Tryptophan quantitative determination

Introduction of microbiological methods for the determination of amino acids made possible the estimation of the amount of both free and combined amino acids in urine. Dunn et al. (D4), Thompson and Kirby (Tl), Eckhard and Davidson (El), and Woodson et al. (W3) estimated the amount of amino acids liberated in the course of acid or, as in the case of tryptophan determination, alkaline hydrolysis. Microbiological and colorimetric methods used for the determination of certain amino acids present very little opportunity for evaluating the proper quantitative relations between free and combined amino acids, since under the applied condition both combined and free amino acids are equally involved in the reaction. In 1949 Albanese et al. (A3) applied such methods to the quantitative determination of free and combined amino acids in the nondiffusible fraction of urine, and subjected the procedures to broad criticism from just this point of view. 

As early as 1905 Abderhalden (Al) isolated from the hydrolyzate of the nondiffusible fraction of human urine four amino acids, i.e., leucine, alanine, glycine, and glutamic acid, and detected two others phenylalanine and aspartic acid. Some amino acids derived from this fraction have been quantitatively determined by Albanese et al. (A3) who found in the amount of the nondiffusible fraction corresponding to one liter of urine as much as 32.8 mg tryptophan, 18.0 mg phenylalanine, 16.2 mg methionine, 15.2 mg cystine, 13.1 mg arginine, 6.7 mg histidine, and 3.9 mg tyrosine. 

T.E. Barman and D.E. Koshland, A colorimetric procedure for the quantitative determination of tryptophan residues in proteins,... 

Experiment 1. Quantitative Determination of Tryptophan in Proteins in 6 M Guanidine... 

Pajot, P. (1976). Fluroescence of proteins in 6-M guanidine hydrochloride. A method for the quantitative determination of tryptophan. European Journal of Biochemistry, 63,263-269. 

For quantitative determinations of bound methionine sulfoxide and tryptophan, 10 cockroaches of each sex were homogenized and were extracted with the above solvents saturated with nitrogen. 

The individual amino acids of a protein can be liberated by hydrolyzing the peptide (amide) bonds (-CO-NH-) that link them. The usual procedure is to dissolve the peptide or protein in 6 N HC1 and heat the solution in a sealed, evacuated tube at 100°C for 8 to 72 hr (or for shorter times at higher temperatures). All amide linkages (including the side chain amides of glutamine and asparagine) are cleaved under these conditions. Certain amino acids are entirely (tryptophan) or partially (serine, threonine, tyrosine, cysteine) destroyed, so that special precautions are required for the quantitative determination of these amino acids. 

In a few cases, alkaline hydrolysis has proved applicable to special problems. Tryptophan is not destroyed in alkali, and analysis of alkaline hydrolyzates forms the basis of one method for quantitative determination of this amino acid (e.g., Dreze, 1960). Despite the fact that tryptophan-containing peptides should be more stable in alkali than acid, partial alkaline hydrolysis has not been employed for identification of this type of peptide. Amino acids often can be regenerated by alkaline hydrolysis from derivatives obtained by the amino-terminal end-group methods. Dinitrophenyl amino acids and phenylthiohydantoin (Fraenkel-Conrat et al., 1955) as well as hydantoin (Stark and Smyth, 1963) derivatives of amino acids can be treated in this manner. 

The urinary excretion of tryptophan, either spontaneous or after tryptophan loading (100 mg/kg), was recently studied in 50 healthy infants 1-20 months of age (V4). The results indicated that 94% of healthy infants excreted spontaneously higher amounts of kynurenine, on the basis of body weight, than do adults. This high value of kynurenine was confirmed by quantitative determination after a tryptophan load and was considered on an average to be 20 times larger than that of healthy adults. 

