Determination of tryptophan as the reduced derivative by acid hydrolysis and chromatography

A new procedure for the analyses of tryptophan and the total amino acid composition of proteins was based on the observations that pyridine borane reduces tryptophan in trifluoroacetic acid, while other amino acids remain intact [M. Kurata, Y. Kikugawa, T. Kuwae, I. Koyama, and T. Takagi (1980) Chem. Pharm. Bull. 28, 2274-2275; W.S.D. Wong, D.T. Osuga, and R.E. Feeney (1984) Anal. Biochem. 139, 58-67]. Concentrated HCl was used instead of trifluoroacetic acid for analytical purposes. The products were stable to hydrolysis in 6 N HCl, and the reduction did not interfere with hydrolysis and subsequent analyses. Quantitative recovery was achieved with most proteins when they were subjected to acid reduction in ice-cooled concentrated HCl with two incremental additions of pyridine borane. The reaction was terminated after 10 min by dilution with an equal volume of H2O, vacuum sealing, and hydrolyzing at 110 degrees C for 22 h. The yields of the expected values for cytochrome c, catalase, bovine serum albumin, subtilisin BPN', trypsin, chymotrypsin, beta-lactoglobulin, lysozyme, and pepsin were obtained. Ovotransferrin and ovalbumin, however, yielded values for tryptophan lower than literature values. With two different ion-exchange methods, the recoveries of all other amino acids were comparable to those obtained by acid hydrolysis with 6 N HCl. Since the same hydrolysate can be analyzed for both tryptophan and all the other amino acids, the procedure is a more convenient method than those requiring separate determinations. Initial results indicate that the method may be applied to high-performance liquid chromatographic procedures with adaptations of the protocols if necessary.     

Acid hydrolysis of protein in a microcapillary tube for the recovery of tryptophan

The acid hydrolysis of proteins was miniaturized and simplified by employing microcapillary tubes (100 microl in volume) with 6 M HCl containing 1% 2-mercaptoethanol and 3% phenol for an amino acid compositional analysis. The method not only eliminated the laborious evacuation step for the hydrolysis tube but also decreased the destruction of tryptophan during hydrolysis. The recovery of tryptophan was 79% by acid hydrolysis at 145 degrees C for 4 h. Since the acid mixture could be removed under vacuum, the hydrolysate was subjected to an amino acid analysis without neutralization or dilution.     

Recovery of tryptophan from 25-minute acid hydrolysates of protein

It was found that thioglycolic acid prevents destruction of tryptophan during rapid hydrolysis of protein with a trifluoroacetic acid/HCl mixture (1:2, v/v) at 166 degrees C for 25 or 50 min. The addition of 5% (v/v) thioglycolic acid gave the maximum tryptophan recovery (88.3%) for a 25-min hydrolysate of lysozyme. Tryptophan recoveries varied slightly among three different proteins; 88% for lysozyme, 73% for alpha-chymotrypsinogen A, and 85% for apomyoglobin. However, when extrapolated to zero time, the values were close to one another: 94, 87, and 88%, respectively. The addition of thioglycolic acid was also advantageous for recovering amino acids other than tryptophan. Particularly, yields of carboxymethylcysteine and methionine were greatly improved. This modified rapid hydrolysis method gave satisfactory results without the need for separate analyses of tryptophan and cysteine, provided proteins were reduced and carboxymethylated prior to hydrolysis.     

5-Methyltryptophan: An internal standard for tryptophan determination by ion-exchange chromatography

The use of 5-methyltryptophan as an internal standard to facilitate tryptophan determination is described. Protein is hydrolyzed in the presence of 5-methyltryptophan for 18 hr at 120° in 5.0n NaOH in the absence of oxygen and in the presence of starch or thiodiglycol as an antioxidant. Ion-exchange chromatography of the hydrolysate on Durrum DC-2 resin using pH 5.43 citrate (0.175n Na⁺) completely resolved tryptophan and 5-methyltryptophan from one another, other amino acids, and artifacts of the alkaline hydrolysate. The chromatographic conditions and stability of tryptophan and 5-methyltryptophan were established initially by demonstrating quantitative recovery of both amino acids that had been added prior to hydrolysis of ribonuclease A, a protein devoid of tryptophan. The tryptophan content of several well-characterized proteins was determined, and the results, after correction to 100% recovery of 5-methyltryptophan, agreed well with values obtained by established procedures.     

