Last week we reviewed some key factors to consider when coupling a label (biotin) to a protein. The main factors considered were:
- Spacer Arms
- Solubility
- Membrane Permeability
- Reversibility
Click here to review last weeks article
This week, we are going to examine the reactive groups and coupling chemistries.
Reactive Groups
Biotin reagents have numerous reactive groups that can couple to amines, sulfhydryls, carboxyls and carbohydrates. The conjugation efficiency of the reactions is dependent on the reaction groups and the buffers used for the reactions as many coupling reactions are sensitive to pH and chemical composition. The following section highlights the key features of the coupling reactions and important buffer information.
Based on the target reactive groups, biotin reagents can be divided into amine reactive, sulfhydryl reactive, carbohydrate reactive, and carboxyl reactive.
Photoreactive biotin reagents react non-specifically upon exposure to UV light and are used when no appropriate reactive target is available on the molecules.
Amine Reactive (-NH2)
Amines, lysine ε-amines and N-terminal α-amines, are the most abundant group in protein molecules and represent the most common target for biotinylation. For example, BSA contains 59 primary amines, of which up to 35 are available on the surface of the molecules and can be reacted with amine reactive esters.
NHS-Esters
The most widely used amine reactive biotinylation reagents are the water insoluble N-hydroxysuccinimide (NHS) esters or the water soluble N-hydroxysulfosuccinimide (sulfo-NHS) esters. The addition of a charged sulfonate (SO3-) on the N-hydroxysuccinimide ring of the sulfo-NHS esters results in their solubility in water (~10mM), but are not permeable to plasma membranes. The solubility and impermeability to plasma membranes makes them ideal for studying cell surface proteins as they will only react with the protein molecules on the outer surface of plasma membranes.
The reaction of the NHS and sulfo-NHS esters with amines are virtually identical leading to the formation of an amide bond and release of NHS or sulfo-NHS.
PFP-Biotin is another reagent that reacts with amines and forms stable amide bonds. PFP-Biotin is more reactive than other NHS esters and can react with both primary and secondary amines at pH 7-9.
Reaction Conditions
NHS esters are soluble in organic solvents and DMSO or DMF are the most commonly used, which are compatible with most proteins in a 20% solution. Sulfo-NHS ester is soluble in water, up to ~10mM and should only be dissolved immediately prior to use.
Reactive pH is neutral pH and above. Competing hydrolysis of the NHS esters and Sulfo-NHS esters in aqueous solution is a major concern as the rate of hydrolysis increases with increasing pH. Half-life of 2-4 hours at pH7.0 increasing to a few minutes at pH 9.0.
Reaction incubation time is a few minutes to a few hours at 4-35°C. For optimal conjugation, we recommend Optimizer Buffer™-I.
General Precautions
Avoid buffers containing amines such as Tris or glycine.
Sulfhydryl Reactive (-SH)
Sulfhydryl reactive reagents are more specific and react only with free sulfhydryl residues (-SH or thiol groups). The side chain of the amino acid cysteine is the most common source of free sulfhydryl groups. If free sulfhydryl residues are not available, they can be generated by the reduction of disulfides (-S-S-) with reducing agents such as mercaptoethylamine, or by modifying lysine ε-amines with Traut’s reagent or SATA. After reduction, excess reducing agent must be removed before coupling. In addition a metal chelating agent (EDTA) (an anti-oxidant) should be used to reduce the chances of reoxidation of sulfhydryls to disulfides.
There are three different reactions employed to couple biotin reagents to sulfhydryl residues and involve either iodoacetyl, maleimide or pyridylthiol groups.
Iodoacetyl Reaction Conditions
Iodoacetyl groups are sulfhydryl reactive reagents that react with thiol groups at pH7.5-8.5 and form stable thioether bonds. For specific reaction with sulfhydryls, limit the reaction to pH 7.5-8.5 and the molar ratio of iodoacetyl-biotin to protein such that the concentration of biotin is only slightly higher than the sulfhydryl concentration. Iodoacetyl reaction should be performed in dark to limit the formation of free iodine, which has the potential to react with tyrosine, tryptophan, and histidine residues. For optimal iodoacetyl conjugation, we recommend Optimizer Buffer™-II.
Maleimide Reaction Conditions
BMMCC is a sulfhydryl reactive reagent that contains a maleimide functional group. The maleimide group is more specific for sulfhydryl residues than iodoacetyl groups, at pH7 maleimide groups are 1000 fold more reactive toward free sulfhydryls than amines. At pH >8.5, maleimide groups favors primary amines. Conjugation is carried out at pH 6.5-7.5 for minimizing the reaction toward primary amine. At higher pH > 8.0, hydrolysis of maleimide to maleamic acid also increases, which can compete with thiol modification. Optimizer Buffer™-III provides ideal conditions for maleimide coupling reactions.
