)
• Horseradish peroxidase (HRP)
• Hydrogen peroxide (H₂O₂)
• Concentrated reaction buffer
• Galactose oxidase from Dactylium dendroides
• Fetuin from fetal calf serum
• Neuraminidase from Clostridium perfringens
• Detailed protocols (Amplex Red Neuraminidase (Sialidase) Assay Kit)

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

Figure 10.2.10 Detection of neuraminidase activity using the Amplex Red Neuraminidase (Sialidase) Assay Kit (A22178). Each reaction contained 50 µM Amplex Red reagent, 0.1 U/mL HRP, 2 U/mL galactose oxidase, 250 µg/mL fetuin and the indicated amount of neuraminidase in 1X reaction buffer. Reactions were incubated at 37°C. After 30 minutes, fluorescence was measured in a fluorescence microplate reader using excitation at 530 ± 12.5 nm and fluorescence detection at 590 ± 17.5 nm. A background fluorescence of 70 fluorescence units was subtracted from each data point. The inset shows the sensitivity and linearity of the assay at low levels of neuraminidase (0–0.8 mU/mL).

Figure 10.2.11 Detection of neuraminidase activity in serum using the Amplex Red Neuraminidase (Sialidase) Assay Kit (A22178). Each reaction contained 50 µM Amplex Red reagent, 0.1 U/mL HRP, 2 U/mL galactose oxidase, 250 µg/mL fetuin and the indicated amount of serum in 1X reaction buffer. Reactions were incubated at 37°C. After 60 minutes, fluorescence was measured in a fluorescence microplate reader using excitation at 530 ± 12.5 nm and fluorescence detection at 590 ± 17.5 nm. A background fluorescence of 112 units was subtracted from each data point.

The EnzChek cellulase substrate (E33953) was developed for simple and rapid fluorescence-based quantitation of cellulase (EC 3.2.1.4). This cellulase assay can be completed in 30 minutes or less using a simple mix-incubate-read format. In contrast to other more complex, multistep assays, the EnzChek cellulase assay is ideal for high-throughput screening, with a detection limit as low as 40 µU/mL using a fluorescence microplate reader (Figure 10.12).

In the presence of cellulase, the EnzChek cellulase substrate exhibits an increase in absorption at 360 nm, which is proportional to the amount of enzyme present and can be used to effectively quantitate enzyme activity using an absorption-based microplate reader or spectrophotometer. In addition, celluase activity can be assayed using the Amplex Red Glucose/Glucose Oxidase Assay Kit (A22189), as described above.

Figure 10.12 Sample standard curves obtained with the EnzChek cellulase substrate. A) Cellulase from Trichoderma reesei was assayed with EnzChek cellulase substrate (E33953) in 100 mM sodium acetate (pH 5.0) digestion buffer using cellulase activities from 0.018 to 11.4 mU/mL. B) This standard curve shows the same experiment at cellulase activities from 0.018 to 0.285 mU/mL. Samples were incubated for 30 minutes at room temperature and fluorescence was measured in a fluorescence microplate reader using excitation/emission wavelengths of 360/460 nm. Background fluorescence has not been subtracted.

α-Amylase is a hydrolytic enzyme that catalyzes the conversion of starch to a mixture of glucose, maltose, maltotriose and dextrins. The levels of α-amylase in various fluids of the human body are of clinical importance in the diagnosis of disease states, including pancreatitis and diabetes. Plant and microbial α-amylases are important enzymes for industrial applications ranging from the manufacture of baked goods and dairy products to the production of ethanol and paper.

