Preparation and characterization of methoxy polyethylene glycol-conjugated phosphotriesterase as a potential catalytic bioscavenger against organophosphate poisoning.
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.
Center of Advanced Studies, Faculty of Military Health Sciences, University of Defence, Hradec Králové, Czech Republic. jun@pmfhk.cz
Daniel
D
Jun
Lucie
L
Musilová
Marek
M
Link
Mélanie
M
Loiodice
Florian
F
Nachon
Daniel
D
Rochu
Frédérique
F
Renault
Patrick
P
Masson
D000447:Aldehydes / Q000737:chemistry; D000931:Antidotes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D055162:Biocatalysis; D042063:Caulobacteraceae / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D062025:Organophosphate Poisoning; D010755:Organophosphates / Q000378:metabolism*; D044345:Phosphoric Triester Hydrolases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D011092:Polyethylene Glycols / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature
D000447:Aldehydes; D000931:Antidotes; D010755:Organophosphates; D011092:Polyethylene Glycols; C028210:monomethoxypolyethylene glycol; D044345:Phosphoric Triester Hydrolases