title: In vitro characterization of cationic copolymer-complexed recombinant human butyrylcholinesterase.
abstract: Effective use of exogenous human BChE as a bioscavenger for organophosphorus toxicants (OPs) is hindered by its limited availability and rapid clearance. Complexes made from recombinant human BChE (rhBChE) and copolymers may be useful in addressing these problems. We used in vitro approaches to compare enzyme activity, sensitivity to inhibition, stability and bioscavenging capacity of free enzyme and copolymer-rhBChE complexes (C-BCs) based on one of nine different copolymers, from combinations of three molecular weights (MW) of poly-L-lysine (PLL; high MW, 30-70 kDa; medium MW, 15-30 kDa; low MW, 4-15 kDa) and three grafting ratios of poly(ethylene glycol) (PEG; 2:1, 10:1, 20:1). Retarded protein migration into acrylamide gels stained for BChE activity was noted with all copolymers as the copolymer-to-protein ratio was increased. BChE activity of C-BCs was lower relative to free enzyme, with the 2:1 grafting ratio showing generally greater reduction. Free enzyme and C-BCs showed relatively similar in vitro sensitivity to inhibition by paraoxon, but use of the 20:1 grafting ratio led to lower potencies. Through these screening assays we selected three C-BCs (high, medium and low MW; 10:1 grafting) for further characterizations. BChE activity was higher in C-BCs made with the medium and low compared to high MW-based copolymer. C-BCs generally showed higher stability than free enzyme when maintained for long periods at 37 °C or following incubation with chymotrypsin. Free enzyme and C-BCs were similarly effective at inactivating paraoxon in vitro. While these results are promising for further development, additional studies are needed to evaluate in vivo performance.
authors.affiliation: Department of Physiological Sciences, Oklahoma State University, Stillwater, OK 74078, United States.
authors.forename: Carey
authors.initials: C
authors.lastname: Pope
authors.affiliation: Department of Physiological Sciences, Oklahoma State University, Stillwater, OK 74078, United States.
authors.forename: Chibuzor
authors.initials: C
authors.lastname: Uchea
authors.affiliation: School of Chemical Engineering, Oklahoma State University, Stillwater, OK 74078, United States.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Flynn
authors.affiliation: Department of Physiological Sciences, Oklahoma State University, Stillwater, OK 74078, United States.
authors.forename: Kirstin
authors.initials: K
authors.lastname: Poindexter
authors.affiliation: Department of Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, MN, United States.
authors.forename: Liyi
authors.initials: L
authors.lastname: Geng
authors.affiliation: Department of Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, MN, United States.
authors.forename: W Stephen
authors.initials: WS
authors.lastname: Brimijoin
authors.affiliation: Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, OK 74078, United States.
authors.forename: Steve
authors.initials: S
authors.lastname: Hartson
authors.affiliation: Department of Physiological Sciences, Oklahoma State University, Stillwater, OK 74078, United States.
authors.forename: Ashish
authors.initials: A
authors.lastname: Ranjan
authors.affiliation: School of Chemical Engineering, Oklahoma State University, Stillwater, OK 74078, United States.
authors.forename: Joshua D
authors.initials: JD
authors.lastname: Ramsey
authors.affiliation: Department of Physiological Sciences, Oklahoma State University, Stillwater, OK 74078, United States. Electronic address: jing.pope@okstate.edu.
authors.forename: Jing
authors.initials: J
authors.lastname: Liu
mesh_terms: D002091:Butyrylcholinesterase / Q000737:chemistry / Q000494:pharmacology*; D002412:Cations; D002800:Cholinesterase Inhibitors / Q000633:toxicity; D004795:Enzyme Stability; D006801:Humans; D066298:In Vitro Techniques; D010261:Paraoxon / Q000633:toxicity; D011108:Polymers / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry / Q000494:pharmacology
chemical_list: D002412:Cations; D002800:Cholinesterase Inhibitors; D011108:Polymers; D011994:Recombinant Proteins; D002091:Butyrylcholinesterase; D010261:Paraoxon
keywords: Bioscavenger; Nerve agent; Organophosphate; Paraoxon