1. Polyionic complexes of butyrylcholinesterase and poly-l-lysine-g-poly(ethylene glycol): Comparative kinetics of catalysis and inhibition and in vitro inactivation by proteases and heat.
- Author
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Hester K, Liu J, Flynn N, Sultatos LG, Geng L, Brimijoin S, Ramsey JD, Hartson S, Ranjan A, and Pope C
- Subjects
- Biocatalysis, Butyrylcholinesterase chemistry, Butyrylcholinesterase genetics, Cholinesterase Inhibitors chemistry, Cholinesterase Inhibitors metabolism, Cholinesterase Inhibitors pharmacology, Enzyme Activation drug effects, Enzyme Assays, Hot Temperature, Humans, Kinetics, Microscopy, Electron, Scanning, Paraoxon chemistry, Paraoxon metabolism, Particle Size, Polylysine chemistry, Recombinant Proteins biosynthesis, Recombinant Proteins chemistry, Recombinant Proteins metabolism, Butyrylcholinesterase metabolism, Peptide Hydrolases metabolism, Polyethylene Glycols chemistry, Polylysine analogs & derivatives
- Abstract
We previously reported that recombinant human butyrylcholinesterase (rhBChE) complexed with a series of copolymers of poly-l-lysine (PLL) with grafted (polyethylene) glycol (PEG) (i.e., PLL-g-PEG) showed reduced catalytic activity but relatively similar concentration-dependent inactivation of the organophosphorus inhibitor paraoxon. Herein, we compared the kinetics of catalysis (using butyrylthiocholine as the substrate) and inhibition (using four different inhibitors) of free and copolymer-complexed rhBChE. Using scanning electron microscopy, polyionic complexes of rhBChE with three different PLL-g-PEG copolymers (based on PLL size) appeared as spheroid-shaped particles with relatively similar particle sizes (median diameter = 35 nm). Relatively similar particle sizes were also noted using dynamic light scattering (mean = 26-35 nm). The three copolymer-complexed enzymes exhibited reduced k
cat (30-33% reduction), but no significant changes in Km . Inhibitory potency (as reflected by the bimolecular rate constant, ki ) was similar among the free and copolymer-complexed enzymes when paraoxon was the inhibitor, whereas statistically significant reductions in ki (16-60%) were noted with the other inhibitors. Sensitivity to inactivation by proteases and heat was also compared. Copolymer-complexed enzymes showed lesser time-dependent inactivation by the proteases trypsin and pronase and by heat compared to the free enzyme. Understanding the unique properties of PLL-g-PEG-BChE complexes may lead to enhanced approaches for use of BChE and other protein bioscavengers., (Copyright © 2017 Elsevier B.V. All rights reserved.)- Published
- 2017
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