Enzyme assays
Dotaz
Zobrazit nápovědu
Miniaturization continues to be one of the leading trends in analytical chemistry and one that brings advantages that can be particularly beneficial in biochemical research. Use of a miniaturized scale enables efficient analysis in a short time and requires very small amounts of samples, solvents, and reagents. This can result in a remarkable decrease in costs of enzyme kinetics studies, especially when expensive or rare enzymes and/or substrates are involved. Free zone electrophoresis is without a doubt the most common microscale separation technique for capillary and on-chip enzyme assays. Progress and applications in this field are reviewed frequently whereas other modes of separation, although successfully applied, receive only marginal interest in such publications. This review summarizes applications of less common modes of separation in capillary or chip formats, namely micellar electrokinetic chromatography, liquid chromatography, gel electrophoresis, isoelectric focusing, and isotachophoresis. Because these techniques are based on separation mechanisms different from those of free zone electrophoresis, they can be, and have been, successfully used in cases where zone electrophoresis fails. Advantages and drawbacks of these alternative separation techniques are discussed, as also are the difficulties encountered most often and solutions proposed by different research groups.
- MeSH
- chromatografie micelární elektrokinetická kapilární metody MeSH
- elektroforéza kapilární metody MeSH
- enzymatické testy metody MeSH
- isoelektrická fokusace metody MeSH
- izotachoforéza metody MeSH
- kapilární elektrochromatografie metody MeSH
- kinetika MeSH
- lidé MeSH
- miniaturizace metody MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
Enzyme assays of β-N-acetylhexosaminidase from Aspergillus oryzae using capillary electrophoresis in the offline and online setup have been developed. The pH value and concentration of the borate-based background electrolyte were optimized in order to achieve baseline separation of N,N',N″-triacetylchitotriose, N,N'-diacetylchitobiose, and N-acetyl-D-glucosamine. The optimized method using 25 mM tetraborate buffer, pH 10.0, was evaluated in terms of repeatability, limits of detection, quantification, and linearity. The method was successfully applied to the offline enzyme assay of β-N-acetylhexosaminidase, which was demonstrated by monitoring the hydrolysis of N,N',N″-triacetylchitotriose. The presented method was also utilized to study the pH dependence of enzyme activity. An online assay with N,N'-diacetylchitobiose as a substrate was developed using the Transverse Diffusion of Laminar Flow Profiles model to optimize the injection sequence and in-capillary mixing of substrate and enzyme plugs. The experimental results were in good agreement with predictions of the model. The online assay was successfully used to observe the inhibition effect of N,N'-dimethylformamide on the activity of β-N-acetylhexosaminidase with nanoliter volumes of reagents used per run and a high degree of automation. After adjustment of background electrolyte pH, an online assay with N,N',N″-triacetylchitotriose as a substrate was also performed.
- MeSH
- Aspergillus oryzae chemie enzymologie MeSH
- automatizace MeSH
- beta-N-acetylhexosaminidasy chemie MeSH
- elektroforéza kapilární metody MeSH
- enzymatické testy metody MeSH
- fungální proteiny chemie MeSH
- hydrolýza MeSH
- kinetika MeSH
- koncentrace vodíkových iontů MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
DNA ligases are essential enzymes in all cells and have been proposed as targets for novel antibiotics. Efficient DNA ligase activity assays are thus required for applications in biomedical research. Here we present an enzyme-linked electrochemical assay based on two terminally tagged probes forming a nicked junction upon hybridization with a template DNA. Nicked DNA bearing a 5' biotin tag is immobilized on the surface of streptavidin-coated magnetic beads, and ligated product is detected via a 3' digoxigenin tag recognized by monoclonal antibody-alkaline phosphatase conjugate. Enzymatic conversion of napht-1-yl phosphate to napht-1-ol enables sensitive detection of the voltammetric signal on a pyrolytic graphite electrode. The technique was tested under optimal conditions and various situations limiting or precluding the ligation reaction (such as DNA substrates lacking 5'-phosphate or containing a base mismatch at the nick junction, or application of incompatible cofactor), and utilized for the analysis of the nick-joining activity of a range of recombinant Escherichia coli DNA ligase constructs. The novel technique provides a fast, versatile, specific, and sensitive electrochemical assay of DNA ligase activity.
- MeSH
- DNA-ligasy chemie MeSH
- DNA chemie MeSH
- elektrochemické techniky přístrojové vybavení metody MeSH
- enzymatické testy přístrojové vybavení metody MeSH
- enzymy imobilizované chemie MeSH
- Escherichia coli chemie enzymologie MeSH
- magnetismus MeSH
- proteiny z Escherichia coli chemie MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
Adenosine triphosphate (ATP) is a crucial substrate and energy source commonly used in enzyme reactions. However, we demonstrated that the addition of this acidic compound to enzyme assay buffers can serve as a source of unnoticed pH changes. Even relatively low concentrations of ATP (up to 5 mM) shifted pH of reaction mixtures to acidic values. For example, Tris buffer lost buffering capacity at pH 7.46 by adding ATP at a concentration higher than 2 mM. In addition to the buffering capacity, the pH shifts differed with respect to the buffer concentration. High ATP concentrations are commonly used in hexokinase assays. We demonstrated how the presence of ATP affects pH of widely used enzyme assay buffers and inversely affected KM of human hexokinase 2 and S0.5 of human glucokinase. The pH optimum of human glucokinase was never reported before. We found that previously reported optimum of mammalian glucokinase was incorrect, affected by the ATP-induced pH shifts. The pH optimum of human glucokinase is at pH 8.5-8.7. Suggested is the full disclosure of reaction conditions, including the measurement of pH of the whole reaction mixtures instead of measuring pH prior to the addition of all the components.