The peculiarities of tryptophan metabolism in the 15 individual members of this family were demonstrated by oral ingestion of 10 g DL-tryp-tophan per test case and quantitative determination of the urinary content of kynurenine, 3-hydroxykynurenine, xanthurenic acid, nicotinic acid amide and its N -methyl derivative, and 4-pyridoxic acid. Of the 15 members of the family examined 6 showed, repeatedly, abnormal levels of... 

CIO. Coppini, D., Benassi, C. A., and Montorsi, M., Quantitative determination of tryptophan metabolites (via kynurenine) in biologic fluids. Clin. Chem. 5, 391-401 (1959). 

This model as given by Equation (41) could be usefully employed for the quantitative determination of tryptophan in the presence of tyrosine. 

Qualitatively, the presence of the nonulosaminic acids is best indicated by the brilliant-red coloration formed on addition of an acidic solution of p-dimethylaminobenzaldehyde (the so-called direct Ehrlich reaction ), and by the bright-purple coloration which develops upon boiling with Bial s reagent for several minutes at 100°. In addition, the nonulosaminic acids give a blue-violet coloration with Dische s diphenylamine reagent for deoxypentoses and a positive reaction in the tryptophan-perchloric acid test. Since no single one of these color reactions is absolutely specific for a nonulosaminic acid, it is advisable to carry out at least two of these for a qualitative analysis. For quantitative determinations, all four reactions have been employed, using either A/ -acetylneuraminic acid (m. p., 183-185° [a] —32.0°) or methoxyneuraminic acid [m. p., 200° (dec.) [a]o —55.0°] as colorimetric standards. 

It was demonstrated that MCD is an excellent tool for the quantitative determination of tryptophan (126, 127). It was found that the xLb transition of tryptophan causes a positive B-term MCD band at 290 nm. At that wavelength no bands of other amino acids interfere with this MCD Cotton effect of tryptophan. Also, the intensity of this band seems to be almost independent of the conformation of a protein and, therefore, is suitable for the calculation of the tryptophan content of proteins. 

With Sibylle Tichy. Higher-Order Derivative Spectrophotometry for the Quantitative Determination of Tryptophan, Tyrosin, and Phenylalanine in T vo- and Three-Component Mixtures, Peptides, and Proteins , in Progress in TYyptophan and Serotonin Research, edited by H. G. Schlossberger, W. Kochen, B. Linzen, H. Steinhart, 95-102 and 129. Berlin, New York Walter de Gruyter u. Co., 1984. 

Diem S., Herderich M. Reaction of tryptophan with carbohydrates identification and quantitative determination of novel P-carboline alkaloids in food. Journal of Agricultural and Food Chemistry, 49 2486-2492 (2001). 

Although the amino acid composition of many proteins has been reported, evaluation of the number of tryptophan residues often has been lacking. This situation reflects the difficulty associated with quantitative determination of tryptophan residues in a protein, since acid hydrolysis usually results in the extensive destruction of tryptophan. Basic hydrolysis is less destructive, but suffers also from limitations ( P7 281). 

In this section, methods for the quantitative determination of tryptophan, free or bound in a polypeptide chain, are discussed in more detail. 

The labelled protein is separated by precipitation or by gel filtration and then the 2-NPS-tryptophan content determined spectro-photometrically at 365 nm. Protein concentration is determined by conventional methods, the most accurate being acid hydrolysis of an aliquot of the solution and subsequent quantitative amino acid analysis. 

Barth, G., W. Voelter, E. Bunnenberg, and C. Djerassi Magnetic Circular Dichroism Studies. XVII. Magnetic Circular Dichroism Spectra of Proteins. A New Method for the Quantitative Determination of Tryptophan. J. Amer. Chem. Soc. 94, 1293-1298 (1972). 

WiTZEMANN, V., R. Koberstein, H. Sund, I. Rasched, H. Jornvall, and K. Noack Studies on Glutamate Dehydrogenase Chemical Modification and Quantitative Determination of Tryptophan Residues. Eur. J. Biochem. 43, 319-325 (1974). 

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