Tryptophan analysis

Aliquots of 50 pmol were pipetted into pre-washed 1.5 ml polypropylene microfuge tubes and vacuum dried. Several aliquots were analyzed by committee members to insure sample quality before distribution. Two aliquots of ABRF-94SEQ were sent to each of the 258 ABl member facilities along with instructions for sample solubilization, a short questionnaire, a data analysis worksheet, and several commonly used methods for cysteine and tryptophan analysis. Members were encouraged to try at least one of the methods described. All results were returned to an independent third party who, after removing all identifying labels, forwarded the anonymous data to the committee for analysis. 

Of the 57 facilities using Applied Biosystems instruments, 35 reported that they added isopropanol (IPA) to solvent B and 21 did not. Because IPA should help separate hyptophan from diphenylurea, tryptophan analysis results are separated into Applied Biosystems users who added isopropanol (IPA+), Applied Biosystems users who did not 0PA ), and users of other instrumentation (Non-Applied Biosystems). 

At the present time the hydrolytic methods of choice for tryptophan analyses appear to be with methanesulfonic acid in the presence of added 3-(2-aminoethyl) indole (see Moore 1972) or with NaOH in the presence of starch (Hugh and Moore 1972). Both methods will be described below, but it should be noted here that hydrolysis with NaOH has the advantage that carbohydrate in the protein sample does not affect the yields of tryptophan as it does in the acid hydrolysates. Other methods of tryptophan analysis involving spectrophotometry (Goodwin and Morton 1946 Edelhock 1967), titration with N-bromo-succinimide (Spande and Witkop 1967), or the formation of colored derivatives (Spies and Chambers 1948 Barman and Koshland 1967 ... 

The superior approach to tryptophan analysis involves the addition of dodecanethiol to HCl acid, es-... 

Quality Control and 5-HTP (5-Hydroxy-L-tryptophan) Analysis of Griffonia Griffonia simplicifolia (DC.) Baill.) Seed Aeeessions Colleeted in Ghana... 

The superior approach to tryptophan analysis involves the addition of dodecanethiol to HCl, especially when combined with automatic vapor-phase hydrolysis. Alternative hydrolysis agents such as methane sulfonic acid, mercap-toethanesulfonic acid, or thioglycolic acid can produce 90% or greater yields. Acid hydrolysis additives and alkaline hydrolysis using 4.2 M NaOH are also used with varying results. 

ALKALINE HYDROLYSIS OF PROTEINS AND PEPTIDES FOR TRYPTOPHAN ANALYSIS... 

As has been stated, tryptophan is completely destroyed by acid hydrolysis. Some methods protect tryptophan with compounds such as 2-mercaptoethanol, but these methods do not always give reproducible results. Other methods for the liberation of tryptophan have included proteolysis by enzymes. An early method developed by Spies and Chambers (1949) did not require freed tryptophan. It is a colorimetric assay, reacting the tryptophan with p-dimethylaminobenzaldehyde (PDBA). This method has been useful in pure proteins, but many food matrices have interfering substances. In addition, tryptophan is one of only two amino acids with a strong extinction coefficient in a usable ultraviolet (UV) range (approximately 280 nm depending on solvent, etc.). However, the most reliable method of tryptophan analysis is to release the amino acid from protein with alkaline hydrolysis (Lucas et al., 1980) and then use chromatography for quantification. 

Because of uncertainties in the present methods for tryptophan analysis, it is obvious that a satisfactory general procedure remains to be found 135, 353). Acid hydrolysis with /7-toluenesulfonic 233, 234) or mercaptoethanesulfonic acid 299) seems to be the most reliable known method, even though the analytical figures for tryptophan have to be checked by different methods. 

Basic hydrolysis of proteins by either Ba(OH)2 or NaOH has been considered preferable to acid hydrolysis for tryptophan analysis unfortunately, the method does not always give satisfactory results. Spies and Chambers (380) studied factors affecting the stability of tryptophan in 5N NaOH. They found that tryptophan is more labile when the amino acid is peptide-linked than in the free form and that losses depend on protein composition. Oelshlegel et ai (281) hydrolyzed proteins with NaOH in the presence of thioglycolic acid, the recoveries of tryptophan being 89-96%. 

Carbohydrate does not interfere in the tryptophan analysis using basic hydrolysis. This allows use of the method in the analysis of samples with appreciable carbohydrate content, including the analysis of tryptophan in foods. 

Slump and Schreuder described a chromatographic method for tryptophan determination, using an automatic amino acid analyzer, which is especially useful for tryptophan analysis in foods. Tryptophan was separated from the other amino acids by means of gel filtration on Sephadex G-25 columns. This separation is ascribed to the property of the dextran gels to adsorb aromatic compounds to a larger extent than aliphatic compounds. 

Bredderman, P. J. Tryptophan Analysis of Proteins in 6M Guanidine Hydrochloride Modification for More General Application. Anal. Biochem. 61, 298-301 (1974). 

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