NHS ester hydrolysis

Dissolve the protein or peptide to be thiolated at a concentration of lOmg/ml in 50 mM sodium phosphate, pH 7.5, containing 1 mM EDTA. Other non-amine containing buffers such as borate, HEPES, and bicarbonate also may be used as the reaction medium. The effective pH for the NHS ester modification reaction is in the range of 7.0-9.0. Conditions closer to neutral pH will limit the degree of NHS ester hydrolysis during the reaction. 

Sulfo-LC-SMPT is not as stable as SMPT. The sulfo-NHS ester is more susceptible to hydrolysis in aqueous solutions and the pyridyl disulfide group is more easily reduced to the free sulfhydryl. Stock solutions of sulfo-LC-SMPT may be prepared in water, but should be used immediately to prevent loss of amine coupling ability. 

Dissolve SANH, SHNH, or SHTH in DMF to prepare a stock solution at a concentration of 2.0-4.0mg in 100-200pi. Use highly pure and dry solvent (H20 content < 0.1 percent or treat with molecular sieves) to prevent hydrolysis of the NHS ester. 

Dissolve mono(lactosylamido) mono(succinimidyl)suberate in dry DMF to prepare a concentrated solution from which an aliquot may be taken and added to a final aqueous reaction medium. The compound is extremely soluble in DMF, and solutions of 100 mg/ ml may be prepared. The use of dry solvent is essential to prevent hydrolysis of the NHS ester. However, make only enough of this stock solution so that a small amount added to the protein reaction will provide the appropriate molar excess desired for the modification reaction. 

Figure 2.1 Three types of fluorophenyl esters have been used for coupling to amine-containing molecules. The PFP and TFP esters are relatively hydrophobic and typically have better stability toward hydrolysis in aqueous solution than NHS esters. The STP ester is water-soluble due to the negatively charged sulfonate group, and it provides better solubility to associated crosslinkers or bioconjugation reagents similar to that of a sulfo-NHS ester group.

Fluorophenyl esters react with amine-containing molecules at slightly alkaline pH values to give the same amide bond linkages as NHS esters (Reaction 15). However, in most cases, the fluorophenyl ester compound will display better stability toward hydrolysis in aqueous solution. It has been reported that a TFP ester has over twice the half-life in basic pH buffers (pH 8) than a corresponding NHS ester on the same compound (Molecular Probes). 

SMPB contains a hydrophobic cross-bridge and relatively nonpolar ends, which allows the reagent to permeate membrane structures. Due to its water-insolubility, it must be dissolved in an organic solvent prior to adding an aliquot to a reaction mixture. The solvents DMF and DMSO work well for this purpose. A concentrated stock solution prepared in these solvents allows for easy addition of a small amount to a conjugation reaction. Long-term storage in these solvents is not recommended due to slow water pickup and possible hydrolysis of the NHS ester end. 

The following operations should be done using standard safety procedures for working with radioactive compounds. All steps involving SASD prior to initiation of the photoreaction should be done protected from light to avoid loss of phenyl azide activity. The radiolabeling procedure should be done quickly to prevent excessive loss of NHS ester activity due to hydrolysis. 

Dissolve sulfo-NHS-LC-SPDP at a concentration of 20mM (5.2mg/ml) in DMSO or water. If water is used, the solution must be used immediately to prevent hydrolysis of the sulfo-NHS ester. 

Dissolve NHS-PEGg-maleimide (MW 601.6) into DMSO at a concentration of 20mM. Short, PEG-type crosslinkers often exist as a thick oily mass, and preparing the solution typically involves dissolving an entire vial of the compound into DMSO to determine accurately the required concentration. Use only dry DMSO to avoid hydrolysis of the NHS ester. 

A water-soluble analog of NHS-biotin containing a negatively charged sulfonate group on its NHS ring structure also is available. Sulfo-NHS-biotin may be added directly to aqueous reactions without the need for organic solvent dissolution. A concentrated stock solution may be prepared in water to facilitate the addition of a small quantity to a reaction, but hydrolysis of the NHS ester will occur at a rapid rate, so the solution must be used immediately. 

Immediately before use, dissolve sulfo-NHS-biotin (Thermo Fisher) in water at a concentration of 20 mg/ml. Alternatively, the compound may be dissolved in organic solvent to prevent hydrolysis prior to a reaction (i.e., dry DMF or DMSO). Adjust the concentration and quantity of this stock solution to be prepared according to the amount of reagent needed to biotinylate the desired amount of protein. If prepared in water, the sulfo-NHS-biotin stock solution must be used immediately, since the NHS ester is subject to hydrolysis in aqueous environments. 

---