CS214546B1 - a method for isolating cholinesterase type enzymes - Google Patents

Abstract

The invention relates to a method for isolating cholinesterase-type enzymes. The essence of the invention lies in the fact that a biological solution containing a cholinesterase-type enzyme, preferably acetylcholinesterase or pseudocholinesterase, after adjustment to pH 7.5 to 9 and an ionic strength below 0.01 M, is allowed to interact in the first stage, preferably on a column with a gel of cross-linked and methylated polyethylenimine, and after subsequent washing of the gel with the same solvent, in the second stage the anchored enzyme is displaced by elution with a solution with pH 8 and an ionic strength higher than 0.1 M or by the effect of a diluted solution of choline or another alkylammonium salt. The invention has application in the isolation or purification of cholinesterase-type enzymes for research or industrial practice.

Description

The present invention relates to a process for the isolation of cholinesterase-type enzymes, namely acetylcholine terrase / acetylcholine hydrolase, EC 3.1.1.7 and pseudocholinesterase / acylcholine acyl hydrolase, EC 3.1.1. In the isolation and purification of cholinesterase-type enzymes, it is common to start with the homogenate of the human / acetylcholinesterase, pseudocholinnesterase / or blood plasma using ethanol fractionation (pseudocholinesterase), Surgenor DM, Ellias DiJAmer.chem.so.76 6049, 1954, possibly further gel chromatography. calcium phosphate, or ion exchange / Malstrom BG, Levin S., Boman H.GtiActa Chem.Sc., 10, 1077, 1956, respectively, additional filtration (Das PK, Liddell J. Biochem. J., 116,875, 1970 /. A rapid method of purifying acetylcholinesterase from bovine erythrocytes by affinity gel chromatography on d-tubukarin-bound Sepharose and eluting with sodium chloride is described by Jung J. and Belleau Bj Mol.Pharmacol., 8, 589, 1972. Trimethyl-m-aminophenyl / ammonium chloride the hydrochloride as an affinity ligand bound to Bio-Gel A describes Berman JD U.S. Pat. No. 68, 395, 1971. The disadvantage of said processes is their attractiveness, or the costly preparation of affinity ligands and, furthermore, the carrier is not easy to bind.

The essence of the invention is that the biological solution containing the cholinesterase-type enzyme, preferably acetylcholinesterase or pseudo-cholinesterterase, after adjusting the pH to 7.5-9 and ionic strength below 0.01M, is preferably interacted with the gel-crosslinked and methylated column in the first stage. polyethyleneimine with a swelling volume of 6 to 12 ml / g and subsequent washing of the gel with the same buffer solution is displaced in the second stage by anchored elution with a buffer of pH 8 and an ionic strength of greater than 0.1 M, or a buffer solution of pH or by the action of a dilute solution of choline, optionally another alkylammonium salt.

The process can be applied in a batchwise manner, or more preferably, the polyethyleneimine modified ligand is loaded in a column through which the biological solution containing cholinesterase enzymes is poured. The carrier capacity reaches several thousand units of enzyme per gram of dry carrier. After anchoring the enzyme, the gel on the column is washed with a 60-fold volume of pH 8 buffer to displace unwanted additives. The washings of the accompanying materials release the bound pure enzyme from the carrier on the column by elution with a pH 8 buffer and an ionic strength greater than 0.1 M, or a pH 6 buffer, or a dilute choline solution or other alkylammonium salt. Purification of the enzyme by almost 50-fold is achieved by this method.

Methylated crosslinked polyethyleneimine is an affinity ligand containing recurring structurally choline units, which is the basis for the superior isolation and purification of choline enzymes. The advantage of the novel process is that the preparation of the affinity ligand is simple and does not require expensive chemicals, allows the batch or column process to be used, the affinity of the ligand is stable and can be used multiple times, allowing simple purification of the isolated enzymes by simple purification. Example 1 To 10 g of an aqueous solution of polyethyleneimine containing 5 g of dry weight of polyethylsulphide was added 1 ml of 25% sodium hydroxide solution and 0.5 g of 2-chloromethyloxirane.

