Thiol-Reactive Probes Excited with Ultraviolet Light—Section 2.3

Page Contents

• Coumarin Derivatives
• Pacific Orange Maleimide
• Pyrene Derivatives
• Naphthalene Derivatives
• Bimanes for Thiol Derivatization
• Polar Reagents for Determining Thiol Accessibility
• 1,10-Phenanthroline Iodoacetamide for Preparing Metal-Binding Conjugates
• Data Table
• Ordering Information

The thiol-reactive dyes described in this section have their longest-wavelength absorption peaks at less than 410 nm (Thiol-reactive dyes excited with ultraviolet light—Table 2.2). Typically, these dyes exhibit blue fluorescence and have much weaker absorption than the dyes described in Thiol-Reactive Probes Excited with Visible Light—Section 2.2, with extinction coefficients often below 20,000 cm^-1M^-1.

The strong environmental dependence of the emission spectra and quantum yields of several of the dyes—especially the coumarin, benzoxadiazole (NBD probes and ABD-F, Thiol-Reactive Probes Excited with Visible Light—Section 2.2), aminonaphthalene (e.g., dansyl) and Dapoxyl fluorophores—makes some of these thiol-reactive probes useful for investigating protein structure and assembly, following protein transport through membranes and studying ligand binding to receptors.

Alexa Fluor 350 Maleimide

Alexa Fluor 350 C₅-maleimide (A30505), a thiol-reactive, sulfonated coumarin derivative, produces protein conjugates that are optimally excited at 346 nm and have bright blue fluorescence emission at wavelengths slightly shorter than AMCA or AMCA-X conjugates (emission maximum ~442 nm versus 448 nm). The shorter emission maximum of Alexa Fluor 350 conjugates reduces their spectral overlap with the emission of fluorescein and Oregon Green 488 dyes. Like our other Alexa Fluor dyes, Alexa Fluor 350 C₅-maleimide offers unrivaled brightness and pH-independent fluorescence, as well as water solubility and a low degree of quenching upon conjugation (The Alexa Fluor Dye Series—Note 1.1).

Pacific Blue Maleimide

The Pacific Blue dye, which is based on the 6,8-difluoro-7-hydroxycoumarin fluorophore, exhibits bright blue fluorescence, with excitation/emission maxima of ~410/455 nm. Significantly, the pK_a value of this 6,8-difluoro-7-hydroxycoumarin derivative is 2–3 log units lower than that of the corresponding 7-hydroxycoumarin. Thus, the thiol-reactive Pacific Blue C₅-maleimide (P30506) yields conjugates that are strongly fluorescent, even at neutral pH. In addition, Pacific Blue conjugates are efficiently excited by the 405 nm spectral line of the violet diode laser developed for fluorescence microscopy and flow cytometry.

Other Coumarin Maleimides and Iodoacetamides

We offer several blue-fluorescent thiol-reactive dialkylcoumarins (Thiol-reactive dyes excited with ultraviolet light—Table 2.2), including 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM, D346) and N-(7-dimethylamino-4-methylcoumarin-3-yl)maleimide (DACM, D10251) and the corresponding iodoacetamides DCIA and DACIA. The dialkylcoumarin fluorophore is an excellent fluorescence resonance energy acceptor from tryptophan and a good donor to fluorescein, NBD, Alexa Fluor 488 dye, Green Fluorescent Protein (GFP) and the nonfluorescent QSY 7, QSY 9 and QSY 35 quenchers, making these thiol-reactive coumarins especially valuable for studying protein structure and for detecting protein–membrane interactions. Fluorescence emission of the dialkylcoumarin conjugates is moderately sensitive to environment.

Unlike MDCC (described below), which is intrinsically fluorescent, the maleimides CPM and DACM are essentially nonfluorescent until they react with thiols, permitting thiol quantitation without a separation step. The environment-sensitive fluorescence of CPM is also a useful indicator of protein folding in thermal-shift assays of ligand- and mutagenesis-dependent protein stability (Monitoring Protein-Folding Processes with Environment-Sensitive Dyes—Note 9.1).

