Determination of the Tryptophan Content of Proteins by Ion Exchange Chromatography of Alkaline Hydrolysates

Abstract A study of the variables in techniques for alkaline hydrolysis of proteins and for chromatographic analysis of the products has led to a method for the accurate determination of tryptophan. Quantitative recoveries of tryptophan are obtained when proteins (1 to 5 mg) are hydrolyzed at 110° or 135° in 0.6 ml of 4.2 n NaOH containing 25 mg of starch. The hydrolysis is performed in polypropylene liners sealed inside glass tubes evacuated to below 50 µm of mercury. Ion exchange chromatography of tryptophan on Beckman PA-35 resin (column height 8 or 12 cm) has been accomplished in 30 to 50 min with pH 5.4 buffer, 0.21 n in Na+. The details in the procedure which make possible complete recovery of tryptophan include: (a) addition of the sample at pH 4.25 instead of at pH 2.2 in order to avoid loss of tryptophan in citrate buffer at acid pH; (b) the use of NaOH instead of Ba(OH)2 to avoid loss of tryptophan by adsorption on BaSO4 or BaCO3; (c) the inclusion of starch as the most effective antioxidant tested; and (d) chromatography with a buffer which separates tryptophan from Ne-(dl-2-amino-2-carboxyethyl)-l-lysine, which can be formed in significant quantities during alkaline hydrolysis. Molar calculations of protein concentrations are based on the results from analysis of an acid hydrolysate run parallel with the alkaline hydrolyses. Integral values (100 ± 3%) have been obtained for the expected number of tryptophan residues in tryptophyl-leucine, human serum albumin, porcine pepsin, sperm whale apomyoglobin, and in bovine α-chymotrypsin, trypsin, deoxyribonuclease, and serum albumin. Since carbohydrate does not interfere, the procedure is applicable to foods and has been tested on normal and opaque-2 maize meals and on wheat flours.