- MeSH
- adenosintrifosfát chemie metabolismus MeSH
- enzymatické testy metody MeSH
- hexokinasa chemie genetika izolace a purifikace metabolismus MeSH
- koncentrace vodíkových iontů * MeSH
- ověření koncepční studie MeSH
- rekombinantní proteiny chemie genetika izolace a purifikace metabolismus MeSH
- substrátová specifita MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Lidský cytomegalovirus (CMV) je nejčastějším etiologickým agens způsobujícím kongenitální infekce. Primoinfekce CMV může vést k těžkému onemocnění a komplikacím také u pacientů, u nichž probíhá imunosupresivní terapie nebo onemocnění charakterizované imunosupresí. Základní možností rozlišit primoinfekci od reaktivace latentní infekce nebo reinfekce je stanovení avidity IgG protilátek. Cílem naší práce bylo stanovení indexu avidity CMV IgG metodikou ELISA s denaturací vazby IgG protilátek na antigen a metodou CMIA (chemiluminiscentní imunoesej na mikropartikulích) na analyzátoru Architect, Abbott. Všechny testované metody prokázaly ve stanovení avidity CMV IgG srovnatelnou shodu. Test firmy Abbott vykázal v některých případech lepší reflexi vyzrávání IgG protilátek v průběhu primoinfekce než metoda chemická používající rozrušení vazby antigenu a protilátky denaturačním činidlem na mikrodestičkách.
Human cytomegalovirus (CMV) is the most common cause of congenital infection. Primary CMV infection can lead to severe disease and complications in patients immunocompromised as a result of disease or therapy. IgG antibody avidity assays make it possible to differentiate between primary infection and reactivation of latent infection or reinfection. The study objective was to determine CMV IgG avidity by enzyme-linked immunosorbent assay (ELISA) with denaturation of IgG antibody binding to the antigen and by chemiluminiscent microparticle immunoassay (CMIA) on an Abbott Architect analyzer. Both methods yielded comparable CMV IgG avidity results. In some cases, the Abbott test was superior in reflecting IgG antibody maturation during primary infection to microplate ELISA using antigen-antibody complex dissociation by a denaturing agent.
Capillary electrophoresis is a modern separation technique characterized by many benefits, which qualify it also for enzyme assays and the study of enzyme kinetics during drug development. Homogeneous or heterogeneous approaches can be followed for the enzymatic incubation. In this study, an immobilization procedure of aldehyde oxidase on magnetic particles was developed considering their integration with capillary electrophoresis. A number of magnetic nano/microparticle types were tested for this purpose, showing that aldehyde oxidase was most active when immobilized on bare silica magnetic nanoparticles. Primarily, the reusability of the enzyme immobilized on bare silica nanoparticles was tested. Three consecutive incubations with substrate could be performed, but the activity considerably dropped after the first incubation. One reason could be an enzyme detachment from the nanoparticles, but no release was detected neither at 4°C nor at 37°C during 5 h. The drop in enzymatic activity observed in consecutive incubations, could also be due to inactivation of the enzyme over time at given temperature. For the immobilized enzyme stored at 4°C, the activity decreased to 83% after 5 h, in contrast with a steep decrease at 37°C to 37%.
- MeSH
- imunologické testy metody MeSH
- lidé MeSH
- protilátky virové MeSH
- virus vztekliny imunologie MeSH
- Check Tag
- lidé MeSH
Biperiden is a drug used in Parkinson disease treatment and it serves also as an antiseizures compound in organophosphates poisoning. It acts as antagonist of muscarinic receptor activated by acetylcholine while the enzyme acetylcholinesterase (AChE) cleaves acetylcholine in synaptic junction into choline and acetic acid. This enzyme is inhibited by various compounds; however there has not been proposed evidence about interaction with biperiden molecule. We investigated this interaction using standard Ellman's assay and experimental findings were critically completed with an in silico prediction by SwissDock docking software. Uncompetitive mechanism of action was revealed from Dixon plot and inhibition constant (Ki ) was calculated to be 1.11 mmol/l. The lowest predicted binding energy was -7.84 kcal/mol corresponding to H-bond between biperiden molecule and Tyr 341 residuum in protein structure of AChE. This interaction seems to be further stabilized by π-π interaction with Tyr 72, Trp 286, and Tyr 341. In conclusion, biperiden appears as a very weak inhibitor but it can serve as a lead structure in a pharmacological research.
- MeSH
- acetylcholinesterasa metabolismus MeSH
- biperiden chemie farmakologie terapeutické užití MeSH
- cholinesterasové inhibitory chemie farmakologie terapeutické užití MeSH
- enzymatické testy MeSH
- lidé MeSH
- molekulární modely MeSH
- Parkinsonova nemoc farmakoterapie enzymologie MeSH
- substrátová specifita účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
