Spectrophotometric estimation of protein concentration in the presence of tryptophan modified by 2-hydroxy-5-nitrobenzyl bromide.

A spectrophotometric method makes it possible to determine the concentration of a protein after covalent modification of tryptophan residues by 2-hydroxy-5-nitrobenzyl bromide. Molar absorption coefficients for the 2-hydroxy-5-nitrobenzyl chromophore, reported here in the pH range from 4.0 to 10.9, can be used to correct the protein absorbance values at 280 nm, which then provides the basis for calculating protein concentration in the usual way. The method was tested with alpha-lactalbumin, beta-lactoglobulin, pepsin, and soybean trypsin inhibitor; spectrophotometrically estimated concentrations of these proteins agreed closely with values obtained by amino acid analysis.