Protein Conjugates - Section 14.7

Unlike the polydisperse dextrans (Fluorescent and Biotinylated Dextrans - Section 14.5), fluorescent protein tracers have molecular weights that are reasonably well defined (bovine serum albumin (BSA) ~66,000 daltons; ovalbumin ~45,000; codfish parvalbumin 12,328 daltons, casein ~23,600 daltons; monomeric subunit B of cholera toxin ~12,000 daltons; horseradish peroxidase (HRP) ~40,000 daltons; soybean trypsin inhibitor ~21,500 daltons; Phaseolus vulgaris leucoagglutinin (PHA-L) ~126,000 daltons). Some of their applications are similar to those of dextran tracers, although protein conjugates may be more susceptible to proteolysis. However, through creative modifications of the proteins, researchers have been able to use these conjugates to target receptors, detect protease activity and measure intracellular pH. Some applications of fluorescent protein conjugates as tracers include:

• Quantitative studies of electroporation
• Reconstitution of functional nuclear pores
• Macromolecule trafficking to endocytic compartments
• Measurement of plasma volume in rats

Tyramide signal amplification (TSA) technology (see Tyramide Signal Amplification (TSA) Technology - Section 6.2 and below) combined with HRP and its conjugates promises to become a major tool for retrograde and anterograde labeling of neurons and other cells ().

Fluorescent BSA, Ovalbumin and Parvalbumin

Molecular Probes supplies bovine serum albumin (BSA) fluorescently labeled with our fluorescein, tetramethylrhodamine, Texas Red and BODIPY FL dyes (A23015, A23016, A23017, A2750). We have also prepared conjugates of BSA with our outstanding Alexa Fluor 488, Alexa Fluor 555, Alexa Fluor 594, Alexa Fluor 647 and Alexa Fluor 680 dyes (A13100, A34786, A13101, A34785, A34786), which provide particularly bright, photostable and pH-insensitive fluorescence.

In addition to the BSA conjugates, Molecular Probes prepares the fluorescein and Texas Red conjugates of ovalbumin (O23020, O23021), as well as the brightly fluorescent Alexa Fluor 488, Alexa Fluor 594 and Alexa Fluor 647 conjugates (O34781, O34783, O34784). Ovalbumin is a protein with a relatively low molecular weight (~45,000 daltons) that has been used to follow endocytic internalization and to estimate microvasculature transport-pathway sizes. We also offer the Alexa Fluor 488 conjugate of codfish parvalbumin (P23012), which is the smallest (molecular weight 12,328 daltons ) fluorescent protein tracer in this section.

Dinitrophenylated Bovine Serum Albumin

We offer BSA that has been conjugated to multiple dinitrophenyl hapten molecules (DNP-BSA, A23018), a reagent that is commonly used to study Fc receptor–mediated immune function and the IgE- and IgG-mediated responses to crosslinking of DNP-specific antibodies. Our dinitrophenylated BSA typically has ~25 dinitrophenyl substituents per molecule of BSA. For these studies, Molecular Probes offers rabbit anti-DNP antibody prepared against DNP-conjugated keyhole limpet hemocyanin (anti–DNP-KLH antibody, A6430; Anti-Dye and Anti-Hapten Antibodies—Section 7.4), as well as the Alexa Fluor 488 dye, fluorescein and biotin-XX conjugates of this important antibody (A11097, A6423, A6435; Anti-Dye and Anti-Hapten Antibodies—Section 7.4).

DQ BSA, DQ Ovalbumin, DQ Collagen and Anti–BSA Antibodies

Our Patented DQ substrates, including the DQ Green BSA and DQ Red BSA (D12050, D12051) and DQ ovalbumin (D12053) are based on the strong fluorescence quenching effect observed when proteins are heavily conjugated with BODIPY dyes or, in the case of DQ collagen conjugates (D12052, D12060), with multiple fluorescein dyes. Upon hydrolysis of the proteins to single dye–labeled peptides by proteases, this quenching is relieved (Figure 10.56). This technology provides an easy continuous assay for numerous proteases (Detecting Peptidases and Proteases—Section 10.4). When complexed to rabbit anti–BSA IgG (A11133), the DQ BSA products are phagocytosed into live cells via the Fc receptor (Probes for Following Receptor Binding and Phagocytosis—Section 16.1, Figure 16.5), where they are broken down into fluorescent peptides (Figure 10.56). DQ Green BSA, which gives bright green fluorescence upon proteolysis, and DQ Red BSA, which yields red-fluorescent hydrolysis products, are packaged in sets of five vials, each containing 1 mg of the substrate. Our Fc OxyBURST Green assay reagent (F2902, Probes for Following Receptor Binding and Phagocytosis—Section 16.1), which is based on a similar concept, comprises BSA that has been conjugated to a dihydrofluorescein derivative and then complexed with anti-BSA IgG (Figure 16.2). Upon binding to the Fc receptor, the immune complex is internalized and oxidized to a fluorescent product within the phagovacuole.

