Comparison of the Coomassie brilliant blue, bicinchoninic acid and Lowry quantitation assays, using non-glycosylated and glycosylated proteins.

The concentrations of several non-glycosylated and glycosylated recombinant and native proteins were determined by three widely used colorimetric methods: Coomassie brilliant blue, bicinchoninic acid and Lowry, and, for comparison, by amino acid composition analysis. The colorimetric methods gave results differing from the values derived from the amino acid analysis, in some cases by up to 60%. For the non-glycosylated recombinant proteins, the results were in relatively good agreement with each other and with the values determined on the basis of the amino acid analysis. The Coomassie blue method was strongly dependent on the hydrophobicity of the individual protein. The bicinchoninic acid method gave results closest to those of the amino acid analysis. For the glycosylated proteins, both recombinant and native, the Coomassie blue assay gave values lower, whereas the two other methods gave values higher than those determined on the basis of the amino acid analysis. The concentration of a recombinant interferon gamma receptor produced in two differently glycosylated forms was underestimated by the Coomassie blue assay and overestimated by the bicinchoninic acid and Lowry methods, while for the non-glycosylated form of the same protein, the three colorimetric methods delivered comparable values. The results suggest a potential interference of protein glycosylation with the colorimetric assays.     

Colorimetric microbial viability assay based on reduction of water-soluble tetrazolium salts for antimicrobial susceptibility testing and screening of antimicrobial substances

The applicability of a colorimetric microbial viability assay based on reduction of a tetrazolium salt {2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt [WST-8]} via 2-methyl-1,4-naphthoquinone (2-methyl-1,4-NQ) as an electron mediator for determining the susceptibility of various bacteria to antibiotics and screening antimicrobial substances was investigated. The measurement conditions, which include the effects of the concentration of 2-methyl-1,4-NQ, were optimized for proliferation assays of gram-negative bacteria, gram-positive bacteria, and pathogenic yeast. In antimicrobial susceptibility testing, there was excellent agreement between the minimum inhibitory concentrations determined after 8 h using the WST-8 colorimetric method and those obtained after 22 h using conventional methods. The results suggest that the WST-8 colorimetric assay is a useful method for rapid determination of the susceptibility of various bacteria to antibiotics. In addition, the current method was applied to the screening of bacteriocin-producing lactic acid bacteria and its efficiency was demonstrated.     

Thin-layer and gas chromatographic deterimination of alpha, epsilon- diaminopimelic acid in Escherichia coli

Both thin-layer (TLC) and gas-liquid chromatographic (GLC) methods are described for determining α,ε-diaminopimelic acid (DAP) in Escherichia coli cell hydrolyzates. The TLC method is both rapid and sensitive whereas the GLC technique is extremely sensitive but more time-consuming. In the latter method, DAP is converted to the trifluoroacetylethylpimelate derivative and then detected by using an electron capture detector. The particular strain of E. coli studied was found to contain 0.68% DAP.     

Ultrasensitive and selective colorimetric detection of uric acid using peroxidase mimetic activity of biogenic palladium nanoparticles

Uric acid (2,6,8-trihydroxypurine) is a metabolic product of purine, which is one of the important markers of human health. The development of a rapid, facile, highly sensitive, and selective method for uric acid detection is critical for the diagnosis of related diseases and is still a strategic challenge. In this study, we developed a highly sensitive and selective colorimetric assay for the detection of uric acid using biogenic palladium nanoparticles (Pd NPs). The synthesized nanoparticles were shown to acquire peroxidase mimetic activity that oxidized 3,3′,5,5′-tetramethylbenzidine and produced a blue colour in an assay. The developed colorimetric assay is instrument-free detection of uric acid with a limit of detection of 0.05 μM and a 1.11 μM limit of quantification (LOQ). This is the first report determining the LOQ for a colorimetric assay that gives the lowest quantity of analyte that can be evaluated with more precision under the specified conditions of the analysis. The developed assay had a linear response at low uric acid concentrations of 0.05 to 1 μM and a 0.99841 linear regression correlation coefficient. This colorimetric detection provides a rapid, cost-effective, and easy-to-use platform for the clinical diagnosis of uric acid biomarkers.     