Tryptophan micro-scale determinations by rapid hydrolysis

A rapid acid-hydrolysis micro method was developed for the accurate determination of the tryptophan contents of proteins. Sample protein (5 micrograms) was hydrolysed in 30 microliters of 3M mercaptoethanesulfonic acid at 166 degrees C for 25 min, resulting in total hydrolysis of the peptide bonds. The subjection of the hydrolysate to an amino-acid analyser revealed the amino-acid composition, including the content of tryptophan with a high recovery of 92%.     

High-performance liquid chromatography and ultraviolet spectrophotometry for quantitation of tryptophan in barytic hydrolysates

A procedure for quantitation of tryptophan in feedstuffs is described. It is based on barytic hydrolysis of material at 125 degrees C for 16 h, acidification of hydrolysate to pH 3 with HCl, high-performance liquid chromatography on Nova Pak C18 (Waters Assoc.), and spectrophotometric determination of tryptophan at 280 nm. The recovery of tryptophan from lysozyme added to samples ranges from 98.7 to 100%.     

Detoxification of dilute acid hydrolysates of lignocellulose with lime

The hydrolysis of hemicellulose to monomeric sugars by dilute acid hydrolysis is accompanied by the production of inhibitors that retard microbial fermentation. Treatment of hot hydrolysate with Ca(OH)(2) (overliming) is an effective method for detoxification. Using ethanologenic Escherichia coli LY01 as the biocatalyst, our results indicate that the optimal lime addition for detoxification varies and depends on the concentration of mineral acids and organic acids in each hydrolysate. This optimum was shown to be readily predicted on the basis of the titration of hydrolysate with 2 N NaOH at ambient temperature to either pH 7.0 or pH 11.0. The average composition of 15 hydrolysates prior to treatment was as follows (per L): 95.24 +/- 7.29 g sugar, 5.3 +/- 2.99 g acetic acid, 1.305 +/- 0.288 g total furans (furfural and hydroxymethylfurfural), and 2.86 +/- 0.34 g phenolic compounds. Optimal overliming resulted in a 51 +/- 9% reduction of total furans, a 41 +/- 6% reduction in phenolic compounds, and a 8.7 +/- 4.5% decline in sugar. Acetic acid levels were unchanged. Considering the similarity of microorganisms, it is possible that the titration method described here may also prove useful for detoxification and fermentation processes using other microbial biocatalysts.     

A procedure for the separation and quantitation of tryptophan and amino sugars on the amino acid analyzer

Tryptophan, 5-methyl tryptophan, glucosamine, and galactosamine can be separated from each other and hydrolysis products including lysinoalanine by chromatography on a 6 × 260-mm column of W-3H resin. The column is developed at 70°C for 20 min with pH 3.95 (0.4 Na⁺) buffer, followed by pH 6.4 (1 Na⁺) buffer for 55 min using a Beckman 119 CL amino acid analyzer. The recovery of the internal standards, 5-methyl tryptophan and galactosamine, can then be used to correct for tryptophan and glucosamine losses, respectively. The procedure uses the column and buffers normally employed for protein hydrolysate analysis and does not require additional resin columns, special buffers, or flow rate changes.     

Determination of deuterium-labeled tryptophan in proteins by means of high-performance liquid chromatography and thermospray mass spectrometry

In order to develop direct methods for determining the extent of metabolic incorporation of isotopically labeled amino acids into a protein, the determination of deuterated tryptophan in [2H5]tryptophan-bacteriorhodopsin was investigated. The isotopically modified protein was subjected to alkaline hydrolysis. After phenyl isothiocyanate derivatization of the hydrolysate, the mixture was separated by reversed-phase liquid chromatography. Field desorption mass spectrometry and thermospray mass spectrometry were investigated for their ability to determine the ratio between [2H5]tryptophan and total tryptophan in the collected fractions. In order to check the procedure a set of known tryptophan/[2H5]tryptophan mixtures were passed through the same derivatization, HPLC separation, and lyophilization procedure as used for the biological samples.     

