Abstract
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.
Highlights
IVateriuls-The n-tryptophan used was chromatographically homogeneous on PA-35 resin (Beckman) and gave values for carbon, hydrogen, and nitrogen that were within 1.0% of the calculated values. n-Tryptophyl-n-leucine obtained from Mann and L-isoleucyl-n-tryptophan obtained from Fox Chemicals gave the correct elementary analyses
3 Since alkaline hydrolysis can give rise to artifacts that overlap the peaks of some of the stable amino acids, we find it more accurate to base all calculations of molar ratios on the analysis of acid hydrolysates than on the measurement of amino acids other than tryptophan in the alkaline hydrolysate
When a mixture of other amino acids, starch, or citrate buffer is present in the solution the t,ryptophan content of the solution decreases significantly with time; losses were observed when a solution of pure tryptophan in 0.005 N NaOH was stored in the presence of air
Summary
A study of the variables in techniquesfor alkaline hydrolysis of proteins and for chromatographic analysis of the products has led to a method for the accurate determination of tryptophan. Liu and Chang [9] have recently made the valuable observation that p-toluenesulfonic acid containing 3-@aminoethyl) indole is much preferable to HCl in terms of the stability of tryptophan Their procedure is capable of giving about 90% recovery of tryptophan in a 22-hour hydrolysate of a purified protein; when the values observed after 22, 48, and 72 hours were extrapolated to zero time, good results were obtained with 10 proteins. We have drawn upon the observation of Dreze [14] that starch is a convenient antioxidant in such a hydrolysate and upon the recent use by Oelshlegel et al [13] of polypropylene liners in Pyrex tubes in order to avoid silicate formation during alkaline hydrolysis The combination of these observations with improvements in the ion exchange chromatography has led to a general procedure which has been tested on a variety of purified proteins and on corn and wheat as examples of foods
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