In this paper, we demonstrate the effectiveness of a new 3D printed magnet holder that enables capture of magnetic microparticles in commercially available capillary electrophoresis equipment with a liquid or air based coolant system. The design as well as the method to capture magnetic microparticles inside the capillary are discussed. This setup was tested at temperature and pH values suitable for performing enzymatic reactions. To demonstrate its applicability in CE- immobilized microenzyme reactors (IMER) development, human flavin-containing monooxygenase 3 and bovine serum albumin were immobilized on amino functionalized magnetic microparticles using glutaraldehyde. These microparticles were subsequently used to perform in-line capillary electrophoresis with clozapine as a model substrate. This setup could be used further to establish CE-IMERs of other drug metabolic enzymes in a commercially available liquid based capillary coolant system. The CE-IMER setup was successful, although a subsequent decrease in enzyme activity was observed on repeated runs.
- MeSH
- aminy chemie MeSH
- design vybavení přístrojové vybavení MeSH
- elektroforéza kapilární přístrojové vybavení MeSH
- enzymy imobilizované chemie MeSH
- glutaraldehyd chemie MeSH
- klozapin chemie MeSH
- lidé MeSH
- magnetické pole MeSH
- magnety chemie MeSH
- mikrosféry * MeSH
- NADP chemie MeSH
- oxid křemičitý chemie MeSH
- oxygenasy chemie MeSH
- povrchové vlastnosti MeSH
- sérový albumin hovězí chemie MeSH
- stabilita enzymů MeSH
- teplota MeSH
- velikost částic MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Mammalian dihydrofolate reductases (DHFRs) catalyze the reduction of folate more efficiently than the equivalent bacterial enzymes do, despite typically having similar efficiencies for the reduction of their natural substrate, dihydrofolate. In contrast, we show here that DHFR from the hyperthermophilic bacterium Thermotoga maritima can catalyze reduction of folate to tetrahydrofolate with an efficiency similar to that of reduction of dihydrofolate under saturating conditions. Nuclear magnetic resonance and mass spectrometry experiments showed no evidence of the production of free dihydrofolate during either the EcDHFR- or TmDHFR-catalyzed reductions of folate, suggesting that both enzymes perform the two reduction steps without release of the partially reduced substrate. Our results imply that the reaction proceeds more efficiently in TmDHFR than in EcDHFR because the more open active site of TmDHFR facilitates protonation of folate. Because T. maritima lives under extreme conditions where tetrahydrofolate is particularly prone to oxidation, this ability to salvage folate may impart an advantage to the bacterium by minimizing the squandering of a valuable cofactor.
- MeSH
- bakteriální proteiny chemie genetika metabolismus MeSH
- dihydrofolátreduktasa chemie genetika metabolismus MeSH
- druhová specificita MeSH
- Escherichia coli chemie enzymologie genetika MeSH
- exprese genu MeSH
- katalytická doména MeSH
- kinetika MeSH
- koncentrace vodíkových iontů MeSH
- kyselina listová chemie metabolismus MeSH
- NADP chemie metabolismus MeSH
- oxidace-redukce MeSH
- protony * MeSH
- sbalování proteinů MeSH
- sekundární struktura proteinů MeSH
- teplota MeSH
- termodynamika MeSH
- tetrahydrofoláty chemie metabolismus MeSH
- Thermotoga maritima chemie enzymologie genetika MeSH
- Publikační typ
- časopisecké články MeSH
FerB from Paracoccus denitrificans is a soluble cytoplasmic flavoprotein that accepts redox equivalents from NADH or NADPH and transfers them to various acceptors such as quinones, ferric complexes and chromate. The crystal structure and small-angle X-ray scattering measurements in solution reported here reveal a head-to-tail dimer with two flavin mononucleotide groups bound at the opposite sides of the subunit interface. The dimers tend to self-associate to a tetrameric form at higher protein concentrations. Amino acid residues important for the binding of FMN and NADH and for the catalytic activity are identified and verified by site-directed mutagenesis. In particular, we show that Glu77 anchors a conserved water molecule in close proximity to the O2 of FMN, with the probable role of facilitating flavin reduction. Hydride transfer is shown to occur from the 4-pro-S position of NADH to the solvent-accessible si side of the flavin ring. When using deuterated NADH, this process exhibits a kinetic isotope effect of about 6 just as does the NADH-dependent quinone reductase activity of FerB; the first, reductive half-reaction of flavin cofactor is thus rate-limiting. Replacing the bulky Arg95 in the vicinity of the active site with alanine substantially enhances the activity towards external flavins that obeys the standard bi-bi ping-pong reaction mechanism. The new evidence for a cryptic flavin reductase activity of FerB justifies the previous inclusion of this enzyme in the protein family of NADPH-dependent FMN reductases.
- MeSH
- aminokyseliny chemie genetika metabolismus MeSH
- bakteriální proteiny chemie genetika metabolismus MeSH
- biokatalýza MeSH
- difrakce rentgenového záření MeSH
- flavinmononukleotid chemie metabolismus MeSH
- flaviny chemie metabolismus MeSH
- flavoproteiny chemie genetika metabolismus MeSH
- katalytická doména genetika MeSH
- kinetika MeSH
- krystalografie rentgenová MeSH
- maloúhlový rozptyl MeSH
- molekulární modely MeSH
- molekulární sekvence - údaje MeSH
- multimerizace proteinu MeSH
- mutageneze cílená MeSH
- NADH, NADPH oxidoreduktasy chemie klasifikace metabolismus MeSH
- NADP chemie metabolismus MeSH
- oxidace-redukce MeSH
- Paracoccus denitrificans enzymologie genetika MeSH
- sekvence aminokyselin MeSH
- sekvenční homologie aminokyselin MeSH
- terciární struktura proteinů * MeSH
- vazba proteinů MeSH
- vazebná místa genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
A 96-well microplate-based HPLC endpoint assay is described for the determination of NADPH-cytochrome P450 reductase (CPR) activity. Novel sampling of NADPH into microplates was optimized. Separation was performed on a Zorbax Eclipse XDB-C₁₈ analytical 4.6 × 150 mm, 5 µm column. To validate the method, recombinant human NADPH-P450 reductase and microsomes with cytochrome P450 CYP1A1 were used. The mobile phase consisted of 80% acetonitrile and 20% water at a flow-rate of 0.8 mL/min. The CPR activity was quantified using NADPH fluorescence at λ(Ex) = 340 nm and λ(Em) = 450 nm. Enzymatic activity was directly proportional to the decrease in NADPH fluorescence. This analytical process enables a highly sensitive endpoint determination for reductase activity in vitro and monitoring of the consumption of NADPH in enzymatic reactions. The method avoids the use of substrates and of organic solvents that may affect CPR and cytochrome P450 activity. In the reaction, molecular oxygen served as a proton source. The method can substitute spectrophotometric detection methods for its accuracy, high reproducibility (~100%) and sensitivity. The lower limit of detection, shown using the Agilent 1200 aparatus, is in the 250 nmol range. In addition, using this method it is possible to set up reactions in a high-throughput format.
- MeSH
- acetonitrily chemie MeSH
- fluorescenční spektrometrie metody MeSH
- kalibrace MeSH
- lidé MeSH
- limita detekce MeSH
- lineární modely MeSH
- NADP analýza chemie metabolismus MeSH
- NADPH-cytochrom c-reduktasa metabolismus MeSH
- rekombinantní proteiny analýza metabolismus MeSH
- reprodukovatelnost výsledků MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