Pyridyldithiol Reaction Conditions
Pyridyldithiol groups (PDA) is a cleavable sulfhydryl reactive reagent. The reactive group is a pyridyldithiol that reacts with free sulfhydryl by disulfide exchange over a wide range of pH, forming a disulfide linkage. The optimal reaction pH is 6-9. Pyridine-2-thione is released, which absorbs light at 343nm. The coupling reaction can be monitored by measuring the absorbance of released pyridine-2-thione at 343nm. The disulfide bonds formed between can be cleaved with a reducing agent, generating the starting protein in its original form. This reagent is suitable for reversible applications. Optimizer Buffer™-III provides the optimized conditions.
General Precautions
Remove reducing agents from the conjugation reaction. Add metal chelating agent EDTA as an anti-oxidant.
Carboxyl Reactive (-COOH)
Carboxyl reactive labeling reagents contain terminal amines and react with carboxyl groups found at the carboxyl termini, aspartate, and glutamate side chains. The reaction is mediated by a water-soluble carbodiimide. The carbodiimide (EDC) activates the carboxyl group and reacts with the amines (-NH2) on the biotinylation agent to form an amide bond. This reaction is rapid and takes just a few minutes to complete. Under these conditions, hydrazide-derivatives of biotin reagents may also react with the carboxyls.
Reaction Conditions
The reaction is mediated by EDC, a water-soluble carbodiimide cross-linking agent. EDC activates carboxyl groups to bind with the -NH2 group from the biotin derivatives. Optimizer Buffer™-IV provides the ideal buffer for EDC and other carbodiimides.
General Precautions
EDC may crosslink protein, decreasing EDC and/or increasing biotin reagent levels minimizing conjugation. Avoid buffers containing amines, such as Tris or glycine, or carboxyls, such as acetate, citrate, etc. These buffers react with aldehydes, quenching the reaction.
Phosphate buffers also reduce the conjugation efficiency.
Carbohydrate Reactive
Some biotin reagents do not bind directly to the protein itself but conjugate to the carbohydrate residues of glycoproteins. Carbohydrate reactive biotin reagents contain hydrazides (-NH-NH2) as a reactive group. The hydrazide reactions require carbonyl groups, such as aldehydes and ketones, which are formed by oxidative treatment of the carbohydrates. Hydrazides react spontaneously with carbonyl groups, forming a stable hydrazone bond. These reagents are particularly suitable for labeling and studying glycosylated proteins, such as antibodies and receptors.
Reaction Conditions
For reaction with glycoproteins, the first step is to generate carbonyl groups that react with hydrazide, under mild oxidizing conditions with sodium periodate (NaIO4). At 1mM periodate and at 0°C, sialic acid residues on the glycoproteins can be specifically oxidized converting hydroxyls to aldehydes and ketones. At higher concentrations of 6-10mM periodate, other carbohydrates in protein molecules will be oxidized. Such oxidation reactions are performed in the dark to minimize unwanted side reactions.
Aldehyde can also be generated by enzymatic reactions. For example, neuraminidase treatment will generate galactose groups from sialic acid residues on glycoproteins and galactose oxidase converts primary hydroxyl groups on galactose and N-acetylgalactosamine to their corresponding aldehydes. For coupling to carbohydrates, Optimizer Buffer™-V is recommended.
General Precautions
Each glycoprotein has an optimal pH for oxidation and optimal pH for the hydrazide reaction. Periodate oxidation is dependent on temperature, pH, as well as concentration. The extent of glycosylation varies for each protein; therefore, optimal condition for each protein must be determined. Avoid buffers containing amines, such as Tris or glycine; these buffers react with aldehydes, quenching their reaction with hydrazides.
Photoreactive
Photoreactive agents on exposure to ultraviolet light become active and bind non-specifically with neighboring molecules. Photoreactive reagents are suitable for labeling molecules that do not contain easily reactable functional groups. There are a variety of photoreactive biotinylation reagents for the labeling of proteins, peptides, nucleic acids, and other molecules. Psoralen, a photoreactive reagent, reacts and labels nucleic acids and protein molecules. When reacted with nucleic acids, it cross-links with pyrimidine bases. Cross-linking does not interfere with hybridization applications.
Reaction Conditions
Photoreactive reagents contain any aryl azide group. Aryl azide groups are chemically inert until exposed to ultraviolet light. Highly reactive and short-lived aryl nitrenes are formed, which rapidly and non-specifically react with electron-rich sites by inserting into double bonds or active hydrogen bonds (insertion into C-H and N-H sites). Uncreated aryl nitrenes undergo ring expansion and become reactive toward primary amines and sulfhydryls. A wide variety of reaction buffer conditions are acceptable for photoreactive reaction, however Optimizer Buffer-I provides excellent buffer conditions.
General Precautions
Avoid acidic and reducing agents since they inactivate aryl azide groups.
