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Reaction mechanism of nhs with amino group

Reaction chemistry of NHS ester

NHS ester is a reactive group formed by carbodiimide activation of carboxylate molecules (see carbodiimide crosslinking agent chemistry). NHS ester-activated crosslinking agents and labeled compounds react with primary amines to form stable amide bonds under physiological to weakly alkaline conditions (pH 7.2 to 9). This reaction releases N- hydroxysuccinimide (NHS).

Hydrolysis of NHS ester competes with primary amine reaction. With the increase of pH value of buffer solution, the hydrolysis rate increases, and the cross-linking efficiency decreases in low concentration protein solution. The hydrolysis half-life of NHS- ester compounds is 4 to 5 hours at pH 7.0 and 0℃. At pH 8.6 and 4 C, the half-life decreases to 10 minute. In aqueous solution without primary amine, the degree of hydrolysis of NHS ester can be measured at 260-280nm, because the by-product of NHS absorbs the peak in this range.

The crosslinking reaction of NHS- ester is usually carried out at room temperature or 4℃ in phosphate, carbonate-bicarbonate, HEPES or borate buffer with pH of 7.2-8.5 (0.5-4 hours). It is incompatible with primary amine buffers (such as Tris and TBS) because they will react competitively. However, in some steps, Tris or glycine buffer can be added at the end of the binding step to quench (terminate) the reaction.

Low concentration of sodium azide (≤3 mM or 0.02%) or thiomersal (≤0.02 mM or 0.0 1%) usually does not significantly interfere with the NHS- ester reaction, but high concentration will. Impure glycerol and high concentration of glycerol (20-50%) will also reduce the reaction efficiency.

Sulfo) -NHS ester is the same as NHS ester, except that it contains sulfonate (--SO3) on the N- hydroxysuccinimide ring. Charged groups have no effect on the whole chemical reaction, and are mainly used to increase the water solubility of crosslinking agents containing them. In addition, charged groups can prevent sulfo -NHS cross-linking agent from crossing cell membrane, making it available for cell surface cross-linking.