Hydroxycinnamic acid esters of isocitric acid: Accumulation and enzymatic synthesis in Amaranthus cruentus

New hydroxycinnamic acid esters have been isolated from the cotyledons of Amaranthus cruentus. (E)-Caffeoylisocitric acid was identified as the major constituent and p-coumaroyl-and feruloylisocitric acids as minor ones on the basis of ¹H and ¹³C NMR spectroscopy. FAB mass spectrometry, high-performance liquid and thin-layer chromatography. The structure of caffeoylisocitric acid was confirmed by chromatographic comparison with synthetic material. The accumulation of the new hydroxycinnamoylisocitric acids and their enzymatic synthesis via the hydroxycinnamoyl-CoA thioesters as acyl donors are described. The caffeoyl-CoA-dependent acyltransferase showed a rapid transient increase in activity reaching ca 5 pkat per cotyledon pair (i.e. ca 390 pkat per mg protein) at day six of seedling development.     

Validation of a simple,rapid and cost effective method for the estimation of caprylic acid and sodium caprylate from biological products using NEFA-C kit

The method for the determination of caprylic acid and sodium caprylate from biological products was systematically validated using NEFA-C kit. The results obtained demonstrated that the kit method was simple, rapid, reliable, sensitive, reproducible and cost effective in comparison to the current methods i.e. colorimetric, High Performance Liquid Chromatography (HPLC) and Gas Chromatography (GC) methods. The assay exhibited excellent linearity, accuracy, precision and robustness. Mean recoveries ranged between 95 and 101.3% (n = 6). The proposed method was linear over the concentration range of 0.05–10 mM of caprylate with values of coefficient of regression being >0.99. Method showed sensitivity of 0.05 mM (7.21 μg/ml for caprylic acid and 8.31 μg/ml for sodium caprylate). The % Relative standard Deviation (%RSD) for intra and interprecision studies was less than 5%. In conclusion the validated method was successfully used in monitoring of processed bulk and final products generated during production of biological products thus laying emphasis on strict control of release criteria for biological products fractionated using caprylic acid.     

Photosensitized covalent organic framework as a light-induced oxidase mimic for colorimetric detection of uric acid

As large numbers of people are suffering from gout, an accurate, rapid, and sensitive method for the detection of gout biomarker, uric acid, is important for its effective control, diagnosis, and therapy. Although colorimetric detection methods based on uricase have been considered, they still have limitations as they produce toxic H₂O₂ and are expensive and not stable. Here, a novel uricase-free colorimetric method was developed for the sensitive and selective detection of uric acid based on the light-induced oxidase-mimicking activity of a new photosensitized covalent organic framework (COF) (2,4,6-trimethylpyridine-3,5-dicarbonitrile–4-[2-(4-formylphenyl)ethynyl]benzaldehyde COF [DCTP–EDA COF]). DCTP–EDA COF has a strong ability to harvest visible light, and it could catalyze the oxidation of 1,4-dioxane, 3,3′,5,5′-tetramethylbenzidine under visible light irradiation to produce obvious color changes. With the addition of uric acid, however, the significant inhibition of the oxidase-mimicking activity of DCTP–EDA COF remarkably faded the color, and thus uric acid could be colorimetrically detected in the range of 2.0–150 μM with a limit of detection of 0.62 μM (3σ/K). Moreover, the present colorimetric method exhibited high selectivity; uric acid level in serum samples was successfully determined, and the recoveries ranged from 96.5% to 105.64%, suggesting the high accuracy of the present colorimetric method, which demonstrates great promise in clinical analysis.     

Rapid and convenient determination of oxalic acid employing a novel oxalate biosensor based on oxalate oxidase and SIRE technology

A new method for rapid determination of oxalic acid was developed using oxalate oxidase and a biosensor based on SIRE (sensors based on injection of the recognition element) technology. The method was selective, simple, fast, and cheap compared with other present detection systems for oxalate. The total analysis time for each assay was 2-9 min. A linear range was observed between 0 and 5 mM when the reaction conditions were 30 degrees C and 60 s. The linear range and upper limit for concentration determination could be increased to 25 mM by shortening the reaction time. The lower limit of detection in standard solutions, 20 microM, could be achieved by means of modification of the reaction conditions, namely increasing the temperature and the reaction time. The biosensor method was compared with a conventional commercially available colorimetric method with respect to the determination of oxalic acid in urine samples. The urine oxalic acid concentrations determined with the biosensor method correlated well (R=0.952) with the colorimetric method.     