A CHROMATOGRAPHIC STUDY OF HYDROLYSIS IN THE FEULGEN NUCLEAL REACTION

In a study on Feulgen hydrolysis of frozen-dried alcohol-fixed lily anthers, a chromatographic technique was developed to analyze the acid hydrolysate for some of the degradation products of nucleic acid. Hydrolysis was accomplished by 10 per cent perchloric acid at 20°C., and a typical hydrolysis time-Feulgen intensity curve was obtained, with maximum staining occurring at 19 hours. Microphotometric measurements indicated that the amount of stain per nucleus was no different from amount in nuclei fixed and hydrolyzed by more conventional procedures. Uracil-containing material (from ribonucleic acid) was almost completely separated from thymine-containing material (deoxyribonucleic acid) of tissue sections by acid treatment for 1½ hours. Adenine (purines), as the base, was effectively all removed from the deoxyribonucleic acid at the time of optimum hydrolysis. Detectable amounts of thymine-containing material appeared in the hydrolysate shortly after the onset of hydrolysis; and the amount increased rapidly with increased hydrolysis time. At the time of optimum hydrolysis approximately two-thirds of the total deoxyribonucleic acid thymine was lost. The removal of these thymine-containing fragments was linear with respect to time during the first 24 hours and occurred at a relatively high rate. Removal after 24 hours was also linear but was at a markedly lower rate. These results would suggest that two kinds of deoxyribonucleic acid exist in lily anthers; an acid-labile fraction amounting to approximately three-fourths of the total, and an acid-resistant fraction making up the remainder. In the Feulgen procedure much of the labile fraction is lost by the time of optimum hydrolysis and is not stained; most of the stable fraction remains in the tissue and is stained. In light of these findings the use of the Feulgen method as a means of determining cytochemically relative amounts of deoxyribonucleic acid in nuclei by measuring their Feulgen dye content was discussed.     

Nature of adenosine triphosphatase accelerating peptide from hydrolysate of fur seal muscle.

Ultrafiltered fur seal muscle hydrolysate was divided into eleven fractions by gel filtration on Sephadex G-15. One of the fractions (Fraction G9) accelerated the ATPase activity of carp myosin B to a rate about two-fold faster than that of the control. Fraction G9 showed a single ninhydrin spot in its silica gel thin layer chromatograph, and gave a positive test for tryptophan by the p-dimethylaminobenzaldehyde method, while tests for tyrosine, and for arginine were negative. The ion exchange amino acid analysis of its acid hydrolysate showed a predominant content of lysine, nearly equivalent to the amount of tryptophan determined from its UV absorbancy and the p-dimethylaminobenzaldehyde method. The N-terminal amino acid analysis gave di-DNP-Lys as the sole DNP-amino acid. The structure of the ATPase accelerating peptide fraction, Fraction G9, was deduced to be Lys-Trp.     

Changes in activity coefficient gamma(w) of water and the foaming capacity of protein during hydrolysis

The changes in the interaction between food proteins and water and in their surface functional property during enzymatic hydrolysis were investigated. Ovalbumin, a soy protein isolate (SPI), and casein were hydrolyzed with trypsin, and the degree of hydrolysis, water activity a(w), and foaming capacity of each hydrolysate were measured. Ovalbumin showed the minimum value for a(w), and the values for SPI and casein progressively decreased during hydrolysis. Therefore, the activity coefficient of water, gamma(w) (=a(w)/x(w), where x(w) is the mole fraction of water) was obtained to remove the influence of mole change and to examine the interaction of protein hydrolysates with water. In order to calculate x(w) in a sample during protein hydrolysis, a method for roughly estimating the number of moles of the protein hydrolysate in a solution was developed. The strategy was to modify the TNBS (2,4,6-trinitrobenzenesulfonic acid) method and to combine this method with the modified Ellman method and the determination of lysine by an amino acid analyzer. During enzymatic hydrolysis, each protein sample showed a minimum gamma(w) value and maximum foaming capacity.     

Digestion of native collagen, denatured collagen, and collagen fragments by extracts of rat liver lysosomes.