Figure 16.2 Fc OxyBURST reagent (F2902) for fluorescent detection of the Fc receptor–mediated phagocytosis pathway. Dichlorodihydrofluorescein (H₂DCF) is covalently attached to bovine serum albumin (BSA), then complexed with a rabbit polyclonal anti-BSA antibody (A11133). Upon binding to an Fc receptor, the nonfluorescent immune complex is internalized and subsequently oxidized to the fluorescent DCF.

We have found our highly quenched DQ ovalbumin (D12053) to be particularly useful for antigen processing and presentation studies. Ovalbumin conjugates are internalized via the mannose receptor–mediated endocytosis pathway and are recognized by antigen-presenting cells. BSA that was heavily labeled with fluorescein (FITC) has been used for these studies. However, fluorescence of the intracellular hydrolysis products of FITC BSA is significantly decreased by the acidity of the phagovacuole. In contrast, the bright fluorescence of the BODIPY FL fluorophore in DQ ovalbumin is completely independent of the pH. The fluorescence generated after DQ collagen (D12052) is hydrolyzed by cellular collagenase has been used to visualize the migratory pathway followed by tumor cells during invasion of a gelatin matrix and to image proteolysis by living breast cancer cells.

Figure 10.56 Principle of enzyme detection via the disruption of intramolecular self-quenching. Enzyme-catalyzed hydrolysis of the heavily labeled and almost totally quenched substrates provided in our EnzChek Protease Assay Kits (E6638, E6639, E33757), EnzChek Ultra Amylase Assay Kit (E33651), EnzChek Gelatinase/Collagenase Assay Kit (E12055), EnzChek Elastase Kit (E12056), EnzChek Lysozyme Assay Kit (E22013) — as well as the stand-alone quenched substrates DQ BSA (D12050, D12051), DQ collagen (D12052, D12060), DQ ovalbumin (D12053) and DQ gelatin (D12054) — relieves the intramolecular self-quenching, yielding brightly fluorescent reaction products.

Alexa Fluor 488 Parvalbumin

Parvalbumin, a stable and protease-resistant protein with a molecular weight of 12,328 daltons, is found in the skeletal muscle of vertebrates and the endocrine glands of mammals. Our Alexa Fluor 488 conjugate of codfish parvalbumin (P23012) has bright green, photostable fluorescence, making this conjugate a potentially useful aldehyde-fixable tracer for microinjection into cells, including neurons.

BODIPY FL casein and BODIPY TR-X casein, which are components of our EnzChek Protease Assay Kits (E6638, E6639, E33757), are substrates for metallo-, serine, acid and sulfhydryl proteases, including cathepsin, chymotrypsin, elastase, papain, pepsin, thermolysin and trypsin (Detecting Peptidases and Proteases - Section 10.4). These casein-based substrates are heavily labeled and therefore highly quenched conjugates; they typically exhibit <3% of the fluorescence of the corresponding free dyes. Protease-catalyzed hydrolysis relieves this quenching, yielding brightly fluorescent peptides (Figure 10.56).

The EnzChek Protease Assay Kit (E6638, E6639) includes:

• BODIPY FL casein (in Kit E6638) or BODIPY TR-X casein (in Kit E6639)
• Concentrated digestion buffer
• Detailed protocols (EnzChek(R) Protease Assay Kit)

Each kit provides sufficient reagents for ~100 assays using 2 mL assay volumes and standard fluorescence cuvettes or ~1000 assays using 200 µL assay volumes and 96-well microplates.

The EnzChek Ultra Protease Assay Kit E33757 includes:

• BODIPY FL casein
• Concentrated digestion buffer
• BODIPY FL propionic acid, for use as a fluorescence standard
• Detailed protocols (EnzChek(R) Ultra Protease Assay Kit)

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

The EnzChek Protease Assay Kits, as well as the DQ BSA and DQ ovalbumin substrates mentioned above, have significant potential as:

• Detectors of protease contamination in culture media
• Fluorogenic substrates for circulating or secreted proteases in extracellular fluids
• Nontoxic, pH-insensitive markers for phagocytic cells, which will ingest and eventually cleave the quenched casein substrates to yield fluorescent BODIPY FL– or BODIPY TR-X–labeled peptides
• Microinjectable tracers to detect enhanced protease activity associated with cell activation and fusion
• Nontoxic markers for assessing various cell-loading and cell-transfection techniques, including electroporation, high-velocity microprojectiles, scrape loading and related methods (Techniques for loading molecules into the cytoplasm - Table 14.1)

