Magnetic circular dichroism studies: XIX. Determination of the tyrosine: Trytophan ratio in proteins

Abstract

Recently (2), we reported the use of magnetic circular dichroism (MCD) for the determination of tryptophan in intact proteins (1). We pointed out that, in comparison to other methods currently in use, MCD has the unique advantage of giving unambiguous results even in cases in which the tyrosine: tryptophan ratio is high, that conformational and environmental effects do not interfere with the accuracy of the method, and that only small amounts of sample are required. Here, we wish to report an important refinement in the procedure that enables the technique to be extended to samples which, due to the presence of unknown quantities of water or inorganic salts, are not of analytical purity. This modification makes use of the fact that, in contradistinction to tryptophan, the quantitative determination of tyrosine by the usual chromatographic procedure subsequent to hydrolytic cleavage of the protein does not present serious difficulties. Consequently, the spectroscopic determination of the tyrosine: tryptophan ratio has the advantage that, given knowledge of the tyrosine content, accurate values for the tryptophan content can be obtained with only rough knowledge of the protein concentration. Thus, tyrosine serves as an internal standard in the determination of tryptophan by the MCD technique. In addition to enabling tryptophan determinations to be made with increased accuracy, this modification illustrates the advantages inherent in a spectroscopic method in which signals may occur with either positive or negative sign.