Modification of Tryptophan with 2-Hydroxy-5-Nitrobenzylbromide
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2-Hydroxy-5-nitrobenzylbromide, Koshland’s reagent (1), reacts rapidly and under mild conditions with tryptophan residues. At low pH (<7.5) this reagent exhibits a marked selectivity for tryptophan; under more basic pH or at higher reagent concentrations, cysteine, tyrosine, and even lysine residues can be modified as well (see Note 1). The reaction is extremely rapid either with the protein or with water. The reagent is relatively insoluble in water; it is therefore necessary to first dissolve it in an organic solvent (e.g., dioxane) and then to add it to the protein solution in buffer. Unlike that of most other modifying reagents, the final organic solvent concentration in the reaction mixture is relatively high (5-15%). Determination of the number of modified tryptophans is done spectrophotometrically.