We also offer 7-diethylamino-3-((((2-maleimidyl)ethyl)amino)carbonyl)coumarin (MDCC, D10253). When conjugated to a mutant phosphate-binding protein, MDCC has proven useful for direct, real-time measurement of inorganic phosphate release during enzymatic reactions. Similarly, environment-sensitive fluorescence of MDCC site-specifically attached to Escherichia coli ParM nucleotide-binding protein provides the basis for a fluorogenic ADP biosensor.

The thiol-reactive Pacific Orange C₅-maleimide yields conjugates with excitation/emission maxima of ~400/551 nm, making it ideal for use with violet diode laser–equipped flow cytometers and fluorescence microscopes. Moreover, Pacific Blue conjugates (described above with the other coumarins) and Pacific Orange conjugates can be simultaneously excited at 405 nm and emit at 455 and 551 nm, respectively, facilitating two-color analysis.

Pyrene Maleimide

Not only is N-(1-pyrene)maleimide (pyrene maleimide, P28) essentially nonfluorescent until it has reacted with thiols, but once excited, pyrene–thiol conjugates can interact to form excited-state dimers (excimers) that emit at longer wavelengths than the excited monomeric fluorophore. Pyrene maleimide conjugates often have very long fluorescence lifetimes (>100 nanoseconds), giving proximal pyrene rings within 6–10 Å of each other ample time to form the spectrally altered excimer (Figure 2.3.1). The excimer-forming capacity of pyrene maleimide can be exploited for detection of conformational changes and subunit assembly of proteins and for analysis of protease activity.

Figure 2.3.1 Excimer formation by pyrene in ethanol. Spectra are normalized to the 371.5 nm peak of the monomer. All spectra are essentially identical below 400 nm after normalization. Spectra are as follows: 1) 2 mM pyrene, purged with argon to remove oxygen; 2) 2 mM pyrene, air-equilibrated; 3) 0.5 mM pyrene (argon-purged); and 4) 2 µM pyrene (argon-purged). The monomer-to-excimer ratio (371.5 nm/470 nm) is dependent on both pyrene concentration and the excited-state lifetime, which is variable because of quenching by oxygen.

Pyrene Iodoacetamides

Fluorescence of the actin monomer labeled with pyrene iodoacetamide (P29) has been demonstrated to change upon polymerization, making this probe a widely utilized tool for following the kinetics of actin polymerization. Conjugates of N-(1-pyrenemethyl)iodoacetamide have the longest excited-state fluorescence lifetimes (>100 nanoseconds) of all reported thiol-reactive probes. Excimer formation can also be a useful indicator of protein folding.

Acrylodan and Badan

As compared with iodoacetamides or maleimides, acrylodan (A433) and 6-bromoacetyl-2-dimethylaminonaphthalene (badan, B6057) generally react with thiols more slowly but they form very strong thioether bonds that are expected to remain stable under conditions required for complete amino acid analysis. The fluorescence emission peak and intensity of these adducts (Figure 2.3.2) are particularly sensitive to conformational changes or ligand binding, making these dyes some of the most useful thiol-reactive probes for protein structure studies. For example, the acrylodan conjugate of an intestinal fatty acid–binding protein, ADIFAB (Probes for Lipid Metabolism and Signaling—Section 17.4), is a sensor for free fatty acids (Figure 2.3.3).

Figure 2.3.2 Fluorescence emission spectra of the 2-mercaptoethanol adduct of badan (B6057) in: 1) toluene, 2) chloroform, 3) acetonitrile, 4) ethanol, 5) methanol and 6) water. Each solution contains the same concentration of the adduct. Excitation of all samples is at 380 nm.

Figure 2.3.3 Ribbon representation of the ADIFAB free fatty acid indicator. In the left-hand image, the fatty acid binding site of intestinal fatty acid–binding protein (yellow) is occupied by a covalently attached acrylodan fluorophore (blue). In the right-hand image, a fatty acid molecule (gray) binds to the protein, displacing the fluorophore (green) and producing a shift of its fluorescence emission spectrum. Image contributed by Alan Kleinfeld, FFA Sciences LLC, San Diego.