Enzymatic hydrolysis and extraction of arachidonic acid rich lipids from Mortierella alpina

A novel method for efficient arachidonic acid rich lipids extraction was investigated. Six different enzymes (papain, pectinase, snailase, neutrase, alcalase and cellulase) were used to extract lipids from Mortierella alpina. The effects of enzyme concentration, temperature and hydrolysis time on oil recovery were evaluated using factorial experimental design and polynomial regression for each enzyme. Hydrolysis time is found to be the most important parameter for all enzymes. The ratios of enzyme mixtures were also studied. It showed that the mixtures of pectinase and papain (5:3, v/v), pectinase and alcalase (5:1, v/v) were better combined effects on oil yields. The effects of hydrolysis time and temperature were then analyzed by response surface methodology, and oil recoveries were satisfactory (104.6% for pectinase and papain and 101.3% for pectinase and alcalase). In the whole process, the lipid composition was not affected by the enzyme treatments according to fatty acid profile.     

Starch determination in Chlorella vulgaris—a comparison between acid and enzymatic methods

Different methods for estimating starch in Chlorella vulgaris were compared with the view of establishing a procedure suitable for rapid and accurate determination of starch content in this microalgal species. A close agreement was observed between methods that use perchloric acid and enzymatic methods that use α-amylase and amyloglucosidase to hydrolyze the starch of microalgae grown under different nitrogen culture conditions. Starch values obtained by these methods were significantly higher than those estimated by using hydrochloric acid as solubilizing and hydrolyzing agent. The enzymatic method (EM1) proved to be the most rapid and precise method for microalgal starch quantification. Furthermore, the evaluation of resistant starch by enzymatic methods assayed in nitrogen-sufficient and nitrogen-starved cells showed that no formation of this type of starch occurred in microalgae, meaning that this should not interfere with starch content determinations.     

Influence of triiodothyronine and dexamethasone on renal amino acid handling in rats loaded with various amino acid mixtures

Summary In adult female rats, the influence of dexamethasone or triiodothyronine on renal amino acid handling was investigated in amino acid loaded animals. Amino acids were administered intravenously as two mixtures, each containing four amino acids to overload amino acid reabsorption capacity. Bolus injections of both mixtures were followed by temporary increase in fractional excretion of the administered amino acids as well of the amino acids which were not covered in the mixtures. The administration of the two mixtures was followed by different interactions between various amino acid carriers.After dexamethasone pretreatment (60µg/100g b.wt. for 3 days, once daily) a stimulation of the renal amino acid handling could be shown. Triiodothyronine (20µg/100g b.wt. for 3 days, once daily) did not increase tubular reabsorption capacity for amino acids. It even increased fractional amino acid excretion in amino acid loaded rats as a sign of enhanced amino acid metabolism in the kidney and/or increased amino acid uptake into the tubular cells from the luminal site.     

LC-MALDI-TOF MS-Based Rapid Identification of Phenolic Acids

This study is the first on combined HPLC and MALDI-TOF MS analysis of phenolic acids. The analyses were carried out for phenolic acid mixtures and showed a unique, individual co-crystalline pattern for each phenolic acid. HPLC could distinguish phenolic acids and MALDI-TOF MS provided comparable mass (m/z) profiles for the samples. This combined study proved to be rapid in the accurate identification and structural analysis of phenolic acids with different masses.     

Antifungal activity of pisiferic acid derivatives against the rice blast fungus

Twenty-eight derivatives of pisiferic acid (1), an antifungal constituent of Chamaecyparis pisifera var. plumosa against Pyricularia oryzae, were prepared and tested for activity. The size of the substituents at C-10 and C-12, as well as the presence and electronegativity of the constituent oxygen atoms, were inferred to be structural determinants for the activity of pisiferic acid derivatives. A computer graphic comparison between the derivatives and probenazole (35) (a commercial antifungal agent of the fungus) revealed the similarity of the size and location of the oxygen-containing substituents, which was supported by the similar mode of action of 1 and 35.     

Determination of Sepharose-bound protein with Coomassie brilliant blue G-250

The colorimetric procedure of Bradford (M. M. Bradford, 1976, Anal. Biochem.72, 248–254) was found to be convenient for determining the content of a protein immobilized on Sepharose. Being simple, sensitive, and rapid, this method appears very useful in studies involving multiple analyses of immobilized protein species present at low concentrations.     

