The most common schemes for forming a well-defined heteroconjugate require the indirect coupling of an amine group on one biomolecule to a thiol group on a second biomolecule, usually by a two- or three-step reaction sequence. The high reactivity of thiols (Thiol-Reactive Probes—Chapter 2) and—with the exception of a few proteins such as β-galactosidase—their relative rarity in most biomolecules make thiol groups ideal targets for controlled chemical crosslinking. If neither molecule contains a thiol group, then one or more can be introduced using one of several thiolation methods. The thiol-containing biomolecule is then reacted with an amine-containing biomolecule using a heterobifunctional crosslinking reagent such as one of those described in Amine–Thiol Crosslinking, below.
Introducing Thiol Groups into Biomolecules
Several methods are available for introducing thiols into biomolecules, including the reduction of intrinsic disulfides, as well as the conversion of amine or carboxylic acid groups to thiol groups:
- Disulfide crosslinks of cystines in proteins can be reduced to cysteine residues by dithiothreitol (DTT, D1532) or tris-(2-carboxyethyl)phosphine (TCEP, T2556; ). However, reduction may result in loss of protein activity or specificity. Excess DTT must be carefully removed under conditions that prevent reformation of the disulfide, whereas excess TCEP usually does not need to be removed before carrying out the crosslinking reaction. TCEP is also more stable at higher pH values and at higher temperatures than is the air-sensitive DTT reagent.
- Amines can be indirectly thiolated by reaction with succinimidyl acetylthioacetate (SATA, S1553), followed by removal of the acetyl group with 50 mM hydroxylamine or hydrazine at near-neutral pH (Figure 5.2.1). This reagent is most useful when disulfides are essential for activity, as is the case for some peptide toxins.
- Amines can be indirectly thiolated by reaction with succinimidyl 3-(2-pyridyldithio)propionate (SPDP, S1531), followed by reduction of the 3-(2-pyridyldithio)propionyl conjugate with DTT or TCEP (Figure 5.2.2). Reduction releases the 2-pyridinethione chromophore, which can be used to determine the degree of thiolation.
- Thiols can be incorporated at carboxylic acid groups by an EDAC-mediated reaction with cystamine, followed by reduction of the disulfide with DTT or TCEP; see Amine–Carboxylic Acid Crosslinking below.
- Tryptophan residues in thiol-free proteins can be oxidized to mercaptotryptophan residues, which can then be modified by iodoacetamides or maleimides.
Our preferred reagent combination for protein thiolation is SPDP/DTT or SPDP/TCEP. We use SPDP to prepare a reactive R-phycoerythrin derivative (P806, Phycobiliproteins—Section 6.4), providing researchers with the optimal number of pyridyldisulfide groups for crosslinking the phycobiliprotein to thiolated antibodies, enzymes and other biomolecules through disulfide linkages. More commonly, the pyridyldisulfide groups are first reduced to thiols, which are then reacted with maleimide- or iodoacetamide-derivatized proteins (Figure 5.2.2). SPDP can also be used to thiolate oligonucleotides and—like all of the thiolation reagents in this section—to introduce the highly reactive thiol group into peptides, onto cell surfaces or onto affinity matrices for subsequent reaction with fluorescent, enzyme-coupled or other thiol-reactive reagents (Thiol-Reactive Probes—Chapter 2). In addition, because the 3-(2-pyridyldithio)propionyl conjugate releases the 2-pyridinethione chromophore upon reduction, SPDP is useful for quantitating the number of reactive amines in an affinity matrix.
Figure 5.2.1 Schematic illustration of the heterobifunctional crosslinker succinimidyl acetylthioacetate (SATA, S1553): A) attachment to an aminosilane-modified surface, B) deprotection with base and C) reaction with a thiol-reactive biomolecule.