IAANS and MIANS

To develop appreciable fluorescence, both the reactive anilinonaphthalenesulfonate iodoacetamide (IAANS) and maleimide (MIANS, also called Mal-ANS) must be reacted with thiols that are located in hydrophobic sites. Often, however, buried unsolvated thiol residues are exceptionally reactive, allowing these sites to be selectively modified by these reagents. The environment-sensitive fluorescence properties of the protein conjugates of MIANS and IAANS are similar to those of the structurally related probes 1,8-ANS and 2,6-TNS (A47, Other Nonpolar and Amphiphilic Probes—Section 13.5). The fluorescence intensity, and to a lesser extent, the emission wavelengths of the conjugates, tend to be very sensitive to substrate binding and folding and unfolding of the protein, as well as the association of the labeled protein with other proteins, membranes or nucleic acids.

IAEDANS

The fluorescence of IAEDANS (I14) is quite dependent upon environment, although less so than that of IAANS and MIANS conjugates. Its conjugates frequently respond to ligand binding by undergoing spectral shifts and changes in fluorescence intensity that are determined by the degree of aqueous solvation. Advantages of this reagent include high water solubility above pH 4 and a relatively long fluorescence lifetime (sometimes >20 nanoseconds, although commonly 10–15 nanoseconds), making the conjugates useful for fluorescence polarization assays (Fluorescence Polarization (FP)—Note 1.4). The emission spectrum of IAEDANS overlaps well with the absorption of fluorescein, Alexa Fluor 488 and Oregon Green 488 dyes, as well as that of Green Fluorescent Protein (GFP). IAEDANS is an excellent reagent for fluorescence resonance energy transfer (FRET) measurements (Fluorescence Resonance Energy Transfer (FRET)—Note 1.2). IAEDANS usually reacts with thiols; however, it has been reported to react with a lysine residue in tropomyosin.

Monobromobimane and Monochlorobimane

Monobromobimane (M1378, M20381), which is essentially nonfluorescent until conjugated, readily reacts with low molecular weight thiols, including glutathione. This reagent, originally described by Kosower and colleagues, is also useful for detecting the distribution of protein thiols in cells before and after chemical reduction of disulfides. Monobromobimane is reportedly susceptible to inactivation by the disulfide reducing agent TCEP (T2556, Introduction to Thiol Modification and Detection—Section 2.1). Both monobromobimane and the more thiol-selective monochlorobimane (M1381MP) have been extensively used for detecting glutathione in live cells (Probes for Cell Adhesion, Chemotaxis, Multidrug Resistance and Glutathione—Section 15.6).

Dibromobimane

Dibromobimane is an interesting homobifunctional crosslinking reagent for proteins because it is unlikely to fluoresce until both of its alkylating groups have reacted. It has been used to crosslink thiols in myosin,Escherichia coli lactose permease and P-glycoprotein. Despite its short length, dibromobimane is also an effective intermolecular crosslinker in some cases.

Bimane Iodoacetamide and Maleimide

Bimane iodoacetamide and bimane C₃-maleimide are blue-fluorescent thiol-reactive fluorophores with excitation/emission maxima of ~375/456 nm. The small size of the bimane fluorophore reduces the likelihood that the label will interfere with the function of the biomolecule, an important advantage for site-selective probes.

Like IAEDANS (I14), the iodoacetamide and maleimide derivatives of stilbene (A484, A485) have high water solubility and are readily conjugated to thiols. Their combination of high polarity and membrane impermeability makes these polysulfonated dyes useful for determining whether thiol-containing proteins and polypeptide chains are exposed at the extracellular or cytoplasmic membrane surface.

The sulfonated stilbene iodoacetamide (A484) was used to label single-cysteine mutants of staphylococcal α-hemolysin in order to determine structural changes that occur during oligomerization and pore formation and of the lipid-binding region of E. coli pyruvate oxidase in order to detect conformational changes upon substrate binding. Similarly, single-cysteine mutants of Escherichia coli Na⁺-glutamate transporter GHS have been probed with the sulfonated stilbene maleimide (A485) to systematically study the topology of this membrane protein.

Conjugation of N-(1,10-phenanthrolin-5-yl)iodoacetamide to thiol-containing ligands confers the metal-binding properties of this important complexing agent on the ligand. For example, the covalent copper–phenanthroline complex of oligonucleotides or nucleic acid–binding molecules in combination with hydrogen peroxide acts as a chemical nuclease to selectively cleave DNA or RNA.

For a detailed explanation of column headings, see Definitions of Data Table Contents