Tryptophan alkaline hydrolysis

The deterruination of amino acids in proteins requires pretreatment by either acid or alkaline hydrolysis. However, L-tryptophan is decomposed by acid, and the racemi2ation of several amino acids takes place during alkaline hydrolysis. Moreover, it is very difficult to confirm the presence of cysteine in either case. The use of methanesulfonic acid (18) and mercaptoethanesulfonic acid (19) as the protein hydroly2ing reagent to prevent decomposition of L-tryptophan and L-cysteine is recommended. En2ymatic hydrolysis of proteins has been studied (20). 

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. 

Methionine, methionine sulfoxide and tryptophan were determined after alkaline hydrolysis, since they are known to degrade during acid hydrolysis. For this prupose, 3N NaOH was used. 

Alkaline hydrolysis (with NaOH, KOH or more seldom with Ba(OH)2) is almost exclusively applied for the determination of tryptophan and phosphoamino acids. Serine, threonine, arginine, and cysteine are completely destroyed by alkaline hydrolysis, while other amino acids are racemized [190]. Since racemization also occurs during acid hydrolysis, when it is important to... 

Alkaline hydrolysis with barium, sodium, or lithium hydroxides (0.2-4 M) at 110°C for 18-70 h126-291 requires special reaction vessels and handling. Reaction mixtures are neutralized after hydrolysis and barium ions have to be removed by precipitation as their carbonate or sulfate salts prior to analysis which leads to loss of hydrolysate. Correspondingly, peptide contents are difficult to perform by this procedure. Preferred conditions for alkaline hydrolysis are 4M LiOH at 145 °C for 4-8 h where >95% of tryptophan is recovered 291 An additional inconvenience of the alkaline hydrolysis procedure is the dilution effect in the neutralization step and thus the difficult application to the analyzer if micro-scale analysis is to be performed. The main advantage is the good recovery of tryptophan and of acid-labile amino acid derivatives such as tyrosine-0-sulfate1261 (Section 6.6) as well as partial recovery of phosphoamino acids, particularly of threonine- and tyrosine-O-phosphate (Section 6.5). 

S Bech-Anderson. Determination of tryptophan with HPLC after alkaline hydrolysis in autoclave using a-methyl-tryptophan as internal standard. Acta Agric Scand 41 305-309, 1991. 

Hymenocardine (58), (32, 40) has a p-hydroxy-w-aminoacetophenone unit (81), instead of the usual styrylamine, in its cyclic system which can be recognized in addition to N,N-dimethylisoleucylvaline and tryptophan in its acid hydrolysate. Mild alkaline hydrolysis results in ring opening via /3 elimination on the hydroxyamino acid and severance of the phenolate to a tetrapeptide 82 whose structure was determined by mass spectrometry and further hydrolysis. It is the only peptide alkaloid in which /3-hydroxy valine is involved in the aryl ether bridge. 

Alkaline Hydrolysis. Protein samples to be analyzed for tryptophan or methionine sulfoxide were hydrolyzed with barium hydroxide (16). Fifty milligrams of protein was the usual quantity hydrolyzed. After barium had been removed from the hydrolyzate (16), the pH of the solution was adjusted to 2.2 and the final volume to 22.5 ml. The entire procedure was conducted under nitrogen to minimize oxidation. 

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 iodometric method was then modified (G23, G24) by changing the final concentration of trichloroacetic acid from 8% to 3.3%, leaving more proteose-like substances in the filtrate, and quantitating the mucosubstances in the filtrate colorimetrically by the Folin-Ciocalteu reaction with the phenol reagent, instead of iodometric titration. This colorimetric reaction determines the tyrosine and tryptophan content after alkaline hydrolysis of the total dissolved mucin. 

