Quantitation of Tryptophan and Tyrosine Residues in Proteins by Fourth-Derivative Spectroscopy

Abstract

A method for quantitation of tryptophan and tyrosine residues in proteins by fourth-derivative ultraviolet spectroscopy is described. The direct quantitation of tryptophan is based on measurement of a tryptophanspecific trough at 292 nm in the fourth derivative of a protein′s ultraviolet absorption spectrum. A peak overlapping the tryptophan and tyrosine signatures at A 282 is used to quantify tyrosine content. The procedure is accomplished by adding back known quantities of tyrosine to the sample and subtracting the contribution of tryptophan to the A 282 peak to obtain an internal calibration curve. This curve is linear, with the ordinate axis intercept relating the quantity (residues/mole) of tyrosine present in the protein. This nondestructive and facile method was used to successfully predict the tryptophan and tyrosine content of a variety of well-characterized proteins. The utility of this method was further demonstrated by resolving the number of tryptophan and tyrosine residues in proteins oxidized by N-bromosuccinimide.