Protein Protocols Protein Protocols

482 Tawfik
4. Notes
1. At neutral and slightly acidic pH the reaction is generally selective. At higher pH, the
major side reaction is the benzylation of sulfhydryl groups; to prevent the modification of
free cysteine residues the protein can be carboxymethylated prior to the modification with
2-hydroxy-5-nitrobenzylbromide.
2. Avoid the use of acetone, methanol, or ethanol as organic co-solvents for this reagent.
Water-free dioxane is commercially available or can be prepared by drying dioxane over
sodium hydroxide pellets.
3. At high reagent concentrations a precipitate of 2-hydroxy-5-nitrobenzyl alcohol is sometimes
observed and can be removed by centrifugation. Purification of the modified protein
from all the remaining alcohol product is sometimes difficult and may require extensive
dialysis in addition to gel filtration.
4. The reaction of hydroxy-5-nitrobenzylbromide is accompanied by the release of hydrobromic
acid. The pH of the reaction should be maintained if necessary by the subsequent
addition of small aliquots of 1 M NaOH solution.
References
1. Horton, H. R. and Koshland, D. E., Jr. (1972) Modification of proteins with active benzyl
halides. Meth. Enzymol. 25, 468–482.