Figure 5.2.2 SPDP derivatization reactions. SPDP (S1531) reacts with an amine-containing biomolecule at pH 7 to 9, yielding a pyridyldithiopropionyl mixed disulfide. The mixed disulfide can then be reacted with a reducing agent such as DTT (D1532) or TCEP (T2556) to yield a 3-mercaptopropionyl conjugate or with a thiol-containing biomolecule to form a disulfide-linked tandem conjugate. Either reaction can be quantitated by measuring the amount of 2-pyridinethione chromophore released during the reaction.
Measuring Thiolation of Biomolecules
To ensure success in forming heterocrosslinks, it is important to know that a molecule has the proper degree of thiolation. We generally find that two to three thiol residues per protein are optimal. Following removal of excess reagents, the degree of thiolation in proteins or other molecules thiolated with SPDP can be directly determined by measuring release of the 2-pyridinethione chromophore (EC343 nm ~8000 cm-1M-1).
Alternatively, the degree of thiolation and presence of residual thiols in a solution can be assessed using 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB, Ellman's reagent; D8451), which stoichiometrically yields the 5-mercapto-2-nitrobenzoic acid chromophore (EC410 nm ~13,600 cm-1M-1) upon reaction with a thiol group. DTNB can also be used to quantitate residual phosphines in aqueous solutions, including TCEP; in this case, two molecules of 5-mercapto-2-nitrobenzoic acid are formed per reaction with one molecule of a phosphine.
Measure-iT Thiol Assay Kit
The Measure-iT Thiol Assay Kit (M30550) provides easy and accurate quantitation of thiol. The kit supplies concentrated assay reagent, dilution buffer, and concentrated thiol standard. The assay has a linear range of 0.05–5 μM thiol (Figure 5.2.3), making it up to 400 times more sensitive than colorimetric methods based on DTNB (Ellman’s reagent).
Each Measure-iT Thiol Assay Kit contains:
- Measure-iT thiol quantitation reagent (100X concentrate in 1,2-propanediol)
- Measure-iT thiol quantitation buffer (50 mM potassium phosphate buffer)
- Measure-iT thiol quantitation standard (reduced glutathione)
- Detailed protocols (Measure-iT Thiol Assay Kit)
Simply dilute the reagent 1:100, load 100 μL into the wells of a microplate, add 1–10 μL sample volumes, mix, then read the fluorescence. Maximum fluorescence signal is attained within 5 minutes and is stable for at least 1 hour. The assay is performed at room temperature, and common contaminants are well tolerated in the assay. The Measure-iT Thiol Assay Kit provides sufficient materials for 500 assays, based on a 100 μL assay volume in a 96-well microplate format; this thiol assay can also be adapted for use in cuvettes or 384-well microplates.
Figure 5.2.3 Linearity and sensitivity of the Measure-iT thiol assay. Triplicate 10 µL samples of glutathione were assayed using the Measure-iT Thiol Assay Kit (M30550) . Fluorescence was measured using excitation/emission of 490/520 nm and plotted versus glutathione concentration. The variation (CV) of replicate samples was <2%.
Thiol and Sulfide Quantitation Kit
Ultrasensitive colorimetric quantitation of both protein and nonprotein thiols can also be achieved using the Thiol and Sulfide Quantitation Kit (T6060). In this assay, which is based on a method reported by Singh, thiols reduce a disulfide-inhibited derivative of papain, stoichiometrically releasing the active enzyme. Activity of the enzyme is then measured using the chromogenic papain substrate L-BAPNA via spectrophotometric detection of p-nitroaniline release at 412 nm (Figure 5.2.4). Although thiols can also be quantitated using DTNB (Ellman's reagent), the enzymatic amplification step in this quantitation kit enables researchers to detect as little as 0.2 nanomoles of a thiol—a sensitivity that is about 100-fold better than that achieved with DTNB. Thiols in proteins and potentially in other high molecular weight molecules can be detected indirectly by incorporating the disulfide cystamine into the solution. Cystamine undergoes an exchange reaction with protein thiols, yielding 2-mercaptoethylamine (cysteamine), which then releases active papain. Thiols that are alkylated by maleimides, iodoacetamides and other reagents are excluded from detection and can therefore be assayed subtractively.