Upon complete mixing of the reaction mixture to form an emulsion, the reaction occurs while heating the mixture. External cooling maintained the temperature of the reaction mixture at 40-50 ° C. Upon completion of the cross-linking (1 h), the crosslinked polyethyleneimine was mixed in the aqueous mixer solution to the required particle size (0.1 to 0.3 mm). The crosslinked polyethyleneimine thus prepared had a swelling volume of 6 ml / g, a cross-linking degree of 0.046 otyleneimine units per transverse poultrypropane bond, and an araldehyde content of 1.65 mol / g, an average outer strand length of 17.4 A, corresponding to 4 ethylene- mine units. The crosslinked polyethyleneimine thus prepared was suspended in 25 ml of water and stirred with electromagnetic stirrer with 5 g of methyl iodide at room temperature for 16 hours. Excess methyl iodide was evaporated in a vacuum evaporator. Obtained cross-linked

Claims (4)

methylated polyethyleneimine with an amino group content of 0.01 mmol / g was washed with 500 ml of water and used previously, washed with 30 ml of 0.1% bovine serum albumin and 50 ml of phosphate buffer (5 mM, pH 8). A 5 ml conical plasma, diluted with 5 mM phosphate pfru / 5 ml, pH 8, with 200 units activity, was blotted onto the 15 µm 1.5 cm column thus prepared. The sana column was washed with about 300 ml of the supernatant phosphate buffer (to a negative reaction to the eluate proteins). Bound butyrylcholinesterase was displaced by 150 ml of 5 mM phosphate buffer (pH 7.6) containing 0.1 M sodium chloride. The displaced fraction contained 12.5 mg of protein and 148 units of butyrylcholinesterase activity, representing 72% of total activity and 48.5% of total activity. multiple refinement. Example 2 The procedure of Example 1, except that a batch process was performed, ie a column and a crosslinked methylated poly (ethyleneimine) gel (5 g) having a swelling volume of 12 ml / g and adjuvanted 30 ml of a 0.1% albumin solution in 50 mM borate buffer / ph 9 /. The gel thus adjusted was mixed with 5 roles of human plasma, diluted to 10 ml with the above borate buffer (total protein content of 442 mg and total activity of 165 units of butyrylcholinesterase activity). Bound butyrylcholinesterase activity was displaced with a 0.2 M choline solution in 5 µM borate buffer (pH 9, 150 mL). The displaced fraction contained 16.8 mg of protein and 105 units of activity, representing a 16.8 fold purification. Example As the source of acetylcholinesterase, a bovine erythrocyte extract prepared from Triton-X-100 was used according to the method of Ciliv G. and Ožand P.T .: Biochem. Biophys. Acta, 248, 136, 1972a, after centrifugation (10 [mu] g, 30 min), the extract (5 ml) was adjusted to pH 8.5 with phosphate buffer (5 ml) and loaded onto a 20 [mu] cm cm column of crosslinked methylated polyethyleneimine. After washing the column with 300 ml of phosphate buffer (ph 8), bound acetylcholinesterase was released in 150 ml of 50 mM phosphate buffer (pH 6). Yield 18.7 mg protein and total enzyme activity 193 units. Example 4 The procedure of Example 2, except that a trimethyl / p-aminophenyl / ammonium chloride solution (0.1 M) was used to displace the bound butyryl choline ester in 5 mM phosphate buffer (pH 8, 150 ml). The displaced fraction contained 12 mg of protein and 110 units of activity. The invention can be applied wherever choline-esterase enzymes are required to be isolated or purified, either in industry or in research. DISCLOSURE OF THE INVENTION The method of isolating cholinesterase-type enzymes by using the affinity chromatography principle, wherein the biological solution containing the cholinesterase-type enzyme, preferably acetylcholinenosterase or pseudocholinesterase, is adjusted to pH 7.5 to 9 and the ionic strength below 0.01 M is left in the first the step of interacting preferably on a cross-linked and methylated polyethyleneimine gel column having a swelling volume of 6 to 12 ml / g and subsequent washing with the same buffer in the second stage displaces the anchored enzyme elution with a buffer solution of pH 8, an ionic strength higher as 0.1 M, or a buffer of pH 6 or a dilute solution of choline or other alkylammonium salt.