CS214443B1 - Method of isolating the enzymes of cholinesteraze type - Google Patents

Abstract

The essence of the invention is that an enzyme-containing biological solution of cholinesterase type are diminished by the attenuation pH 7.5 to 8.5 per 1% concentration protein and loaded onto the column from the pelysaccharide goal, with a view from cellulose containing bound p- / w aminophenyl / boronic acid adjusted to pH 7.5 with a buffer of 0.5 M glycerol and purified enzyme choline etherase type elutes at further washing the dolons with the buffers o jH 7.5 with 0.5 M glycerol.

Description

The invention also relates to the isolation of enzymes of the sholeneetherase type, namely, ethyl ethyl choline esterase (acetyloholine hydrolase, EC 3.1.1.7) and peeudecholine etherase (aoethyloholine acyl hydrolase, EC 3.1.1.8).

The isolated enzymes of the sholinneetherase-type are based on tissue homogenate (a-oetloholine etherase, peeudeoholinetherase) or ethanol-fractionated blood plasma (pseudocholino-esterase), Surgenor P.M., Ellias D.t J Amer. Chem., Sec. 76, 6049, 1954, and subsequent chromatography on a calcium phosphate gel, reep. ion exchangers (Maletrom BO, Lavin 0, Bomaa HGs Aota Chem. Scand., 10, 1077), optionally by another gel filter (Dae PK, Llddell Jt Blochem. <RTI ID = 0.0> J, </RTI> 116, 875, 1970). bovine erythrocytes by Sepharose affinity gel chromatography and coupled d-tubucarin and elution and sodium chloride are described by Jung J. and Belleau Bs Mol. Pharmaool., 8, 589, 1972.

Tri-ethylC &lt; RTI ID = 0.0 &gt; h-amiaofeayl &lt; / RTI &gt; ammonium chloride, hydrochloride as an affinity ligand bound to Bio-Cel A is described by Berman J.D. and Young M. Proe. Nat. Acad. Soi. US 68, 395 (1971). The disadvantages of these processes are that they are wasteful, costly to prepare affinity ligands and, in the following, not simply binding to a carrier.

These disadvantages are solved by the process according to the invention.

SUMMARY OF THE INVENTION The biological solution comprising a choline etherase-type enzyme, preferably α-ethylcholine esterase or pseudocholine esterase, is diluted with a buffer solution of pH 7.5 to 8.5 to a 1% protein concentration and loaded onto a polysaoharide gel column, preferably cellulose. containing bound β- (β-aminophenyl) boronic acid adjusted to pH 7,5 with β buffer containing 0,5 M glycerol in a volume ratio of: not more than 1/20 of the volume of the biological solution to the volume of the polysaohaldic gel and was eluted from the column with a further wash of the column with buffer (50 µM) pH 7.5 with 0.5 M glycerol eluate fraction equal to 2-4 times the volume of polysaccharide gel used.

The procedure can be applied in a column, because the enzyme hololine etherase type is specifically retained relative to other accompanying proteins in the formo complex with immobilized borate due to its specific interaction and its esterotic center per column. Said process is useful in combination with an affinity method for isolating Eholine esterase-type enzymes based on at least any other anionic center where a high degree of purification of the enzyme can be achieved, even in the presence of other sorine-type enzymes. This procedure is a peculiar breast isolation of the active cholinesterase-type enzymes from tissue homogonate, erythrocytes or blood plasma, since the enzyme portion inhibited by organophosphate substances does not interact with said affinity ligand.

The advantage of this procedure is to go

- simple preparation of affinity ligand

- allows the purification of the fraction of the enzymes of the holiethereterase type, neinhi2

214 443 with organophosphate substances

- the affinity league is stable and can be used multiple times

- allows multiple purifications of the active cholinesterases to be achieved in a simple manner.

Example 1

Prepared in a column made of cross-linked cellulose formate and 50 / mol bound p- (n-aminophenyl) boronic acid (20 ml), 0.1 to 0.3 mm in size, adjusted with 50 mM phosphate buffer pH 7.5 containing 0, 5 M glycerol was loaded with 0.25 ml of horse plasma diluted with 1 ml of phosphate buffer pH 8. The active pseudocholinesterase (6.5 units) was eluted by eluting with 50 ml of phosphate buffer with 0.5 M glyoerol, ranging from 52 to 52 ml. 95 ml, with a maximum activity in 82 ml.

The displaced fraction contained 0.68 mg of protein to achieve a degree of purification of 10.5.

Example 2

As in Example 1, except that a diluted plasma solution containing pseudocholine esterase partially inhibited by malatine was applied to the column. Affinity chromatography on said column refrained the proportion of active pseudocholinesterase (4.3 units), which was eluted with 50 mM e 0.5 M glyoerol.

The degree of purification of the pseudocholine esterase was the same as in Example 1.

Example 3

The bovine erythrocyte extract prepared from Triten-X-100 according to the procedure of Ciliv G. and Ozand PT: Biochem was used to isolate ateetylcholinesterase. Biophys. Acta 284, 136, 1972 and after centrifugation (100,000g, 30 minutes), said extract (0.5 ml) was adjusted to pH 7.5 with phosphate buffer (1.5 ml, 50 mM) containing 0.5 M glycerol and applied to the prepared with the mesh of silice pectic acid 40 ml, (swelling volume of 6 ml / g) of p-bound (w aminophenyl) boronic acid (62 _/ ol / g), adjusted to pH 7.5, 50 mM phosphate buffer with 0.5 M glyoerol.

Active acetylcholinesterase (16.5 units) was released from the column by elution with 80-165 ml of the above phosphate buffer with 0.5 M glyoerol with a total protein content of 1.95 mg, representing a 37.2-fold purification of the enzyme.

The invention can be applied wherever it is necessary to purify the cholinesterase-type enzymes actively, either in industrial practice or in research, and to isolate them from the organophosphate-inhibited fraction.

Claims (1)

A method of isolating cholinesterase-type 8 enzymes, preferably acetyloholinesterase or pseudecholinesterase, characterized in that a dilute biological solution containing enzymes shaves a non-esterase type of pH 7.5 and a protein content of nio greater than 1% by weight is applied to a column prepared from polysaccharide crosslinked. cellulose containing covalently bound β - (β-aminophenyl) boronic acid adjusted to pH 7,5 with a buffer and containing 0,5 M glycerol in a volume ratio not exceeding 1/20 of the volume of the biological solution to the volume of polysaccharide gel and purified by cholinesterase type enzyme not inhibited and eluting from the column by further washing the column with a pH 7.5 buffer containing 0.5 M glyeerol in an eluate fraction equal to 2-4 times the volume of the gelled polysaccharide gel used.