The Thiol and Sulfide Quantitation Kit contains:
- Papain–SSCH3, the disulfide-inhibited papain derivative
- L-BAPNA, a chromogenic papain substrate
- DTNB (Ellman's reagent), for calibrating the assay
- L-Cysteine, a thiol standard
- Detailed protocols for measuring thiols, inorganic sulfides and maleimides (Thiol and Sulfide Quantitation Kit)
Sufficient reagents are provided for approximately 50 assays using 1 mL assay volumes and standard cuvettes or 250 assays using a microplate format.
Figure 5.2.4 Chemical basis for thiol detection using the Thiol and Sulfide Quantitation Kit (T6060): A) the inactive disulfide derivative of papain, papain–SSCH3, is activated in the presence of thiols; B) active papain cleaves the substrate L-BAPNA, releasing the p-nitroaniline chromophore; C) protein thiols, often poorly accessible, exchange with cystamine to generate 2-mercaptoethylamine (cysteamine), which is easily detected.
Thiol residues in close proximity can be oxidized to disulfides by either an intra- or intermolecular reaction. In many circumstances, however, this oxidation reaction is reversible and difficult to control.
Fluorescent Thiol–Thiol Crosslinkers
Dibromobimane (bBBr, D1379; ) is an interesting 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, actin, hemoglobin, Escherichia coli lactose permease and mitochondrial ATPase. It has also been shown to intramolecularly crosslink thiols in a complex of nebulin and calmodulin. In addition, dibromobimane has been used to probe for the proximity of dual-cysteine mutagenesis sites in ArsA ATPase and P-glycoprotein. Dibromobimane, a stimulator of the ATPase activity of a cysteine-free P-glycoprotein, was used with cysteine-scanning mutagenesis to identify amino acid residues important for function.
In addition to dibromobimane, we offer the thiol-reactive homobifunctional crosslinker bis-((N-iodoacetyl)piperazinyl)sulfonerhodamine (B10621), which is derived from a relatively rigid rhodamine dye (). This crosslinker is similar to a thiol-reactive rhodamine-based crosslinking reagent that was used to label regulatory light-chains of chicken gizzard myosin for fluorescence polarization experiments. Researchers have attached bis-((N-iodoacetyl)piperazinyl)sulfonerhodamine to the kinesin motor domain and determined the orientation of kinesin bound to microtubules in the presence of a nonhydrolyzable ATP analog by fluorescence polarization microscopy. Images of single molecules of chicken calmodulin crosslinked between two engineered cysteines by bis-((N-iodoacetyl)piperazinyl)sulfonerhodamine have been used to generate comparisons of experimental and theoretical super-resolution point-spread functions (PSF).
The scientific literature contains numerous references to reagents that form crosslinks between amines of biopolymers. Homobifunctional amine crosslinkers include glutaraldehyde, bis(imido esters), bis(succinimidyl esters), diisocyanates and diacid chlorides. These reagents, however, tend to yield high molecular weight aggregates, making them unsuitable for reproducibly preparing well-defined conjugates between two different amine-containing biomolecules. For example, glutaraldehyde is still used by some companies and research laboratories to couple horseradish peroxidase, which has only six lysine residues, to proteins with a larger number of lysine residues. Unfortunately, this practice can result in variable molecular weights and batch-to-batch inconsistency.
Well-defined conjugates between two amine-containing molecules are more reliably prepared by thiolating one or more amines on one of the biomolecules and converting one or more amines on the second biomolecule to a thiol-reactive functional group such as a maleimide or iodoacetamide, as described below in Amine–Thiol Crosslinking. For example, we prepare our horseradish peroxidase conjugates (Secondary Immunoreagents—Section 7.2, Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices—Section 7.6) using SPDP- and SMCC-mediated reactions (Figure 5.2.2, Figure 5.2.5).
