Assay Of Tryptophan Research Articles

Rapid Tryptophan Assay as a Screening Procedure for Quality Protein Maize.

Tryptophan is an essential amino acid deficient in cereals, especially maize. However, maize (Zea mays L.) is the main source of protein in some developing countries in Africa and Latin America. In general, the nutritional profile of cereals is poor, because they are deficient in essential amino acids such as tryptophan and lysine due to a relatively higher proportion of alcohol-soluble proteins. Quality protein maize (QPM) has been developed through genetic manipulation for the nutritional enrichment of maize to address these problems. Nevertheless, methods for protein, lysine and tryptophan are time-consuming and require relatively large amounts of samples. Therefore, we have advanced here a simple, cheap, fast, reliable and robust procedure for the determination of protein and tryptophan in the same biuret supernatant, which can also be used for chemical characterization of other cereals. Samples of 50 mg maize ground to pass through a 0.1 mm screen were sonicated for 5 min. in eppendorf vials with 1.5 mL of a biuret reagent each. After centrifugation and protein determination by biuret, 0.2 mL of supernatant was treated with 0.8 mL of a tryptophan reagent. Both total protein and tryptophan can be determined in microplates at 560 nm to speed up the measurements. The main advantage of the new micro-method is the rapid estimation of the nutrient quality of maize samples by a single weighing of a small amount of valuable plant materials.

Identification and validation of the mode of action of the chalcone anti-mycobacterial compounds

ObjectivesThe search for new TB drugs has become one of the great challenges for modern medicinal chemistry. An improvement in the outcomes of TB chemotherapy can be achieved by the development of new, shorter, cheap, safe and effective anti-TB regimens. MethodsChalcones (1a-1o) were synthesized and evaluated for their antimycobacterial activity against Mycobacterium bovis BCG using growth inhibition assays. Compound 1a was selected as a ‘hit’ compound. The mode of action of compound 1a, was identified by mycolic acid methyl esters (MAMEs) and fatty acid methyl esters (FAMEs) analysis using thin layer chromatography. Dose dependent experiments were conducted by over-expressing components of FAS-II in M. bovis BCG to confirm the target. Ligand binding using intrinsic tryptophan assay and molecular docking were used to further validate the target. ResultsMAMEs and FAMEs analysis showed dose-dependent reduction of MAMEs with the overall abundance of FAMEs suggesting that compound 1a targets mycolic acid biosynthesis. Direct binding of 1a to InhA was observed using an intrinsic tryptophan fluorescence binding assay, and a 2-fold IC50 shift was observed with an InhA overexpressing strain confirming InhA as the cellular target. ConclusionThe chalcone 1a exhibits potent antimycobacterial activity, displays a good safety profile and is a direct inhibitor of InhA, a key component in mycolic acid synthesis, validating this series for further anti-TB drug development.

Protein engineering for improving the thermostability of tryptophan oxidase and insights from structural analysis.

l-Tryptophan oxidase, VioA from Chromobacterium violaceum, which has a high substrate specificity for tryptophan, is useful for quantitative assay of tryptophan. However, stability of wild type VioA is not enough for its application in clinical or industrial use. To improve the thermal stability of the enzyme, we developed a VioA (C395A) mutant, with higher stability than wild type VioA. The VioA (C395A) exhibited similar specificity and kinetic parameter for tryptophan to wild type. Conventionally, the quantity of tryptophan is determined by instrumental methods, such as high-performance liquid chromatography (HPLC) after pre-column-derivatization. Using the mutant enzyme, we succeeded in the tryptophan quantification in human plasma samples, to an accuracy of <2.9% when compared to the instrumental method, and to a precision of CV <3.2%. To analyse the improvement in storage stability and substrate specificity, we further determined the crystal structures of VioA (C395A) complexed with FAD, and with FAD and tryptophan at 1.8 Å resolution.

Determination of Tryptophan in Pharmaceutical Formulations Using a Sequential Injection–Zone Fluidic–Chemiluminescence Tubular Reactor

ABSTRACTTryptophan is an important amino acid for humans with a significant role in cell metabolism. Depletion of tryptophan in the human body may contribute to diseases and development of disorders among the human population. It is, therefore, very important to have a reliable, stable, sustainable, and cost-effective analytical method for the determination of tryptophan. Tryptophan was determined using sequential injection–zone fluidics analysis with luminol–hydrogen peroxide and the Firefly with its unique liquid core waveguide flow-cell design as chemiluminescence tubular reactor with a high-sensitivity photomultiplier tube. This was based on an intense chemiluminescence formation of tryptophan in luminol–hydrogen peroxide inside the tubular reactor for measurement. The chemiluminescence intensity was linear with tryptophan in the range of 1.0 × 10−6 to 1.0 × 10−3 mol/L, and the limit of detection was 7.5 × 10−7 mol/L. The precision for the method was 3.6% (relative standard deviation) for six measurements of 1.0 × 10−4 mol/L tryptophan. The proposed method has been used to determine tryptophan in pharmaceutical formulations. The system is relatively fast for online assays. Eighty seconds are required to complete one cycle providing a throughput of 45 samples/h. The proposed sequential injection analysis–zone fluidics–chemiluminescence system for the assay of tryptophan in certain specific pharmaceutical capsules is simple, reliable, sustainable, and convenient with relatively low-cost consumption of reagents.

