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Comparative Kinetics of Inhibition, Proteolytic Sensitivity and Heat Inactivation of Free & Nanocomplexed Butyrylcholinesterase

Comparative Kinetics of Inhibition, Proteolytic Sensitivity and Heat Inactivation of Free & Nanocomplexed Butyrylcholinesterase

Name:
Kirstin Poindexter

Department:
Physiological Sciences

Abstract:
We previously reported that nanoformulation of recombinant human BChE with a series of poly-L-lysine (PLL) grafted poly(ethylene glycol) (PEG) co-polymers (PLL-g-PEG) led to loss of 30-50% enzyme activity but relatively little effect on sensitivity to in vitro inhibition by the OP anticholinesterase, paraoxon. Importantly, the nanocomplexed enzyme acted as a bioscavenger in vitro, inactivating paraoxon in a similar concentration-dependent manner as the free enzyme. We now report the in vitro effects of nanoformulation on comparative kinetics of catalysis and inhibition, heat inactivation, and sensitivity to proteases. Human recombinant BChE was nanocomplexed with one of three PLL-g-PEG copolymers using different sizes of PLL (30-70 kDa, 15-30 kDa, 4-15 kDa), all with 10:1 PEG:PLL grafting ratio. Inhibition kinetics was done with four covalent inhibitors (paraoxon, diisopropylfluorophosphate, pyridostigmine and echothiophate). Enzyme activity was monitored using a modified Ellman assay with butyrylthiocholine as substrate. Inhibition constants (ki values) for the free and nanocomplexed enzymes were relatively similar with paraoxon (0-6% reduction), whereas more extensive reductions (23-60% reduction) were noted with other inhibitors. Nanocomplexed enzymes were less sensitive to heat inactivation, and showed greater stability to proteases. Together, our data suggest that nanocomplex formation of BChE with PLL-g-PEG reduces catalytic activity but has little effect on interaction with paraoxon. In contrast, inhibition kinetics with DFP, pyridostigmine and echothiophate suggested changes in enzyme-inhibitor interactions with nanoformulation, potentially influencing bioscavenger efficacy. Nanocomplexed BChE exhibited increased stability to both temperature- and protease-mediated inactivation. Understanding the unique properties of BChE nanoformulated with PLL-g-PEG copolymers may lead to enhanced approaches for use of BChE and other protein bioscavengers. Supported by grant HDTRA1-13-1-0042 from the Defense Threat Reduction Agency.