Direct amine–amine crosslinking routinely occurs during fixation of proteins, cells and tissues with formaldehyde or glutaraldehyde. These common aldehyde-based fixatives are also used to crosslink amine and hydrazine derivatives to proteins and other amine-containing polymers. For example, lucifer yellow CH (L453, Polar Tracers—Section 14.3) is nonspecifically conjugated to surrounding biomolecules by aldehyde-based fixatives in order to preserve the dye's staining pattern during subsequent tissue manipulations. Also, biotin hydrazides (Biotinylation and Haptenylation Reagents—Section 4.2) have been directly coupled to nucleic acids with glutaraldehyde, a reaction that is potentially useful for conjugating fluorescent hydrazides and hydroxylamines to DNA.
Indirect crosslinking of the amines in one biomolecule to the thiols in a second biomolecule is the predominant method for forming a heteroconjugate. If one of the biomolecules does not already contain one or more thiol groups, it is necessary to introduce them using one of the thiolation procedures described above in Thiolation of Biomolecules. Thiol-reactive groups such as maleimides are typically introduced into the second biomolecule by modifying a one or more of its amines with a heterobifunctional crosslinker containing both a succinimidyl ester and a maleimide. The maleimide-modified biomolecule is then reacted with the thiol-containing biomolecule to form a stable thioether crosslink (Figure 5.2.5). Chromatographic methods are usually employed to separate the higher molecular weight heteroconjugate from the unconjugated biomolecules.
Introducing Maleimides at Amines
Succinimidyl trans-4-(maleimidylmethyl)cyclohexane-1-carboxylate (SMCC, S1534) is our reagent of choice for introducing thiol-reactive groups at amine sites because of the superior chemical stability of its maleimide and its ease of use (Figure 5.2.5).
Figure 5.2.5 Two-step reaction sequence for crosslinking biomolecules using the heterobifunctional crosslinker SMCC (S1534).
Introducing Disulfides at Amines
Our preferred method for preparing heteroconjugates employs the thiolation reagent SPDP (S1531). The pyridyldisulfide intermediate that is initially formed by reaction of SPDP with amines can form an unsymmetrical disulfide through reaction with a second thiol-containing molecule (Figure 5.2.2). The thiol-containing target can be a molecule such as β-galactosidase that contains intrinsic thiols or a molecule in which thiols have been introduced using one of the thiolation procedures described above in Thiolation of Biomolecules. In either case, it is essential that all reducing agents, such as DTT and TCEP, are absent. The heteroconjugate's disulfide bond is about as stable and resistant to reduction as disulfides found in proteins; it can be reduced with DTT or TCEP to generate two thiol-containing biomolecules.
Protein–Protein Crosslinking Kit
Our Protein–Protein Crosslinking Kit (P6305) provides all of the reagents and purification media required to perform three protein–protein conjugations in which neither protein contains thiol residues. The chemistry used to thiolate the amines of one of the proteins with SPDP and to convert the amines of the second protein to thiol-reactive maleimides with SMCC is shown in Figure 5.2.2 and Figure 5.2.5, respectively. Included in the kit are:
- SPDP, for thiolating amines
- SMCC, for converting amines to thiol-reactive maleimides
- TCEP, for reducing the pyridyldisulfide intermediate
- N-ethylmaleimide (NEM), for capping residual thiols
- Six reaction tubes, each containing a magnetic stir bar
- Spin columns plus collection tubes
- Dimethylsulfoxide (DMSO)
- Detailed crosslinking protocols (Protein–Protein Crosslinking Kit)
The Protein–Protein Crosslinking Kit was designed to prepare and purify protein–protein conjugates; however, it can be readily modified for generating peptide–protein or enzyme–nucleic acid conjugates or for conjugating biomolecules to affinity matrices.
We have considerable experience in preparing protein–protein conjugates and will apply this expertise to a researcher's particular application through our custom synthesis service. We provide custom conjugation services on an exclusive or nondisclosure basis when requested. For more information or a quote, please contact Invitrogen Custom Services.