The reaction rate doubles with every 10°C increase, so that hydrolysis at 145°C for 4 h gives results comparable to the conventional method. Microwave hydrolysis reduces analysis time to 30-45 min. Alternative hydrolysis agents include sulfonic acid, which often gives better recovery but is nonvolatile, and alkaline hydrolysis, used in the analysis of tryptophan, proteoglycans, and proteolipids. 

There are numerous protocols for protein hydrolysis, involving minor variants of the standard procedure, that are intended to minimise the destruction of particular amino acids (tryptophan and cysteine/cystine in particular) through the sensitivity of their side-chains to the reaction conditions, especially when access of oxygen is not prevented. Tryptophan largely survives alkaline hydrolysis (but other coded amino acids, particularly serine and threonine, but also arginine and cysteine, do not). 

In the case of indole, alkaline hydrolysis and putrefaction of proteins result in its formation. This formation in the putrefaction of proteins is presumed to be result of decomposition of tryptophan. The formation of indole from albumin may be stopped by the addition of lactose, while other sugars have varying effects on its production. Indole frequently accompanies pus formation and is found in the human liver, pancreas, brain, and bile. Indole, accompanied by its p-methyl homolog, skatole, is found in the feces of humans and animals and in the contents of the intestines. ... 

Hymenocardine (mp 261° [ ]j) — 124°) from this plant was shown to have structure LXX largely because of its acid and alkaline hydrolysis to tryptophan, V-dimethylisoleucylvaline, and p-hydroxy-oi-aminoaceto-phenone. Exhaustive spectral analyses confirmed this structure and aided in the identification of the hydrolytic fragments 109). 

Alkaline hydrolysis offers a possible advantage for the investigation of tryptophan peptides since tryptophan itself is more stable in alkali than in acid (Lugg, 1938 Brand and Kassell, 1939). 

Amino acids Acid hydrolysis (HCI 6N) followed by chromatography. There are many methods with variants in duration (24-48 hours) and temperature (110-I45°C). Methionine and cystine are obtained after performic acid oxidation and tryptophan after alkaline hydrolysis. 

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 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. 

Since biological value is dependent primarily upon essential amino acid constitution, it would seem logical to assess the nutritive value of a protein by determining its essential amino acid constitution and then comparing this with the known amino acid requirements of a particular class of animal. Application of modern chromatographic techniques coupled with automated procedures allows relatively quick and convenient resolution of mixtures of amino acids. However, the acid hydrolysis used to produce such mixtures from protein destroys practically all the tryptophan and a considerable proportion of the cystine and methionine. Tryptophan has to be released by a separate alkaline hydrolysis, and cystine and methionine have to be oxidised to cysteic acid and methionine sulphone to ensure their quantitative recovery. Losses of amino acids and the production of artefacts, which are greater with foods of high carbohydrate content, are reduced if the hydrolysis is carried out in vacuo. Evaluations of proteins in terms of each individual amino acid would be laborious and inconvenient, and several attempts have been made to state the results of amino acid analyses in a more useful and convenient form. 

Synthesis. DL-Tryptophan is synthesized in 70% over-all yield from crude indole by the method of Albertson and Tullar (50). Ethyl o-acetamido-a-cyano-/3-(3-indolyl)-propionate (A) is prepared in 98% yield from ethyl acetamidocyanoacetate , gramine (3-dimethylamino-methyl indole) , sodium, ethanol and ethyl iodide according to the procedure described by Albertson et al. (49). OL-Tryptophan is prepared by alkaline hydrolysis of (A). Other practicable syntheses of tryptophan which have been reported recently are outlined in the following equations. 

Cross-Hnked proteins produce unusual amino acids by proteolysis. Bound lysine yields lysinoalanine (2-121), cysteine lanthionine (2-122) and ornithine ornithinoalanine (2-123). Bound ornithine forms in proteins (together with urea) by alkaline hydrolysis of arginine. In the same way, many other amino acids (arginine, histidine, threonine, serine, tyrosine and tryptophan) and also amines and ammonia react with dehydroprotein. 

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. 

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