A convenient spectrophotometric assay for the determination of L-ergothioneine in blood.
Ontology highlight
ABSTRACT:
SUBMITTER: Carlsson J
PROVIDER: S-EPMC1166272 | biostudies-other | 1974 Apr
REPOSITORIES: biostudies-other
Similar Datasets
Project description:We report the development and optimisation of an assay for quantitating iron from iron oxide nanoparticles in biological matrices by using ferene-s, a chromogenic compound. The method is accurate, reliable and can be performed with basic equipment common to many laboratories making it convenient and inexpensive. The assay we have developed is suited for quantitation of iron in cell culture studies with iron oxide nanoparticles, which tend to manifest low levels of iron. The assay was validated with standard reference materials and with inductively coupled plasma-mass spectrometry (ICP-MS) to accurately measure iron concentrations ?1?×?10-6?g in about 1?×?106 cells (?1?×?10-12?g Fe per cell). The assay requires preparation and use of a working solution to which samples can be directly added without further processing. After overnight incubation, the absorbance can be measured with a standard UV/Vis spectrophotometer to provide iron concentration. Alternatively, for expedited processing, samples can be digested with concentrated nitric acid before addition to the working solution. Optimization studies demonstrated significant deviations accompany variable digestion times, highlighting the importance to ensure complete iron ion liberation from the nanoparticle or sample matrix to avoid underestimating iron concentration. When performed correctly, this method yields reliable iron ion concentration measurements to ?2?×?10-6?M (1?×?10-7?g/ml sample).
| S-EPMC5871594 | biostudies-literature
Project description:BackgroundThe availability of a sensitive and robust activity assay is a prerequisite for efficient enzyme production, purification, and characterization. Here we report on a spectrophotometric assay for lytic polysaccharide monooxygenase (LPMO), which is an advancement of the previously published 2,6-dimethoxyphenol (2,6-DMP)-based LPMO assay. The new assay is based on hydrocoerulignone as substrate and hydrogen peroxide as cosubstrate and aims toward a higher sensitivity at acidic pH and a more reliable detection of LPMO in complex matrices like culture media.ResultsAn LPMO activity assay following the colorimetric oxidation of hydrocoerulignone to coerulignone was developed. This peroxidase activity of LPMO in the presence of hydrogen peroxide can be detected in various buffers between pH 4-8. By reducing the substrate and cosubstrate concentration, the assay has been optimized for minimal autoxidation and enzyme deactivation while maintaining sensitivity. Finally, the optimized and validated LPMO assay was used to follow the recombinant expression of an LPMO in Pichia pastoris and to screen for interfering substances in fermentation media suppressing the assayed reaction.ConclusionsThe biphenol hydrocoerulignone is a better substrate for LPMO than the monophenol 2,6-DMP, because of a ~ 30 times lower apparent K M value and a 160 mV lower oxidation potential. This greatly increases the measured LPMO activity when using hydrocoerulignone instead of 2,6-DMP under otherwise similar assay conditions. The improved activity allows the adaptation of the LPMO assay toward a higher sensitivity, different buffers and pH values, more stable assay conditions or to overcome low concentrations of inhibiting substances. The developed assay protocol and optimization guidelines increase the adaptability and applicability of the hydrocoerulignone assay for the production, purification, and characterization of LPMOs.
| S-EPMC6894463 | biostudies-literature
Project description:A new simple spectrophotometric method was developed for the simultaneous determination of drugs with interfering spectra in binary mixtures without previous separation. The new method is based on a simple modification for the ratio subtraction method. This modification enabled wider range of application. The proposed ratio difference method was applied for the determination of brimonidine and timolol in laboratory prepared mixtures with mean percentage recoveries 100.40±2.29 and 101.23±1.30 respectively, and in their pharmaceutical formulation with mean percentage recoveries 101.08±0.44 and 100.66±0.52 respectively. The suggested ratio difference method was validated according to USP guidelines and can be applied for routine quality control testing.