A tryptophan assay based on the glassy carbon electrode modified with a nano-sized tryptophan-imprinted polymer and multi-walled carbon nanotubes

A new nanostructured molecularly imprinted polymer (nano-MIP), possessing tryptophan compatible cavities, was synthesized using 1-(allyloxy)-4-nitrobenzene as an innovative functional monomer.

VPO1 mediates oxidation of LDL and formation of foam cells.

Deposition of oxidized-LDL in vascular walls is essential in the initiation of atherosclerosis. Oxidation of LDL has been attributed to myeloperoxidase as its generation of potent oxidants. However, the exact mechanism of LDL oxidation and foam cell formation in atherosclerosis remains to be elucidated. Vascular peroxidase-1 (VPO1), a newly-identified heme-containing peroxidase, is primarily expressed in cardiovascular systems, and secreted into the circulation. The present study evaluates VPO1-mediated LDL oxidation and its role in atherosclerosis. VPO1 was first demonstrated binding to LDL. VPO1-mediated oxidation of proteins and lipids in LDL was verified by a variety of methods including immunoblot analysis, free tryptophan assay, UV absorbance, and thiobarbituric acid assay. VPO1-oxidized LDL caused accumulation of LDL in monocyte-like cells and promoted formation of foam cells. Administration of inflammation factors, LPS or TNF-α, induced increasing expression of VPO1 in aorta and secretion to plasma. TNF-α also promoted formation and retention of VPO1-oxidized LDL in aortic walls. Our data suggest that VPO1 contributes to oxidation and retention of LDL in vessel walls, and formation foam cells, indicating VPO1 as a novel potential mediator of atherosclerosis.

Selective tryptophan determination using tryptophan oxidases involved in bis-indole antibiotic biosynthesis

A novel tryptophan assay was developed using tryptophan oxidases. Although many l-amino acid oxidases (LAAOs) have been reported to catalyze tryptophan oxidation, most of them have broad substrate specificity and oxidize multiple amino acids besides tryptophan. To obtain a tryptophan-specific LAAO, we focused on bis-indole antibiotic biosynthesis, a bacterial secondary metabolic pathway. A putative LAAO from Streptomyces sp. TP-A0274, StaO involved in staurosporine biosynthesis, was heterologously expressed, biochemically characterized, and shown to serve as a selective tryptophan oxidase for the first time. In addition, another LAAO, VioA involved in violacein biosynthesis in Chromobacterium violaceum, was characterized for comparison with StaO. Interestingly, StaO and VioA share similar properties, namely narrow substrate specificity and high affinity for l-tryptophan, despite the phylogenetic distance between these enzymes. Owing to these features, uncommon among known LAAOs, StaO and VioA assays can be used for selective and accurate quantification of l-tryptophan via a coupled colorimetric reaction. Indeed, StaO and VioA assays provided tryptophan concentrations in human plasma as accurately as those obtained by high-performance liquid chromatography. Therefore, these enzymes were clearly shown to offer an effective method for determining tryptophan in biological samples rapidly, inexpensively, and accurately. The results shown here also suggest the possibility of metabolism-oriented screening as a strategy to obtain enzymes highly selective for individual biomolecules.

Highly sensitive and selective tryptophan colorimetric sensor based on 4,4-bipyridine-functionalized silver nanoparticles

This paper reports a novel colorimetric sensor for tryptophan based on 4,4-bipyridine-functionalized silver nanoparticles (4-DPD-Ag NPs) which were characterized by IR spectroscopy, ultraviolet–visible spectroscopy (UV–vis), and transmission electron microscopy (TEM). The newly synthesized 4-DPD-Ag NPs are yellow in color due to the intense surface plasmon absorption band centered at 390 nm. In the presence of tryptophan, the yellow 4-DPD-Ag NPs solution turns to red accompanying a dramatic surface plasmon absorption band centered at 556 nm. However, the sensitivity of 4-DPD-Ag NPs towards other neutral amino acids, including leucine, threonine, valine, phenylalanine, tyrosine, proline, serine, pyroglutamic acid and cysteine can be negligible. This highly selective sensor allows a direct and rapid quantitative assay of tryptophan with a colorimetric limited detection concentration of 2 × 10−5 M. The possible underlying mechanism is discussed.

