Preparation and characterization of methoxy polyethylene glycol-conjugated phosphotriesterase as a potential catalytic bioscavenger against organophosphate poisoning
Language English Country Ireland Media print-electronic
Document type Journal Article, Research Support, Non-U.S. Gov't
PubMed
20230809
DOI
10.1016/j.cbi.2010.03.017
PII: S0009-2797(10)00180-8
Knihovny.cz E-resources
- MeSH
- Enzyme Activation MeSH
- Aldehydes chemistry MeSH
- Antidotes chemistry isolation & purification metabolism pharmacology MeSH
- Biocatalysis * MeSH
- Caulobacteraceae enzymology MeSH
- Electrophoresis, Polyacrylamide Gel MeSH
- Phosphoric Triester Hydrolases chemistry isolation & purification metabolism pharmacology MeSH
- Hydrogen-Ion Concentration MeSH
- Organophosphates metabolism MeSH
- Organophosphate Poisoning * MeSH
- Polyethylene Glycols chemistry MeSH
- Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization MeSH
- Enzyme Stability MeSH
- Temperature MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Aldehydes MeSH
- Antidotes MeSH
- Phosphoric Triester Hydrolases MeSH
- monomethoxypolyethylene glycol MeSH Browser
- Organophosphates MeSH
- Polyethylene Glycols MeSH
Bioscavengers are considered as promising antidotes against organophosphate poisoning. We focused on a bacterial phosphotriesterase (PTE) expressed in Escherichia coli. The main disadvantage of this non-human catalytic bioscavenger is its relatively short half-life in the organism and strong immunogenicity after repeated administration. Therefore, we prepared different methoxy polyethylene glycol (MPEG)-conjugated recombinant PTE as a potential catalytic bioscavenger with the aim to improve its biological properties. Enzyme was modified with two linear monofunctional MPEG derivatives with reactive aldehyde group of molecular weight 2 kDa and 5 kDa. We optimized reaction conditions (reagent ratios, temperature and duration of modification reaction) and we prepared homogeneous population of fully modified recombinant PTE with molecular weight around 52 kDa and 76 kDa, respectively. Modified PTE was characterized using SDS-PAGE and MALDI-TOF and by determining K(m) and V(max). We also investigated thermal stability of modified enzyme at 37 degrees C. Based on our results, for future in vivo evaluation of pharmacokinetics and pharmacodynamics properties, we selected recombinant PTE modified with 5 kDa MPEG aldehyde for its superior thermal stability.
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