| S-EPMC5760779 | biostudies-literature
Project description:Favipiravir (FAV) has been approved as an antiviral drug used in pandemic corona virus to treat covid-19. It has an amide moiety susceptible to hydrolysis and degradation in acid medium. Therefore, four simple, sensitive, and accurate stability indicating spectrophotometric methods have been developed for the determination of FAV in presence of its acid induced degradation product. The first method describes direct determination of FAV at 323 nm. Dual wavelength method was the second developed one for FAV quantitation by recording the absorbance difference at 322.7 and 270 nm. The third method involves using first derivative peak to peak amplitude at 338.0 and 308.0 nm, while difference spectrophotometry was the fourth suggested method, and it was based on recording the spectral changes at 361.3 nm as pH changes. The obtained calibration curves were linear over 4.0-22.0 µg/mL. Accuracy of the suggested procedures ranged from 99.11 to100.06, while precision results were from 0.80 to1.68. The developed methods were used to determine FAV in pure powdered form, laboratory-prepared mixtures with their degradation product, and pharmaceutical formulation without interference from its acidic degradation product.The greenness was assessed based on GAPI and ACREE metric and was found to be compatible and in reconciliation with green analytical chemistry concepts.
| S-EPMC10542695 | biostudies-literature
Project description:Ubiquitination is a widely studied regulatory modification involved in protein degradation, DNA damage repair, and the immune response. Ubiquitin is conjugated to a substrate lysine in an enzymatic cascade involving an E1 ubiquitin-activating enzyme, an E2 ubiquitin-conjugating enzyme, and an E3 ubiquitin ligase. Assays for ubiquitin conjugation include electrophoretic mobility shift assays and detection of epitope-tagged or radiolabeled ubiquitin, which are difficult to quantitate accurately and are not amenable to high-throughput screening. We have developed a colorimetric assay that quantifies ubiquitin conjugation by monitoring pyrophosphate released in the first enzymatic step in ubiquitin transfer, the ATP-dependent charging of the E1 enzyme. The assay is rapid, does not rely on radioactive labeling, and requires only a spectrophotometer for detection of pyrophosphate formation. We show that pyrophosphate production by E1 is dependent on ubiquitin transfer and describe how to optimize assay conditions to measure E1, E2, and E3 activity. The kinetics of polyubiquitin chain formation by Ubc13-Mms2 measured by this assay are similar to those determined by gel-based assays, indicating that the data produced by this method are comparable to methods that measure ubiquitin transfer directly. This assay is adaptable to high-throughput screening of ubiquitin and ubiquitin-like conjugating enzymes.
| S-EPMC3178097 | biostudies-literature
Project description:In order to develop a simple, reliable and low cost enzymatic method for the determination of phenolic compounds we studied polyphenol oxidase activity of crude eggplant (S. melongena) extract using 13 phenolic compounds. Catechol, caffeic and chlorogenic acids, and L-DOPA have been rapidly oxidized with the formation of colored products. Monophenolic compounds have been oxidized at a much slower speed. Ferulic acid, quercetin, rutin, and dihydroquercetin have been found to inhibit polyphenol oxidase activity of crude eggplant extract. The influence of pH, temperature, crude eggplant extract amount, and 3-methyl-2-benzothiazolinone hydrazone (MBTH) concentration on the oxidation of catechol, caffeic acid, chlorogenic acid, and L-DOPA has been investigated spectrophotometrically. Michaelis constants values decrease by a factor of 2 to 3 in the presence of MBTH. Spectrophotometric (cuvette and microplate variants) and smartphone-assisted procedures for phenolic compounds determination have been proposed. Average saturation values (HSV color model) of the images of the microplate wells have been chosen as the analytical signal for smartphone-assisted procedure. LOD values for catechol, caffeic acid, chlorogenic acid, and L-DOPA equaled 5.1, 6.3, 5.8 and 30.0 µM (cuvette procedure), 12.2, 13.2, 13.2 and 80.4 µM (microplate procedure), and 23.5, 26.4, 20.8 and 120.6 µM (smartphone procedure). All the variants have been successfully applied for fast (4-5 min) and simple TPC determination in plant derived products and L-DOPA determination in model biological fluids. The values found with smartphone procedure are in good agreement with both spectrophotometric procedures values and reference values. Using crude eggplant extract- mediated reactions combined with smartphone camera detection has allowed creating low-cost, reliable and environmentally friendly analytical method for the determination of phenolic compounds.