The EnzChek Ultra Amylase Assay Kit (E33651) provides a solution-based assay with the speed, high sensitivity and convenience required for measuring amylase activity or for screening amylase inhibitors in a high-throughput format. This EnzChek Kit contains DQ starch, a starch derivative that is labeled with BODIPY FL dye to such a degree that the fluorescence is quenched. Amylase-catalyzed hydrolysis of this substrate relieves the quenching, yielding brightly fluorescent BODIPY FL dye–labeled fragments. The accompanying increase in fluorescence is proportional to amylase activity and can be monitored with a fluorescence microplate reader or fluorometer, using standard fluorescein filters. In tests using α-amylase from Bacillus sp. and 200 µg/mL of the DQ starch substrate (30-minute incubation at room temperature), the EnzChek Ultra Amylase Assay Kit could be used to detect α-amylase activity down to a final concentration of  0.002 U/mL, where one unit is defined as the amount of enzyme required to liberate 1 mg of maltose from starch in 3 minutes at 20°C, pH 6.9 (Figure 10.2.13).

The EnzChek Ultra Amylase Assay Kit provides:

• DQ starch—a heavily labeled, highly quenched BODIPY FL conjugate of starch from corn
• Concentrated reaction buffer
• Substrate solvent
• BODIPY FL propionic acid in dimethylsulfoxide (DMSO), for use as a fluorescence standard
• Detailed protocols (EnzChek Ultra Amylase Assay Kit)

Each kit provides sufficient reagents for 500 assays using 100 µL assay volumes in a 96-well microplate assay format.

Figure 10.2.13 Assay of α-amylase from Bacillus sp. using the EnzChek Ultra Amylase Assay Kit (E33651). Reactions contained 200 µg/mL DQ starch and the indicated amount of α-amylase in 100 mM MOPS (pH 6.9). After incubating at room temperature for 30 minutes, the fluorescence was measured in a microplate reader using excitation at 485 ± 12 nm and fluorescence detection at 520 ± 12 nm. The fluorescence of the blank sample (without enzyme) was subtracted from each value.

The hydrolysis of xylosidic linkages in hemicellulose polysaccharides by xylanase (EC 3.2.1.8) is important in a wide range of industrial processes, including baking, pulp and paper manufacturing and animal feed production. Xylanases occur in a variety of bacteria and fungi and are classified into two families—glycosyl hydrolase families 10 and 11. Family 11 xylanases are more specific for xylans; family 10 xylanases also exhibit cellulase activity. Existing xylanase assay methods typically require separation or heating steps. The chromogenic substrate o-nitrophenyl-β-D-xylobioside can be used in a simpler homogeneous assay format; however, o-nitrophenol is pH sensitive (pK_a = 7.2) and therefore less than ideal for spectrophotometric assays of xylanases, which exhibit a wide range of pH optima (from pH 2 to 9).

The EnzChek Ultra Xylanase Assay Kit (E33650) provides a quick and convenient mix-and-read assay for measuring xylanase activity or for screening xylanase inhibitors in a high-throughput format using a fluorescence microplate reader or a standard fluorometer (excitation/emission maxima ~358/455 nm). This kit can be used for continuous detection of xylanase activity and offers broad dynamic and pH ranges (1.5 to 200 mU/mL and pH 4–10, respectively; Figure 10.2.14), high sensitivity (as low as 1.5 mU/mL) and excellent temperature tolerance. It has been tested with xylanases from Trichoderma viride, T. longibrachiatum, Thermomyces lanuginosus, Aspergillus niger and other bacterial and fungal species.

The EnzChek Ultra Xylanase Assay Kit contains:

• Xylanase substrate
• Concentrated reaction buffer
• Fluorescence standard
• Detailed protocols (EnzChek Ultra Xylanase Assay Kit)

Each kit provides sufficient reagents for 500 assays using 100 µL assay volumes in a 96-well microplate assay format.

Figure 10.2.14 Detection of xylanase activity using the EnzChek Ultra Xylanase Assay Kit (E33650). The EnzChek Ultra xylanase assay was performed at pH 4.6 under standard conditions (30-minute incubation) as described in the experimental protocol, using Aspergillus niger xylanase and the EnzChek xylanase substrate. The fluorescence signal for 500 mU/mL of xylanase is equivalent to 49 µM fluorescence standard, indicating 94% turnover of the xylanase substrate (initially 52 µM).

