Spectrophotometric determination of microgram quantities of protein without nucleic acid interference

Abstract A spectrophotometric method which eliminates interference due to nucleic acid absorbance has been developed for determining protein concentration over the range of 5 to 180 μg/ml. Absorbance measurements are made in the 2240 to 2400 A range where light absorbance by polypeptide results from the presence of tryptophan, tyrosine, phenylalanine, histidine, methionine, cysteine, and cystine as well as some contribution apparently from peptide bond. The variation of absorbance as a function of primary structure has been studied using pure proteins with widely differing quantities of the above amino acids. With egg white lysozyme and bovine serum albumin, the results obtained by the present method have been compared to those obtained with the Folin-Lowry and microbiuret methods.