Extracts of highly purified lysosomes from rat liver were examined for their ability to degrade native collagen and thermally denatured collagen at pH values between 3.5 and 7.0. After a 24-h digestion at 36 degrees with the lysosomal extract at a pH of 5.5 or lower (collagen/lysosomal protein; 2/1 or 8/1), both native and denatured collagen were degraded to an extent equivalent to 60 to 70% of that observed upon total acid hydrolysis in 6 N HCl as measured by the ninhydrin reaction (570 nm). At a pH of 6.0, native collagen and denatured collagen were degraded by the mixture of lysosomal proteinases to 11% and 40% of total acid hydrolysis, respectively. At pH 6.5 AND 7.0, the corresponding values were 3% versus 33% and 0.3% versus 11%, respectively. Fragments of collagen (TCA and TCB) are produced when mammalian collagenase degrades native collagen at 25 degrees. These fragments were degraded by the lysosomal extract at 36 degrees to an extent equivalent to 28% and 8% of total acid hydrolysis at pH 6.5 and 7.0, respectively. The experiments at pH 6.5 and 7.0 were done using a collagen/lysosomal protein ratio of 2/1. At pH 5.0 (a pH which is found within secondary lysosomes), the lysosomal extracts degraded collagen to a mixture of free amino acids and small peptides. Amino acid analysis established that approximately 30% of the amino acid residues of the collagen appeared in the lysosomal hydrolysate as free amino acids. Hydroxyproline and perhaps hydroxylysine were the only amino acids found in collagen which did not appear at least to some extent as the free amino acid in this hydrolysate.     

A new acid hydrolysis method for determining tryptophan in peptides and proteins

Three different methods for hydrolysis and determination of amino acid composition of peptides and proteins were compared. We found, that the method of Matsubara and Sasaki (using 6N HCl and thioglycolic acid) gives comparatively low recoveries for tryptophan, while Liu and Chang's method, using p-toluenesulfonic acid and tryptamine, is more suitable. To eliminate the difficulties of the latter method, we used mercaptoethane-sulfonic acid, which, in the concentration used, results in total hydrolysis of peptide bonds within 22 hr and gives very high tryptophan recoveries. Both sulfonic acid methods were used for hydrolysis of the pentapeptide “pentagastrine” as well as of the proteins lysozyme, cytochrome c, and chymotrypsine. Their amino acid composition was determined using an automatic amino acid analyzer. Similarly to the p-toluenesulfonic acid method, the results of our method are totally reliable only for pure peptides and proteins, though the results obtained with our method using samples containing carbohydrates are better than those of all earlier methods.     

Recovery of tryptophan in peptides and proteins by high-temperature and short-term acid hydrolysis in the presence of phenol

The addition of 3% (w/v) phenol to 6 M HCl largely prevented the destruction of tryptophan during rapid hydrolysis of peptides and proteins at 166 degrees C for 25 min or at 145 degrees C for 4 h. This hydrolysis procedure was advantageous for amino acid microanalysis using conventional high-performance liquid chromatography with a precolumn derivatization technique. The recovery of tryptophan from proteins was at least 80%. The addition of phenol also improved the recovery of methionine and carboxymethylcysteine. The amount of tryptophan in proteins electroblotted onto a polyvinylidene difluoride membrane was determined by this method.     

A simple colorimetric method for the determination of tryptophan

A simple, sensitive, and reproducible colorimetric method for the determination of tryptophan in amounts as low as 2 μg is described. It is based on the oxidation of tryptophan by sodium nitrite and the coupling of the oxidized product to the leucodye N-1-(naphthyl)ethylenediamine dihydrochloride. The purple-pink product has an absorption maximum at 550 nm. There is no interference by carbohydrates, other amino acids, neutral salts, or a number of other compounds likely to be found in tissue hydrolysates. A number of indole derivatives including indole-3-acetic acid also react to give a colored product. Dipeptides containing tryptophan are much less reactive than free tryptophan; hence proteins must be hydrolyzed completely for the method to be useful. The assay is carried out at room temperature and can be modified easily to increase or decrease its sensitivity. It has been employed to determine the tryptophan content of a number of proteins following alkaline hydrolysis. Generally, values obtained were in close agreement with values reported in the literature.     