Substrate specificity and pH dependence of homogeneous wheat germ acid phosphatase.

The broad substrate specificity of a homogeneous isoenzyme of wheat germ acid phosphatase (WGAP) was extensively investigated by chromatographic, electrophoretic, NMR, and kinetic procedures. WGAP exhibited no divalent metal ion requirement and was unaffected upon incubation with EDTA or o-phenanthroline. A comparison of two catalytically homogeneous isoenzymes revealed little difference in substrate specificity. The specificity of WGAP was established by determining the Michaelis constants for a wide variety of substrates. p-Nitrophenyl phosphate, pyrophosphate, tripolyphosphate, and ATP were preferred substrates while lesser activities were seen toward sugar phosphates, trimetaphosphate, phosphoproteins, and (much less) phosphodiesters. An extensive table of Km and Vmax values is given. The pathway for the hydrolysis of trimetaphosphate was examined by colorimetric and 31P NMR methods and it was found that linear tripolyphosphate is not a free intermediate in the enzymatic reaction. In contrast to literature reports, homogeneous wheat germ acid phosphatase exhibits no measurable carboxylesterase activity, nor does it hydrolyze phenyl phosphonothioate esters or phytic acid at significant rates.     

Thermal degradation and isomerisation kinetics of triolein studied by infrared spectrometry and GC-MS combined with chemometrics

Triolein, a triglyceride containing oleic acid as the only acid moiety in the glyceride molecules has been isothermally treated at 280, 300, and 325 °C in glass vials under nitrogen atmosphere. The products formed during the thermal treatment at each temperature have been analysed both by infrared spectrometry and GC-MS. The GC-MS analysis was performed after derivatisation of the fatty acids into their methyl esters (FAMEs).Chemometric tools were used in determining the concentrations of the main products namely triolein and trieaidin in the thermally treated mixtures. The concentration profiles of the trielaidin formed during thermal treatment at the above three temperatures were used in determining activation energy for the cis-trans isomerisation of triolein.The combined analysis reveals that the thermal treatment induces not only cis-trans isomerisation but also fission and fusion in the molecules. Furthermore, migration of the double bond in oleic and elaidic acids forming cis and trans isomers of the 18:1 acid was also observed. The heat-induced isomerisation in triolein follows a zeroth order reaction with an activation energy 41 ± 5 kcal/mol.     

Colorimetric estimation of 3-deoxy-D-manno-octulosonic acid in oligosaccharides with diphenylamine

A method for the colorimetric estimation of 3-deoxy-d-manno-octulosonic acid with diphenylamine is described. The aldulosonic acid can be estimated in the presence of a tenfold excess of other sugars.     

A eudesmane acid from Montanoa speciosa

The major constituent of a leaf-surface methylene dichloride wash of Montanoa speciosa was a eudesmane acid, 1,2-dehydro-3-oxo-costic acid.     

Determination of absolute configuration of salvic acid,an ent-labdane from Eupatorium salvia,by vibrational circular dichroism

The relative stereochemistry at C13 and the absolute configuration of salvic acid, a constituent of the leaves of Eupatorium salvia, were established as the 13-(R)-ent-labdane 1. The results follow from vibrational circular dichroism measurements of the derived O-methyl ether methyl ester 3 which were compared to DFT B3LYP/DGDZVP calculated spectra. The relative stereochemistry of salvic acid at C13 was independently verified by single crystal X-ray diffraction measurements of 1, and of its derived diol 4.     

Growth characteristics of Geotrichum ingens on propionic acid and acetic acid in batch and continuous culture

Growth of Geotrichum ingens in batch cultures was completely inhibited by 47 g acetic acid/l or 33 g propionic acid/I. With mixtures of acetic and propionic acids, however, growth only ceased at 55 g/l. Acetic acid inhibited growth linearly, whereas propionic acid inhibited growth non-linearly. In continuous culture, two steady states at each dilution rate were observed at high dilution rates for acetic acid and propionic acid. The highest yield coefficient (0.69 g cells/g substrate) was achieved with propionic acid as substrate. On both substrates and their mixtures, the protein content of the biomass increased when the dilution rate was increased.The authors are with the Department of Microbiology and Biochemistry, University of the Orange Free State, P.O. Box 339, Bloemfontein 9300, South Africa