CS258359B1 - A method for the isolation and purification of mitochondrion maltosehydrogenase - Google Patents

Abstract

The essence of the solution is that the isolation and purification of mitochondrial malate dehydrogenase is carried out by sorption of the enzyme from buffer solutions with pH 6 to 8 and a certain concentration onto a modified macroporous gel copolymer of 2-hydroxyethyl methacrylate with ethylene dimethacrylate, which contains 1,4-diamino-9,10-anthraquinone-3-sulfonic acid in the side chains. Simultaneously sorbed accompanying substances are removed by washing with buffers, which desorb the enzyme after the addition of sodium or potassium salt of hydrochloric or sulfuric acid and optionally nicotinamidenine dinucleotide, nicotinamide-adenine-dinucleotide reduced or the same in a mixture with sulfurous acid salts.

Description

The present invention relates to the isolation and purification of mitochondrial malate dehydrogenase (MDH) and animal hearts by affinity chromatography techniques on modified anthraquinone-bound hydroxyethyl methacrylate gels.

Maleate dehydrogenase is an important enzyme that is widely used in clinical diagnostics and food analytics (transaminases, malic acid, etc.). The enzyme is isolated from various sources, most commonly from animal hearts. The extracts and concentrates prepared from these sources contain a variety of ballast substances from which malate dehydrogenase needs to be separated.

A variety of procedures have been developed for enzyme isolation and purification. State-of-the-art techniques include affinity chromatography on immobilized anthraquinone dyes (T. Atkinson et al., Biochem. Soc. Trans., 9, 290 (1981), R. Bittner et al., GDR Pat. No. 152,359 (1,981)) and an azo dye (K. Smith et al. Biochem. Biophys. Acta, 708, 17, (1982)). However, the disadvantageous mechanical properties of the agarose matrix on which the dyes have been anchored prevent the wider use of these processes.

These disadvantages are overcome by the method of the invention. It is based on the fact that the enzyme is absorbed from a buffer solution of pH 6-8 at a concentration of 0.002 to 0.1 mol / l to a modified macroporous gel copolymer of 2-hydroxyethyl methacrylate with ethylenedimethacrylate containing 1,4-diamino- 9,10-anthraquinone-3-sulfonic acid covalently bound via arylidene sulfonylethoxy groups, simultaneously sorbed accompanying substances are removed from the sorbent by washing with a buffer of pH 6-8 and a concentration of 0.002 to 0.2 mol / l and finally the entrapped enzyme is desorbed with the above defined washing buffers to which 0.2 to 1 mol of sodium or potassium salt of hydrochloric or sulfuric acid and optionally 0.1 to 10 mmol / l of nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide reduced, or nicotinamide adenine dinucleotide in a mixture with a 3 to 5 molar excess of sulfuric acid salts.

Hydroxyethyl methacrylate gels with 1,4-diamino-9,10-anthraquinone-3-sulfonic acid linked in the side chains via the arylidene sulfonylethoxy group are described in AO 255 416. These materials * retain all the beneficial properties of the starting hydroxyethyl methacrylate gels, in particular their mechanical strength and volume stability and can, therefore, unlike agarose-based derivatives, be used at higher flow rates and pressures.

Their advantage over materials with 1,4-diamino-9,10-anthraquinone-3-sulfonic acid bound in a manner other than arylidenesulfonylethoxy groups is their high selectivity to mitochondrial malate dehydrogenase. For example, lactate dehydrogenase is not adsorbed onto them so that the modified gel can be used to separate both enzymes. Similarly, cytoplasmic malate dehydrogenase is not absorbed or can be washed out with dilute buffer without any addition.

The method of isolation and purification of the mitochondrial malate dehydrogenase of the invention is illustrated by the following Examples.

Example 1 g of acetone powder from pig hearts was extracted with 100 ml of 0.1 M phosphate buffer pH 7.4. The extract was mixed with ammonium sulfate to 45% saturation, the precipitate formed was centrifuged and the supernatant saturated with ammonium sulfate to 70% saturation.

After centrifugation, the precipitate was dissolved in water and the solution dialyzed against a 0.01 mol / l phosphate buffer, pH 7.1. After dialysis 30 ml of a solution was obtained which, according to the analysis, contained 595 mg of protein; MDH activity was 45,000 U. To the solution was added 5 g of modified gel prepared from the starting copolymers obtained from 61% by weight of 2-hydroxyethyl methacrylate and 39% by weight. Ethylenedimethacrylate with a molecular weight exclusion of 10 Daltons and a grain size of 63-100 µm and contained 199 µmol / g 1,4-diamino-9,10-anthraquinone-3-sulfonic acids covalently bound via arylidene sulfonylethoxy groups, previously equilibrated with phosphate buffer, pH 7.1 and the suspension was shaken for 30 minutes.

