Tryptophan spectrophotometric determination

Fio. 125. Spectrophotometrically determined titration curves of the carboxyl group of tryptophan at the temperatures and wavelength indicated. The curve is a theoretical one for the pK indicated by the vertical stroke. The reference pH s are approximately 6 (Hermans et al., 1960). 

The reaction is used for the selective cleavage of peptide chains and the spectrophotometric determination of tryptophan. 

Price, M.L. and Butler, L.G., Rapid visual estimation and spectrophotometric determination of tannin content of sorghum grain, J. Agric. Food Chem., 25, 1268, 1277, 1977. Folin, O. and Ciocalteu, V., On tyrosine and tryptophan determinations in proteins, J. Biol. Chem., 27, 627, 1927. 

JW DeVries, CM Koski, DC Egberg, PA Larson. Comparison between a spectrophotometric and a high-pressure liquid chromatography method for determining tryptophan in food products. J Agric Food Chem 28 896-898, 1980. 

A small peptide was subjected to hydrolysis and amino acid analysis. In addition, because acid hydrolysis destroys tryptophan, the tryptophan content was estimated spectrophotometrically. From the following data, determine the empirical formula of the peptide. 

Table I gives the amino acid composition of bovine chymotrypsinogen A. Quite similar results for most amino acids have been obtained for this protein in two laboratories by the usual chromatographic technique in its manual or automatic (83) form involving all the necessary corrections and extrapolations to zero time. Cystine has been determined as cysteic acid after pcrformic acid oxidation. Values of tryptophan are probably less satisfactory sin( e they are derived from spectrophotometric or microbiological determinations. The protein contains only 2 histidines, 2 methionines, 4 arginines, 4 tyrosines, and 5 cystines per mole.

Tompsett (T3) achieved a separate elution from cation or anion resin columns of several tryptophan metabolites which were then determined colorimetrically. Finally, Boyland and Williams (B18) quantitatively adsorbed on inactivated charcoal anthranilic acid, kynurenine, 3-hy-droxyanthranilic acid, 3-hydroxykynurenine, and the sulfuric acid ester derivatives of the two latter compounds from urine of normal controls and of patients with bladder cancer. After elution, the compounds were separated by gradient chromatography on Celite columns and determined colorimetrically or spectrophotometrically. 

The tryptophan value reported by Harrison et al. (110) is less than half that found by us (107). The estimation by Harrison et al. (110) was by modification (118) of the method of Spies and Chambers (119) using the >-dimethylaminobenzaldehyde reagent with samples of apoferritin which had first been denatured. Bryce and Crichton (107) determined tryptophan both by the spectrophotometric method of Edelhoch (120) in 6M guanidine hydrochloride and using 2-nitrophenylsulphenyl chlo-... 

Apart from this the interest and application of ultraviolet spectra of proteins are analytical. On a microscale the absorption spectrum may be the simplest and best evidence for the recognition of a protein. It is possible that, with care, it will be the best means of obtaining an estimate of tyrosine and tryptophan in a protein. The instability of tryptophan under the conditions required for protein hydrolysis gives weight in favor of a method such as the spectrophotometric which allows a direct determination of tryptophan to be made (on a protein) without hydrolysis. 

There are several recorded determinations of the absorption curves of the aromatic amino-acids. Most of these were obtained with photographic methods of spectrophotometry which have been superceded by more accurate photoelectric methods. It will be shown that in the spectrophotometric analysis of tyrosine and tryptophan in proteins, the photometric error is magnified in the final estimate of tyrosine and tryptophan contents. This fact is inevitably bound up with the form of the equations of mixture analysis. It is therefore important that the absorption constants be measured as accurately as possible. 

It is evident from the published data and from data privately collected that considerable variations exist between the values of these constants as determined in different laboratories. This applies particularly to tryptophan. The question is discussed separately for each amino acid but it is relevant here to comment generally on the accuracy of spectrophotometric methods. 

Finally, several of the amino acids listed have partial lability during acid hydrolysis and require appropriate corrections, usually derived from kinetic analyses (Smith and Stockell, 1954 Wilcox et al, 1957). One amino acid generally considered to be extremely labile to acid is tryptophan. However, its destruction is actually a function of other contaminants present in the sample (Olcott and Fraenkel-Conrat, 1947). Pure samples of this amino acid are fully stable to the standard conditions of acid hydrolysis. Nonetheless, the observed lability is encountered sufficiently often that the preferred determination of this amino acid is carried out by spectrophotometric analysis (Edelhoch, 1967) or colorimetric methods (Koshland et a/., 1964 Scoffone et al, 1968) on unhydrolyzed samples or by carefully controlled alkaline hydrolysis (Hugh and Moore, 1972) (vide infra). 

The principal disadvantages of acid hydrolysis are the destruction of some amino acids, notably serine, threonine, cystine, and tryptophan, and the slow release of amino acids from some dipeptide combinations, notably those involving isoleucine and valine (Hill, 1965). For most of these, timed analyses allow the extrapolations that give excellent estimations of the original amino acid content of the sample. Cystine can be readily estimated as one of several derivatives vide supra), and tryptophan can be either analyzed after alternative hydrolysis procedures or determined directly on the intact protein or peptide by spectrophotometric techniques (Edelhoch, 1967). Two amino acids, glutamine and asparagine, are quantitatively destroyed and can be determined only on enzymatic hydrolysates. 

The spectrophotometric method has been used with reasonable success, but great difficulties can be encountered in the determination of tryptophan by the spectrophotometric method when this amino acid is present in the protein at a low level or when the tyrosine content is high. 

A sensitive spectrophotometric method for the determination of free or bound tryptophan has been proposed by Messineo and Musarra 258). The method utilizes the reaction of fructose with cysteine in sulfuric acid to give a green chromophore (A, max 415 nm), which upon the addition of tryptophan or other indole derivative forms a pink chromophore (A max 518 nm). Once the pink chromophore is formed, it is stable for more than 24 hours. The exact nature of the reactions involved is not known. The method offer advantages, since it does not require previous hydrolysis of the protein, and is simple and quite sensitive. 

Messineo, L., and E. Musarra A Sensitive Spectrophotometric Method for the Determination of Free or Bound Tryptophan. Intern. J. Biochem. 3, 700-704... 

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