Ion-exchange HPLC assay of tryptophan in breast milk of mothers with three months breast-feeding

Tryptophan is essential for the biosynthesis of biogenic amines. The correct ingestion of this amino acid is vital in the newborn for correct growth. An ion-exchange HPLC method is described for the assay of tryptophan in breast milk. The protein was hydrolysed by barium hydroxide and then assayed by the indicated chromatographic technique, establishing the conditions for the analysis. The values obtained confirmed the utility of the method for determining the tryptophan content in breast milk. The concentration of tryptophan found in the study population in mothers who had been breast-feeding for three months was 10.02 mgl−1 g−1 of protein.

Microbial assay for tryptophan using silicon-based transducer

A potentiometric microbial assay for tryptophan was devised using silicon-based transducer and auxotrophic bacteria, and the chemical performances are discussed. Escherichia coli WP2, an auxotrophic mutant requiring tryptophan for its growth, was used as tryptophan recognition element, and extracellular pH changes resulting from metabolic activity was monitored by the light-addressable potentiometric sensor (LAPS). The potentiometric sensor detected the acidification of the medium by E. coli WP2, and the acidification rate was dependent on tryptophan concentration. The acidification rate was approximately linear between 0 and 15 μM of tryptophan. Furthermore, the cells were immobilized on the electrode, and the tryptophan sensor was characterized. The sensor responded to the addition of tryptophan, and the sensor could measure tryptophan in the range of 0–12 μM.

In-vitro effects of antimicrobial agents on Pseudomonas aeruginosa biofilm formation.

The in-vitro effects of ten antimicrobial agents on the biofilm formation of Pseudomonas aeruginosa were investigated. The production of alginic acid by mucoid P. aeruginosa cells cultured in agar media with sub-MICs of antimicrobial agents was quantified by high-performance liquid chromatography. Alginic acid production was inhibited by 1/4 MIC of minocycline (P < 0.002) and tobramycin (P < 0.02), and by 1/256-1/1/64 MIC of macrolides (erythromycin, clarithromycin, roxithromycin, and rokitamycin) and clindamycin (P < 0.02), compared with drug-free controls. Piperacillin, ceftazidime, and ofloxacin did not inhibit alginic acid production. The production of exopolysaccharide by non-mucoid P. aeruginosa cells grown on silicone plates in sub-MICs of antimicrobial agents was determined by quantitative tryptophan assay. Exopolysaccharide production was inhibited by 1/16 MIC of macrolides and clindamycin, but not by other antimicrobial agents. Electron microscopy showed that biofilm formation by mucoid and non-mucoid type P. aeruginosa strains was inhibited by sub-MICs of erythromycin and correlated with the in-vitro production of alginic acid and exopolysaccharide. These results suggest that sub-MICs of macrolides and clindamycin suppress biofilm formation by P. aeruginosa and that intractable chronic respiratory tract infections due to P. aeruginosa might be prevented.

Quantitative determination of tryptophan in food and feedstuffs: Practical considerations on autoclaving samples for hydrolysis

Hydrolysis of wheat flour and soybean meal in the presence of 2.7 N barium hydroxide at 110 o C for 16 h, at 125 o C for 8 h, or at 140 o C for 4 h led to a complete release of tryptophan, as determined by a conventional procedure involving hydrolysate neutralization by HCl, reversed-phase high-performance chromatography, and quantitation by fluorometry. These data together with the features of two types of benchtop autoclaves used for hydrolysis allow one to specify the more practical conditions for this process, constituting an essential step for the quantitative assay of tryptophan

Assay of tryptophan 2,3-dioxygenase using liver slices and high-performance liquid chromatography

Liver tryptophan 2,3-dioxygenase (TDO) activity was determined by high-performance liquid chromatography. The enzyme activity was expressed as the sum of N-formyl- l-kynurenine (FK) and l-kynurenine (KYN) produced from l-tryptophan (TRY) by liver slices. FK and KYN were detected spectrophotometrically at 254 nm after their separation on a reversed-phase C 18 column. KYN formation proceeded according to zero-order kinetics for at least 4 h with 15 m M TRY at 37°C. The apparent Michaelis constant was 1.2 m M TRY with a maximum velocity of 59 pmol min −1 mg −1 wet weight. The method was applied for TDO assay in mice treated with the organophosphorus acid triester diazinon. Kynurenine formamidase inhibition by diazinon resulted in reduced KYN formation, FK accumulation, and moderate TDO increase.