Lysozyme (muramidase) hydrolyzes β-1-4-glycosidic linkages between N-acetylmuramic acid and N-acetyl-D-glucosamine residues present in the mucopolysaccharide cell wall of a variety of microorganisms. Lysozyme is present in human serum, urine, tears, seminal fluid and milk. Serum and urine lysozyme levels may be elevated in acute myelomonocytic leukemia (FAB-M4), chronic myelomonocytic leukemia (CMML) and chronic myelocytic leukemia (CML). Increased serum lysozyme activity is also present in tuberculosis, sarcoidosis, megaloblastic anemias, acute bacterial infections, ulcerative colitis and Crohn disease. Elevated levels of urine and serum lysozyme occur during severe renal insufficiency, renal transplant rejection, urinary tract infections, glomerulonephritis and nephrosis.

We have developed a simple and sensitive fluorescence-based EnzChek assay that can continuously measure lysozyme activity in solution. This assay is based on a DQ lysozyme substrate comprising M. lysodeikticus cell walls that are labeled with fluorescein to such a degree that fluorescence is quenched. Lysozyme action relieves the quenching, yielding a dramatic increase in fluorescence that is proportional to lysozyme activity. This increase in fluorescence can be measured with any spectrofluorometer, mini-fluorometer or fluorescence microplate reader that can detect fluorescein (excitation/emission maxima ~490/525 nm). Our fluorescence-based EnzChek Lysozyme Assay Kit (E22013) permits the detection of as little as 30 U/mL of lysozyme (Figure 10.2.15). One unit of lysozyme is the quantity of enzyme that produces a decrease in turbidity of 0.001 optical density units per minute at 450 nm measured at pH 7.0 (25°C) using a 0.3 mg/mL suspension of Micrococcus lysodeikticus cells as substrate.

The EnzChek Lysozyme Assay Kit (E22013) contains:

• DQ lysozyme substrate—a heavily labeled, highly quenched fluorescein conjugate of Micrococcus lysodeikticus
• Reaction buffer
• Lysozyme from chicken egg white, for use as a positive control
• Detailed protocols (EnzChek Lysozyme Assay Kit)

Each kit contains sufficient materials for approximately 400 assays of 100 µL in a fluorescence microplate reader.

Figure 10.2.15 Detection of lysozyme activity using the EnzChek Lysozyme Assay Kit (E22013). Increasing amounts of lysozyme were incubated with the DQ lysozyme substrate for 60 minutes at 37°C. The fluorescence was measured in a fluorescence microplate reader using excitation/emission wavelengths of ~485/530 nm. Background fluorescence, determined for a no-enzyme control, was subtracted from each value.

The widely used β-galactosidase substrate—5-bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-Gal, B1690)—yields a dark blue precipitate at the site of enzymatic activity. X-Gal is useful for numerous histochemical and molecular biology applications, including detection of lacZ activity in cells and tissues. In contrast to β-glucuronidase as a gene marker, β-galactosidase can be fixed in cells and tissues with glutaraldehyde without loss of activity and detected with high resolution with X-Gal.

The chromogenic substrate 5-bromo-4-chloro-3-indolyl β-D-glucuronic acid (X-GlcU, B1691) forms a dark blue precipitate. X-GlcU is routinely used to detect GUS expression in transformed plant cells and tissues. X-GlcU can also be used to detect E. coli contamination in food and water.

Phenylethyl β-D-Thiogalactopyranoside (PETG)

Phenylethyl β-D-thiogalactopyranoside (PETG, P1692) is a cell-permeant inhibitor of β-galactosidase activity. We provide PETG in our FluoReporter, DetectaGene Green, ImaGene Green and ImaGene Red lacZ Gene Expression Kits for stopping the enzymatic reaction.