The peptide hydrolysis products of BODIPY FL casein exhibit green fluorescence that is optimally excited by the argon-ion laser at 488 nm, permitting flow sorting of the cells. The red-fluorescent BODIPY TR-X–labeled peptides, with excitation and emission spectra similar to those of the Texas Red fluorophore, should be useful for multilabeling experiments or measurements in the presence of green autofluorescence. Following intracellular hydrolysis, some of the lower molecular weight fluorescent peptides may diffuse into other organelles or pass thorough gap junctions.

Cholera toxin comprises two subunits, A and B, arranged in an AB₅ conformation. Subunit A is an ADP-ribosyltransferase, which disrupts the proper signaling of G proteins and eventually leads to dehydration of the cell. The 12,000-dalton nontoxic subunit B ("choleragenoid"), which is assembled into a 60,000-dalton pentamer above pH 2, allows the protein complex to bind to cellular surfaces via the pentasaccharide chain of ganglioside G_M1.

Cholera toxin subunit B and its conjugates are emerging as superior tracers for retrograde labeling of neurons. Researchers have used cholera toxin subunit B in a variety of applications, including tracing of rat forebrain afferents, projections of the parabrachial region and neurons of the urinary bladder wall. Cholera toxin subunit B conjugates bind to the pentasaccharide chain of ganglioside G_M1 on neuronal cell surfaces and are actively taken up and transported; alternatively, they can be injected by iontophoresis. Unlike the carbocyanine-based neuronal tracers such as DiI (D282, D3911, V22885; Tracers for Membrane Labeling - Section 14.4), cholera toxin subunit B conjugates can be used on tissue sections that will be fixed and frozen.

Fluorescent cholera toxins are also markers of lipid rafts — regions of cell membranes high in ganglioside G_M1 that are thought to be important in cell signaling (, ). The Vybrant Lipid Raft Labeling Kits (V34403, V34404, V34405; Probes for Lipid Metabolism and Signaling - Section 17.4) provide the key reagents, including fluorescent cholera toxin subunit B conjugates, for fluorescently labeling lipid rafts in vivo with our bright and extremely photostable Alexa Fluor dyes (, ). All of Molecular Probes' cholera toxin subunit B conjugates (Cholera Toxin Subunit B (CT-B) Conjugates) are prepared from recombinant cholera toxin subunit B, which is completely free of the toxic subunit A, thus eliminating any concern for toxicity or ADP-ribosylating activity. Our Alexa Fluor 488 (C22841, C34775), Alexa Fluor 555 (C22843, C34776), Alexa Fluor 594 (C22842, C34777) and Alexa Fluor 647 (C34778) conjugates of cholera toxin subunit B combine this versatile tracer with the superior brightness of our Alexa Fluor dyes to provide sensitive and selective receptor labeling and neuronal tracing. We also offer biotin-XX (C34779) and horseradish peroxidase (C34780) conjugates of cholera toxin subunit B, which, in conjunction with sensitive secondary detection methods such as diaminobenzidine (DAB) and tyramide signal amplification (TSA), can be used as retrograde tracers in neurons.

Phycobiliproteins make effective polar tracers because they are intensely fluorescent, are very soluble in water and have a low tendency to bind nonspecifically to cells. These intrinsically fluorescent biomolecules are stable proteins that are monodisperse and substantially larger than albumins, with molecular weights of 240,000 daltons (B- and R-phycoerythrin, P800, P801; Phycobiliproteins) and 104,000 daltons (allophycocyanin, A803, or crosslinked allophycocyanin, A819; Phycobiliproteins). Phycobiliproteins have been employed as tracers to:

• Follow nucleocytoplasmic transport of proteins
• Measure permeability of reconstituted nuclear pores
• Assess nuclear exclusion of nonnuclear proteins and soluble cytoplasmic factors involved in protein import
• Observe transport through single transporters in cells

Phycoerythrin has also been conjugated to nuclear-localization peptide sequences to follow nuclear import processes. B-phycoerythrin can pass through pores in erythrocyte membranes created by streptolysin O. Single molecules of R-phycoerythrin have been imaged and tracked within mammalian cells. Unlike our DQ and EnzChek protease substrates (Detecting Peptidases and Proteases - Section 10.4), proteolysis of phycobiliproteins results in the loss of most of their intrinsic fluorescence.