SU1388792A1 - Method of isoelectric focusing of ampholyte - Google Patents

Abstract

This invention relates to an isoelectric focusing method of ampholytes and can be used in a chromo graphic technique for the separation of protein mixtures. The invention allows to increase the separation capacity with simultaneous desalting (separation of ampholytes into fractions differing in pH by 1-2). The effect is achieved by using as the solid phase cation exchangers in hydrogen form and anion exchangers in hydroxyl form and desorption of ampholytes with cation exchangers with alkali solution and with anion exchangers with acid solution. 1 il.

Description

The invention relates to biochemistry, in particular, to methods for separating protein mixtures, and can be used in the production of narrow fractions of amphipolytes of carriers, amino acids, and for separating petids and proteins according to their isoelectric points.

The aim of the invention is to increase

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fractions 4-9 and glutamic acid in fractions 9-13.

Example4. A 5 ml solution containing 0.5 mmol of glutamic acid, serine, and lysine hydrochloride is applied to a column containing 9 ml of Separon HEMAQ anion exchange resin in OH-form. Provo

separation capacity and ynpo-jg injection with 0.1 M HC1 solution, separation technology separation.

The drawing shows optical absorption diagrams and pH fractions.

Example. A 10 ml column with a cation-exchange resin Agaro-15 ZOY 4B containing IO µmol / ml SOj-groups in protonated form is added with 50 ml of a solution containing 0.1% ampholytes (for isoelectric focusing in the pH 4 range). -7), which are a mixture of poly-aminopolycarboxylic acids with iso-electric points lying in the pH range 3.5–10.0, 0.5 mg of egg protein, and then displace the fractions with a 6 mM solution.

Example2. A 17-mm column with a cation-exchange resin SP-Sephadex C-25 in the protonated form, capacity for Na ions 2.5 mmol / g of dry resin (a polysaccharide with sulfopropyl fragments) is added with 5 ml of a solution containing 5% ampholytes. carriers in the range of pH 4-9 and 1% NaCl. Squeezed out with a 10 mM KOH solution. In the pH range 4-9, 10 fractions of 7 ml each are collected. Fractions free from

20

25

thirty

35

while lysine is in effluent from 6.0 to 12.7 ml, series from 10.6 to 24.8 ml, glutamic acid from 24 to 29.8 ml.

Example 5 (comparative). The experiment was carried out similarly to that described in Example 4, but the applied probe was further eluted with a solution of glutamic acid and lysine (0.75 mM each). Lysine is from 5 to 60 m of effluent, serine from 15 to 28 ml and glutamic acid from 26 to 60 ml.

From the results it can be seen that, by a known method, only the first part of lysine can be obtained in pure form, all other components are permanently present in the mixture.

PRI me R 6 (comparative). The experiment was carried out similarly to that described in Example 4, but anion exchanger in the CHjCOO - form was used as the sorbent. The effluent contains, in addition to a mixture of ampholytes (lysine, glutamic acid and serine), and eluent ions.

Claims (1)

Invention Formula Salts that have an electrical conductivity lower than 11000 with isoelectric focusing create pH gradients in dia-40 ion-exchange resins in a column with 1-2 pH units. PRI me R 3. In a 9 mm column with anion-exchange resin Separon HEMAQ in OH-form, capacitance C1 the subsequent desorption and selection of ampholyte fractions, characterized in that, in order to increase the separation ability and 300 µmol / ml simultaneously (spherical desalting, 5 ml of ionoliglycolmeacrylate gel with quaternary ammonium groups) are added, containing About, 5 mmol of lysine, serine and glutamic acid. The extrusion was carried out with 100 mM HC1, collecting 1 ml fractions. Lysine is present in fraction 1-4, serine in 50 cation exchangers in hydrogen form or hydroxy-anion exchangers are used as exchangers with resins, and desorption of ampholytes is carried out with an aqueous solution of an acid when using anion exchangers or bases as the sorbent when using cation exchangers.  pressing with 0.1 M HC1 solution, while lysine is in the effluent from 6.0 to 12.7 ml, series from 10.6 to 24.8 ml, glutamic acid from 24.1 to 29.8 ml. Example 5 (comparative). The test was carried out as described in Example 4, but the applied sample was then eluted with a solution of glutamic acid and lysine (0.75 mM each). Lysine is from 5 to 60 ml of effluent, serine from 15 to 28 ml and glutamic acid from 26 to 60 ml. From the results it can be seen that, by a known method, only the first part of lysine can be obtained in pure form, all other components are permanently present in the mixture. PRI me R 6 (comparative). The experiment was carried out as described in Example 4, but anion exchange resin in the CHjCOO form was used as the sorbent. Effluent contains in addition to a mixture of ampholytes (lysine, glutamic acid and serine) and eluent ions. Invention Formula The method of isoelectric focusing of ampholytes by sorption on the subsequent desorption and selection of ampholytic fractions, characterized in that, in order to increase the separation capacity and, at the same time, desalting, as an ion cation exchangers in hydrogen form or hydroxy-anion exchangers are used as exchangers with resins, and desorption of ampholytes is carried out with an aqueous solution of an acid when using anion exchangers or bases as the sorbent when using cation exchangers. 0.30 .20 .1 Compiler V. Mkrtychan Editor T. Parfenova Tehred M. Khodich Corrector O. Kundrik Order 1575/46 Circulation 847 VNIIPI USSR State Committee for inventions and discoveries 113035, Moscow, Zh-35, Raushsk nab., 4/5 PH at Y6 20 O f Subscription