Influences of Various Amino Acids on Tryptophan-Mediated Control of the Tryptophan Biosynthetic Enzymes in Escherichia coli

Lysates of Escherichia coli Ymel obtained from cultures grown in the absence of tryptophan in minimal medium supplemented with 0.1% casein hydrolysate show an approximate fivefold increase in steady-state specific activity of both anthranilate synthetase and tryptophan synthetase A protein relative to cultures grown in nonsupplemented medium. In the presence of repressing levels of exogenous tryptophan, growth of cultures in casein hydrolysate-supplemented medium results in a noncoordinate enhancement of repression of 10-fold for anthranilate synthetase and twofold for tryptophan synthetase A protein. Similar, but less pronounced, effects are shown for strain W3110. Strains possessing tryptophan regulator gene mutations do not exhibit this first effect, but do yield an approximate twofold decrease in specific activity of both enzymes when grown in medium supplemented with tryptophan and casein hydrolysate. A stimulation of derepression of both enzymes in strain Ymel equivalent to that induced by casein hydrolysate can be reproduced by growth in minimal medium supplemented with threonine, phenylalanine, tyrosine, serine, glutamic acid, and glutamine. Doubling time in this medium is not significantly different from that in minimal medium. An enhancement of repression which partially mimics that observed on growth in medium supplemented with tryptophan plus casein hydrolysate is obtained when Ymel is grown on medium supplemented with tryptophan plus methionine. Threonine or phenylalanine plus tyrosine as separate medium supplements are independently capable of producing a 1.4-fold or 3.4-fold stimulation, respectively, but in combination only the phenylalanine plus tyrosine effect is manifested unless serine and glutamic acid or glutamine are included. Our data show that expression of the tryptophan biosynthetic enzymes can be significantly influenced in vivo as a result of growth in medium supplemented with a variety of amino acids.     

Protein hydrolysate from visceral waste proteins of Catla (Catla catla): optimization of hydrolysis conditions for a commercial neutral protease

Protein hydrolysate was prepared from visceral waste proteins of an Indian freshwater major carp, Catla catla. Hydrolysis conditions (viz., time, temperature and enzyme to substrate level) for preparing protein hydrolysates from the fish visceral waste proteins using in situ pH of the visceral mass were optimized by response surface methodology (RSM) by employing a factorial design. The regression coefficient close to 1.0, observed during both experimental and validation runs, indicated the validity of prediction model. An enzyme to substrate level of 1.25 % (v/w), temperature of 55 degrees C and a hydrolysis time of 165 min were found to be the optimum conditions to obtain a higher degree of hydrolysis of >48% using multifect-neutral. The amino acid composition of the protein hydrolysate prepared using the optimized conditions revealed that the protein hydrolysate was similar to FAO/WHO reference protein. The chemical scores computed indicated methionine to be the most limiting amino acid. The protein hydrolysate has the potential for application as an ingredient in balanced fish diets.     

HPLC determination of tryptophan in foodstuffs using barium hydroxide hydrolysis at 140°C

Summary Hydrolysis of samples of food and feedstuffs with 2.7 N Ba(OH)₂ at 140°C for 4 h was tested for the recovery of tryptophan. On the basis of 100% yield for the tryptophan content, corresponding to samples determined after 16h-hydrolysis at 125°C, the recovery averaged 98.7 ± 0.9% SD or 99.4 ± 2% depending on how the bulk or aliquot of hydrolysate was analysed (conventional or simplified procedure). Tryptophan can be assayed by high performance liquid chromatography and fluorescence detection within 8h, 4h-hydrolysis at 140°C and a simplified procedure.     

Optimization of enzymatic hydrolysis of visceral waste proteins of Catla (Catla catla) for preparing protein hydrolysate using a commercial protease

Protein hydrolysate was prepared from visceral waste proteins of Catla (Catla catla), an Indian freshwater major carp. Hydrolysis conditions (viz., time, temperature, pH and enzyme to substrate level) for preparing protein hydrolysates from the fish visceral waste proteins were optimized by response surface methodology (RSM) using a factorial design. Model equation was proposed with regard to the effect of time, temperature, pH and enzyme to substrate level. An enzyme to substrate level of 1.5% (v/w), pH 8.5, temperature of 50 degrees C and a hydrolysis time of 135 min were found to be the optimum conditions to obtain a higher degree of hydrolysis close to 50% using alcalase. The amino acid composition of the protein hydrolysate prepared using the optimized conditions revealed that the protein hydrolysate was similar to FAO/WHO reference protein. The chemical scores computed indicated methionine to be the most limiting amino acid. The protein hydrolysate can well be used to meet the amino acid requirements of juvenile common carp and hence has the potential for application as an ingredient in balanced fish diets.