Quantitation of tryptophan and tyrosine residues in proteins by fourth-derivative spectroscopy

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 tryptophan-specific trough at 292 nm in the fourth derivative of a protein's ultraviolet absorption spectrum. A peak overlapping the tryptophan and tyrosine signatures at A282 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 A282 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.