Streptavidin Conjugate of β-Galactosidase

We also offer the streptavidin conjugate of β-galactosidase (S931), a reagent used in a variety of ELISAs. β-D-Galactosidase streptavidin reportedly provided enhanced sensitivity over that obtained with the avidin conjugate of horseradish peroxidase in the detection of a variety of mammalian interleukins and their receptors by ELISA. This reagent has also been used in fluorometric-reverse (IgE-capture) and fluorescence-sandwich ELISAs.

Rabbit Anti–β-Galactosidase Antibody

We offer a polyclonal antibody to the widely used reporter gene product, β-galactosidase. Our rabbit anti–β-galactosidase antibody (A11132) is raised against E. coli–derived β-galactosidase and demonstrates high selectivity for the enzyme. Whether it is being used as a reporter gene or to generate fusion proteins, anti–β-galactosidase provides an easy tool for detecting the enzyme. The antibody is suited to a variety of techniques, including immunoblotting, ELISA, immunoprecipitation and most immunological methods. β-Galactosidase has been used as a tag for quantitative detection of molecules expressed on a cell surface in unfixed, live cells, using anti–β-galactosidase and a β-galactosidase substrate for detection. This novel "cell-ELISA" technique is reported to be applicable to adherent cells and nonadherent cells and to have utility for large-scale screening for expression of cell-surface molecules and of hybridomas for production of antibodies to cell-surface epitopes.

Rabbit Anti–β-Glucuronidase Antibody

In combination with a fluorophore- or enzyme-labeled anti–rabbit IgG secondary antibody (Secondary Immunoreagents—Section 7.2, Summary of Molecular Probes secondary antibody conjugates—Table 7.1), our anti–β-glucuronidase antibody (A5790) can be used to detect the GUS enzyme in transformed plant tissue and in transfected animal cells using western blotting or immunohistochemical techniques. Furthermore, this antibody, which is raised in rabbits against E. coli–type X-A β-glucuronidase, can be immobilized in microplate wells in order to capture the GUS enzyme from cell lysates. The enzymatic activity can subsequently be determined using any of our fluorogenic or chromogenic β-glucuronidase substrates.

Related Products for Carbohydrate Research

We offer an extensive assortment of reagents for detection and analysis of carbohydrates that are described in other sections of this Handbook. These products include:

• Hydrazine, hydroxylamine and aromatic amine reagents for derivatization and analysis of reducing sugars and periodate-oxidized carbohydrates by electrophoretic methods (Reagents for Modifying Aldehydes and Ketones—Section 3.3, Molecular Probes hydrazine, hydroxylamine and amine derivatives—Table 3.1)
• Lectins and fluorescent lectin conjugates (Lectins and Other Carbohydrate-Binding Proteins—Section 7.7, Molecular Probes lectin conjugates—Table 7.10)
• Pro-Q Glycoprotein Blot and Gel Stain Kits (Detecting Protein Modifications—Section 9.4)
• Pro-Q Emerald 300 Lipopolysaccharide Gel Stain Kit (P20495, Reagents for Modifying Aldehydes and Ketones—Section 3.3)
• Fluorescent lipopolysaccharides (Probes for Following Receptor Binding and Phagocytosis—Section 16.1, Fluorescent lipopolysaccharide conjugates—Table 16.1)
• Fluorescent glycolipids, including BODIPY FL C₅-ganglioside G_M1 (B13950, Sphingolipids, Steroids, Lipopolysaccharides and Related Probes—Section 13.3)
• Fluorescent and biotinylated dextrans (Fluorescent and Biotinylated Dextrans—Section 14.5, Molecular Probes dextran conjugates—Table 14.4)
• NBD-glucosamine derivatives for glucose-transport studies (N13195, N23106; Viability and Cytotoxicity Assay Reagents—Section 15.2)
• Fluorescein insulin (I13269, Probes for Following Receptor Binding and Phagocytosis—Section 16.1)