Assaying Maleimide- and Iodoacetamide-Modified Biomolecules
The potential instability of maleimide derivatives and the photosensitivity of iodoacetamide derivatives may make it advisable to assay the modified biomolecule for thiol reactivity before conjugation with a thiol-containing biomolecule. SAMSA fluorescein (A685, ), which is currently our only fluorescent reagent that can generate a free thiol group, was designed for assaying whether or not a biomolecule is adequately labeled with a heterobifunctional maleimide or iodoacetamide crosslinker. Brief treatment of SAMSA fluorescein with NaOH at pH 10 liberates a free thiol (SAMSA Fluorescein). By adding base-treated SAMSA fluorescein to a small aliquot of the crosslinker-modified biomolecule, the researcher can check to see whether the biomolecule has been sufficiently labeled before proceeding to the next step. The degree of modification can be approximated from either the absorbance or the fluorescence of the conjugate following quick purification on a gel-filtration column.
Alternatively, thiol reactivity of the modified biomolecule can be assayed using the reagents provided in our Thiol and Sulfide Quantitation Kit (T6060), a product that is described above. Once unconjugated reagents have been removed, a small aliquot of the maleimide- or iodoacetamide-modified biomolecule can be reacted with excess cysteine. Thiol-reactive groups can then be quantitated by determining the amount of cysteine consumed in this reaction with the Thiol and Sulfide Quantitation Kit.
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC, E2247) can react with biomolecules to form "zero-length" crosslinks, usually within a molecule or between subunits of a protein complex. In this chemistry, the crosslinking reagent is not incorporated into the final product. The water-soluble carbodiimide EDAC crosslinks a specific amine and carboxylic acid between subunits of allophycocyanin, thereby stabilizing its assembly; we use EDAC to stabilize allophycocyanin in its allophycocyanin conjugates (Phycobiliproteins—Section 6.4). EDAC has also been used to form intramolecular crosslinks in myosin subfragment-1, intermolecular crosslinks in actomyosin, intersubunit crosslinks of chloroplast subunits and DNA–protein crosslinks. Addition of N-hydroxysuccinimide or N-hydroxysulfosuccinimide (NHSS, H2249) is reported to enhance the yield of carbodiimide-mediated conjugations, indicating the in situ formation of a succinimidyl ester–activated protein (Figure 5.2.6). EDAC has been reported to be impermeant to cell membranes, which should permit selective surface labeling of cellular carboxylic acids with fluorescent amines.
Reaction of carboxylic acids with cystamine (H2NCH2CH2S–SCH2CH2NH2) and EDAC followed by reduction with DTT results in thiolation at carboxylic acids. This indirect route to amine–carboxylic acid coupling is particularly suited to acidic proteins with few amines, carbohydrate polymers, heparin, poly(glutamic acid) and synthetic polymers lacking amines. The thiolated biomolecules can also be reacted with any of the probes described in Thiol-Reactive Probes—Chapter 2.Figure 5.2.6 Stabilization of an unstable O-acylisourea intermediate by N-hydroxysuccinimide in a carbodiimide-mediated (EDAC, E2247) modification of a carboxylic acid with a primary amine.
The succinimidyl ester of 6-((acryloyl)amino)hexanoic acid (acryloyl-X, SE; A20770; ) reacts with amines of proteins, amine-modified nucleic acids and other biomolecules to yield acrylamides that can be copolymerized into polyacrylamide matrices or onto surfaces, such as in microarrays and in biosensors. For example, streptavidin acrylamide (S21379, Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices—Section 7.6) copolymerizes with acrylamide on polymeric surfaces to create a uniform monolayer of the immobilized protein. The immobilized streptavidin can then bind biotinylated ligands, including biotinylated hybridization probes, enzymes, antibodies and drugs.
|A685||521.50||F,D,L||pH >6, DMF||491||78,000||515||pH 9|
|D1379||350.01||L||DMF, MeCN||391||6100||see Notes||MeOH||2|
|D8451||396.35||D||pH >6||324||18,000||none||pH 8||3|