Large-scale purification and long-term stability of human butyrylcholinesterase: a potential bioscavenger drug

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Butyrylcholinesterase from human plasma (HuBChE) is a potential drug candidate for detoxification of certain harmful chemicals that contain carboxylic or phosphoric acid ester bonds. Large quantities of purified HuBChE, displaying a high stability upon long-term storage, are required for the evaluation of its therapeutic capacity and its pharmaceutical properties. Several modifications of a previously reported procedure enabled us to purify the enzyme >15,000-fold from pools of up to 100 l of human plasma. The three-step procedure is based on precipitation of plasma proteins by ammonium sulfate (step I) and batch adsorption of HuBChE on procainamide–Sepharose 4B gel (step II). Ammonium sulfate was also employed in the third stage to fractionate the final product from procainamide-containing HuBChE solution. The overall yield (63%) of electrophoretically pure enzyme was significantly higher than that previously reported (34%) for the purification of HuBChE from 12.5 l of plasma or from 5 kg of Cohn fraction IV-4. Purified HuBChE was stored at 5°C in 10 mM phosphate buffer (pH 7.4) containing 1 mM EDTA and 0.02% NaN 3. The specific activity, protein migration on gel electrophoresis, thermostability at 54°C and the mean residence time in the circulation of mice remained essentially constant for at least 46 months. The modifications introduced can provide large quantities of purified enzyme that maintains its activity and bioavailability properties for several years.