After gel separation, 6,100 U of cytoplasmic MDH remained in the remaining solution, and all mitochondrial dehydrogenase remained adsorbed to the dye-bound gel. After washing with a buffer of pH 7.1 and a concentration of 0.01 mol / l, the captured mitochondrial MDH was eluted with the same buffer to which 0.5 mol / l of potassium chloride was added. The eluate activity was 35,800 U (92% based on adsorbed enzyme).

Example 2 ml of pork heart extract prepared in a similar manner to Example 1 containing 40 mg of protein and 3000 U MDH were applied to a 0.7 x 5.5 cm column containing a gel similar to Example 1 but with a grain size of 40 to 63 µm and a content of bound dye of 67 µmol / g, which had been previously equilibrated with phosphate buffer, pH 7.1 and a concentration of 0.01 mol / l. The sorbent was washed with the same buffer. Cytoplasmic MDH was eluted (690 U,% of total activity). The sorbed mitochondrial MDH buffer was then eluted to which 0.5 mol / L potassium chloride was added. A 12 ml fraction was obtained which contained 0.3 mg protein / ml and 180 U mitochondrial MDH (ml), ie a total activity of 2,160 U, which is 93.5% of the sorbed activity; spec. activity 600 U / mg protein, purification factor - 8x).

Example 3

The procedure was analogous to Example 2, except that the same material as in Example 1 was used as the column filler. The captured enzyme was eluted with 1 mM NAD in phosphate buffer, pH 7.1, 0.1 mol / l. The desorption yield was 89% (based on captured mitochondrial MDH, preparation specific activity 640 U / mg. *

Example 4

The procedure was as in Example 3, but the elution solution contained, in addition to 1 mmol / l NAD, 5 mmol / l sodium sulfite. The yield and specific activity were similar to Example 3, but the captured enzyme eluted at a narrower peak (9 ml fraction volume).

Example 5

The procedure was analogous to Example 3, except that the elution solution contained 1 mmol / l NADH. The desorption yield was 95% (based on captured activity), the specific activity of mitochondrial MDH in the eluate was 700 U / mg.

Example 6

The procedure was analogous to Example 3 except that the batch was half and the phosphate buffer used had a concentration of 0.1 mol / l and a pH of 6.8. The desorption was performed with the same buffer to which 10 mmol / l NAD and 0.1 mol / l sodium sulfate were added. The yield was 74%, specific activity 620 U / mg protein, fraction volume 8 ml.

Example 7

The procedure was analogous to Example 3, except that 0.05 mol / l Tris buffer pH 7 (a solution of 6.05 g of tris / hydroxymethyl / aminomethane in a pH of 1 v / v water) was used instead of phosphate buffer pH 7.1 was adjusted by adding hydrochloric acid to the indicated value). The captured enzyme was desorbed with Tris buffer of the above pH and concentration to which 0.2 mol / l potassium chloride and 0.5 mmol / l NADH were added. The desorption yield was 85% (based on captured mitochondrial MDH), specific activity 670 U / mg, fraction volume 11 ml.

Claims (1)

object of the invention Method for isolation and purification of mitochondrial malate dehydrogenase from extracts and concentrates prepared from animal hearts, characterized in that the enzyme is sorbed from buffer solutions of pH 6-8 at 0.002-0.1 mol / l to a modified macroporous gel copolymer of 2-hydroxymethylmethacrylate with ethylenedimethacrylate , which contains 1,4-diamino-9,10-anthraquinone-3-sulfonic acid in the side chains covalently bound by arylidenesulfonylethoxy groups, simultaneously sorbed accompanying substances are removed from the sorbent by washing with a buffer of pH 6 to 8 and a concentration of 0,002 to 0,2 mol And finally the captured enzyme is desorbed with the above-defined wash buffers to which 0.2 to 1 mol of sodium or potassium hydrochloric or sulfuric acid and optionally 0.1 to 10 mmol / l of nicotinamide-adenine dinucleotide, nicotinamide-adenine have been added. reduced-nucleotide, or nicotinamide-adenine-dinucleotide mixed with 3 to 5 us with a molar excess of sulfuric acid salts.