Purification of soluble acetylcholinesterase from sheep liver by affinity chromatography

Kasim Abass Askar, Caleb Kudi, John Moody. School of Biomedical and Biological Sciences, Faculty of Science and Technology, University of Plymouth, Plymouth, PL4 8AA, United Kingdom.

Background: The purification of a soluble acetylcholinesterase (AChE, E.C.3.1.1.7) from sheep liver using affinity chromatography on Concanavalin A–Sepharose 4B and edrophonium–Sepharose 6B is studied. The affinity matrix was synthesized by coupling an inhibitor edrophonium to epoxy-activated Sepharose at flow rate of 0.5 ml/min. AChE is a pivotal enzyme in the cholinergic nervous system. Its primary function is to catalyze hydrolysis of released acetylcholine and thus maintain homeostasis of this neurotransmitter in the central and peripheral nervous systems. Hence, AChE is important in both pharmacological and toxicological mechanism (1). The purpose of this study was to provide proof for the existence of AChE enzyme and to develop a purification method for further enzyme characterization. A further aim was to study the edrophonium’s pharmacologic action is due primarily to the inhibition or inactivation of AChE at sites of cholinergic transmission.

Materials and Methods: Liver from sheep was obtained from local abattoirs and transported in a cool box to the laboratory. To extract AChE (liver) was cut into small pieces (3-5 mm3), homogenized using a mechanically-driven homogenizer (Model X520-D, T6 probe, Bennett and Company, Weston-super-Mare, UK) with sodium phosphate buffer (0.1 M, pH 8) containing 0.5 M NaCl at a ratio of 1 part of tissue to 9 parts of buffer, and a speed of 10000 rpm. The homogenate was then centrifuged in 50 ml tubes by using (MES, T8 probe, Europa 284) at 30,000 g for 1 h, at 4°C (1). Enzyme activity was determined using the Ellman (1961) method (2), adapted for a plate reader (1). The protein content was quantified either by measuring the absorbance at 280 nm (3) or by Bradford Method (4).

Results: The purification of soluble AChE from sheep liver is summarized in Table 1. Sodium dodecyl sulphate (SDS) electrophoresis resulted in a monomeric molecular weight of 67.1 kDa, while on gel chromatography using Sephacryl S-200 under nondenaturing conditions to be 201.5 kDa. Based on the molecular weight obtained by gel filtration the purified AChE was assumed to be a tetrameric form.

Table 1: Purification of AChE from sheep liver.

The specific activity of AChE, expressed as μmol hydrolyzed/min/mg of protein. The recovery (%) of protein and activity was based on the total protein and AChE activity, respectively.

Conclusions: We succeeded in establishing a gentle solubilization technique that provided a favourable SDS during further purification procedure by stabilizing the native form of this fragile protein. Secondly, we could purify AChE by a two-step separation on Concanavalin A–Sepharose 4B column followed by edrophonium–Sepharose 6B column. This protocol, in our opinion, (combined use of Concanavalin A-Sepharose 4B and edrophonium affinity 6B chromatography) could be a useful resource for purifying soluble AChE from sheep liver, which is readily applicable to the purification of soluble AChE from other sources.

(1) Son JY et al, 2002. Int. J. Biochem. Cell Biol. 34:204-210.

(2) Ellman GL et al, 1961. Biochem. Pharmacol. 7:88-90.

(3) Warburg, O., and Christian, W. 1942. Biochem. 310:384-421.

(4) Bradford, M. M. 1976. Anal. Biochem. 72:248-254.