| S-EPMC6930448 | biostudies-literature
Project description:A sensitive assay to determine the activity of leucine aminopeptidase (EC 3.4.11.1), using L-leucine thiobenzyl ester as substrate, was developed. Hydrolysis of the ester by leucine aminopeptidase can be monitored in the presence of 5,5-dithiobis-(2-nitrobenzoic acid) by continuous spectrophotometric measurement at 412 nm. Comparison with some amide substrates showed that the thiol ester provides a much more sensitive assay, its specificity constant (Vmax./Km) being some 3000-fold higher than that of leucine p-nitroanilide.
| S-EPMC1147694 | biostudies-other
Project description:A new method for the assay of aminopropyltransferase activity is described. The method measures the formation of [methyl-14C]methylthioadenosine from decarboxylated S-adenosyl[methyl-14C]methionine in the presence of an amine acceptor. When used with extracts from rat ventral prostate, kidney, liver or brain, and with putrescine or spermidine as amines, the method gave results in excellent agreement with those obtained by the much more time-consuming conventional method. It was found that 1,3-diamino-propane and 1,8-diamino-octane were not acceptors for the prostatic enzyme fraction, but 1,5-diaminopentane (cadaverine) was active and 1,9-diaminononane and 1,12-diaminododecane also lead to the production of [methyl-14C]methylthioadenosine.
| S-EPMC1183845 | biostudies-other
Project description:Foods contaminants pose a challenge for food producers and consumers. Due to its spontaneous formation during heating and storage, hydroxymethylfurfural (HMF) is a prevalent contaminant in foods rich in carbohydrates and proteins. Colorimetric assays, such as the Seliwanoff test, offer a rapid and cost-effective method for HMF quantification but require careful optimization to ensure accuracy. We addressed potential interference in the Seliwanoff assay by systematically evaluating parameters like incubation time, temperature, and resorcinol or hydrochloric acid concentration, as well as the presence of interfering carbohydrates. Samples were analyzed using a UV-Vis spectrophotometer in scan mode, and data obtained were validated using HPLC, which also enabled quantification of unreacted HMF for assessing the protocol's accuracy. Incubation time and hydrochloric acid percentage positively influenced the colorimetric assay, while the opposite effect was observed with the increase in resorcinol concentration. Interference from carbohydrates was eliminated by reducing the acid content in the working reagent. HPLC analyses corroborated the spectrophotometer data and confirmed the efficacy of the proposed method. The average HMF content in balsamic vinegar samples was 1.97 ± 0.94 mg/mL. Spectrophotometric approaches demonstrated to efficiently determine HMF in complex food matrices. The HMF levels detected in balsamic vinegars significantly exceeded the maximum limits established for honey. This finding underscores the urgent need for regulations that restrict contaminant levels in various food products.
| S-EPMC11313681 | biostudies-literature
Project description:A new spectrophotometric assay was developed to measure, continuously and specifically, phospholipase A1 (PLA1) or phospholipase A2 (PLA2) activities using synthetic glycerophosphatidylcholines (PCs) containing α-eleostearic acid, either at the sn-1 position [1-α-eleostearoyl-2-octadecyl-rac-glycero-3-phosphocholine (EOPC)] or at the sn-2 position [1-octadecyl-2-α-eleostearoyl-rac-glycero-3-phosphocholine (OEPC)]. The substrates were coated onto the wells of microtiter plates. A nonhydrolyzable ether bond, with a non-UV-absorbing alkyl chain, was introduced at the other sn position to prevent acyl chain migration during lipolysis. Upon enzyme action, α-eleostearic acid is liberated and then solubilized into the micellar phase. The PLA1 or PLA2 activity was measured by the increase in absorbance at 272 nm due to the transition of α-eleostearic acid from the adsorbed to the soluble state. EOPC and OEPC differentiate, with excellent accuracy, between PLA1 and PLA2 activity. Lecitase(®), guinea pig pancreatic lipase-related protein 2 (known to be a PLA1 enzyme), bee venom PLA2, and porcine pancreatic PLA2 were all used to validate the assay. Compared with current assays used for continuously measuring PLA1 or PLA2 activities and/or their inhibitors, the development of this sensitive enzymatic method, using coated PC substrate analogs to natural lipids and based on the UV spectroscopic properties of α-eleostearic acid, is a significant improvement.
| S-EPMC4959851 | biostudies-literature