Association of cell-adherent glycocalyx and endocarditis production by viridans group streptococci

To assess the role of glycocalyx production in the pathogenesis of endocarditis caused by viridans group streptococci in adult patients, glycocalyx production was examined for 49 blood culture isolates. The tryptophan assay, a quantitative spectrophotometric test, was used to measure cell-adherent glycocalyx production. Absorbance values of the isolates that produced endocarditis were significantly higher (means, 0.166 versus 0.060 [P less than 0.001]). At a breakpoint of absorbance of 0.120, the sensitivity of the test was 0.83, the specificity was 0.96, and the predictive value was 0.95. These data suggest that the in vitro tryptophan assay of glycocalyx production by viridans group streptococci has potential value as a predictor of clinical pathogenicity.

The use of Streptococcus zymogenes for estimating tryptophan and methionine bioavailability in 17 foods.

As part of a cooperative study assessing amino acid bioavailability and/or protein quality, the provisional method of Boyne et al. (Brit J Nutr 21: 181-206) was used to assay 17 protein sources for methionine and tryptophan availability with S. zymogenes. Pronase was used as the predigesting enzyme. Product composition was found to affect reproducibility. The microbial assay results correlated positively with results from rat growth studies on the same foods (p = 0.05), and were generally accurate in identifying products of lower protein quality. Defatting four high-fat products increased microbial values in the methionine assay, but only the chicken franks and the sausage values in the tryptophan assay. Heating non-fat milk increased methionine values slightly. Low values for rolled oats were further reduced by finer grinding.

A convenient and highly sensitive spectrophotometric assay for tryptophan 2,3-dioxygenase

A highly sensitive assay for tryptophan dioxygenase based on the formation of an azo-dye derivative of kynurenine is described combining simplicity and excellent reproducibility. The assay procedure provides optimal conditions for enzyme activation, diazotization of kynurenine, and color development. Furthermore, it renders the measurement of tryptophan dioxygenase activity independent of formamidase, in contrast to many other methods reported, which thus may lead to misinterpretations, if no additional precautions against rate limitation of that enzyme are taken. These improvements make the assay particularly suited for small samples of any tissue, irrespective of endogenous formamidase activity, as well as for more purified enzyme preparations, even at very low activities.

A new method for the assay of tryptophan 2,3-dioxygenase

The enzyme is found in many organisms, but in mammals is restricted to the liver. As a frequent with regulatory enzymes in liver, tryptophan 2,3dioxygenase concentrations change markedly with variations in dietary and hormonal status [ 11. Basal activities are, however, very low and have presented problems in assaying. A number of procedures have been used for quantitating the enzyme, but are open to criticism on practical grounds. The formation of kynurenine (produced by the cleavage of formylkynurenine by an active formylase) can be followed spectrophotometrically in both continuous [2] and stopped [3] assays, but the sensitivity is low, and relatively large amounts of tissue are required. The presence of apeand holoforms of the enzyme necessitates a preincubation period with a haem compound, ascorbate and tryptophan before assay [4 1. This preincubation increases blank values and the assay is further complicated by absorbance increases attributable to reaction between ascorbate and other components [5]. A radiochemical assay based on D,L-[ring-2-r4C]-

Surfactant ion-pair high-performance liquid chromatography of tryptophan and some of its metabolites in biological fluids

A rapid, sensitive assay for tryptophan and some of its metabolites in urine, plasma and saliva has been developed using sodium dodecylsulphate as a pairing ion in a surfactant ion-pair high-performance liquid chromatography technique. The method is highly selective for tryptophan which is separated from its main indoleamine metabolites, 5-hydroxytryptophan, 5-hydroxytryptamine and 5-hydroxyindoleacetic acid, and from kynurenine. The usefulness of the assay has been demonstrated in plasma level and urinary excretion studies of orally administered tryptophan.

Quenching effects of various compounds on fluorometric assay of tryptophan.

Quenching effects of various compounds on fluorometric assay of tryptophan.

Indoleamine compartmentation in human blood

Improved methods are described for the fluorimetric determination of 5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), and tryptophan (TRP) in plasma, platelet and RBC blood compartments. The methods for 5-HT and 5-HIAA utilize ion exchange chromatography and allow simultaneous determinations to be made on the same blood sample. The tryptophan assay is a modification of an existing extraction method. All of the 5-HT in whole blood was found to be associated with the platelet fraction (122.6 ng/ ml), whereas all 5-HIAA was distributed between the plasma (78.3 ng/ml) and RBC (124.0 ng/ml) compartments. Tryptophan was found in all three compartments although the majority of this amino acid was in the plasma (4.3 μg/ml) and RBC (1.7 μg/ml) fractions.