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

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Abstract

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% NaN3. 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.

Introduction

Blood contains esterases that protect physiologically important sites by enhancing the hydrolysis of certain toxicants in the circulation. Human butyrylcholinesterase from plasma (EC 3.1.1.8; HuBChE), has been suggested to participate in the endogenous scavenging of naturally occurring (e.g., physostigmine, cocaine) and synthetic drugs (e.g. succinylcholine), and of anti-cholinesterase-based pesticides 1, 2, 3. The observations that in healthy subjects that carry the normal HuBChE genotype, inhibition of plasma BChE does not cause toxic symptoms, and the fact that individuals who lack BChE activity in blood are healthy [1], supports the contention that one of the principal functions of HuBChE is to detoxify ester bond-containing drugs. For example, there is a relationship between the low activity of plasma HuBChE in people carrying the atypical or the silent variants and the observed prolonged apnea caused by succinylcholine. Well-timed administration of exogenous HuBChE in patients treated with succinylcholine shortened the duration of the induced apnea [4].

It has been suggested that a deficiency in HuBChE might be involved in the susceptibility of certain individuals to cocaine [5]. Mattes et al. [6] have recently indicated that exogenous administration of HuBChE in cocaine-intoxicated patients with low endogenous HuBChE activity would be expected to shift the metabolism of cocaine from norcocaine and benzoylecgonine to the less toxic metabolites, benzoic acid and ecgonine methylester, which are rapidly excreted from the circulation. HuBChE was also demonstrated to be an efficient prophylactic antidote against nerve agent toxicity 7, 8, 9.

The growing interest in HuBChE as a bioscavenger drug is expected to increase future efforts in defining the scope of its therapeutic capacity. This will require continuous supply of large quantities of purified enzyme that is suitable for long-term studies. Three methods were previously described for processing of 10 l [1] and 12.5 l [10] of human plasma, with an overall yield of approximately 34% of pure enzyme. These procedures were based on an initial step that decreased the plasma concentration of the bulk protein, followed by affinity chromatography on procainamide–Sepharose 4B affinity gel.

We describe a modified method for the purification of HuBChE from large volumes of human plasma, with as high as 63% overall yield. Concentrated solutions of the purified enzyme were monitored over several years for changes in specific activity, migration on gel electrophoresis, thermostability and rate of clearance from blood of mice following i.v. injection. The activity and physical properties of HuBChE were practically unchanged after almost 4 years at 5°C, suggesting that this purification method can guarantee the supply of large quantities of enzyme for long-term studies.

Section snippets

Caution: CNBr (solid and solutions) is a hazardous material and should be handled with gloves, mask and only in protected hood

5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), S-butyrylthiocholine (BTC) and cyanogen bromide (CNBr) were obtained from Aldrich (Milwaukee, WI, USA). S-Acetylthiocholine (ATC) was purchased from Fluka (Buchs, Switzerland). Procainamide hydrochloride, 1-ethyl-3(3-dimethylaminopropyl)carbodiimide hydrochloride, ε-aminocaproic acid and sodium azide (NaN3) were obtained from Sigma (St. Louis, MO, USA). Sepharose 4B was purchased from Pharmacia (Upssala, Sweden). A biochemically pure grade of ammonium

Purification of HuBChE

The use of procainamide–Sepharose 4B gel for the purification by affinity chromatography of BChE from human plasma was introduced by Lockridge and La Du [12] in the final stage of a purification method that was preceded by chromatography on DEAE–cellulose. This procedure was scaled-up to 7.5 and 10 l, yielding 8–12 mg/batch of pure HuBChE, with a specific activity of 704 units/mg [1]. Assuming an average concentration of 3.5 mg/l HuBChE in normal plasma, the overall yield was approximately 33%.

Conclusions

The three-step procedure was 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 a procainamide-containing HuBChE solution. The method described here differs from previous works with large volumes of human plasma by the choice of batch adsorption [25], rather than passing the plasma through columns of affinity

Acknowledgements

This work was supported by the U.S. Army Medical Research and Development Command under Contract No. DAMD17-90-0033 (to Y.A.). Opinions, interpretations, conclusions and recommendation are those of the authors and are not necessarily endorsed by the U.S. Army. The authors acknowledge the skilful technical assistance of Mr. Y. Alfasi and Mr. D. Alkalai.

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