CS242391B1 - Method of cystathionines,cysteine proteinases' natural inhibitors insulation from solutions won from natural materials by means by means of immobilized cysteine proteinases - Google Patents

Abstract

The method consists in isolating cystatin from solution using cysteine proteinase immobilized by glutaraldehyde polymerization to a polymeric carrier based on polyethylene terephthalate in a batch configuration at pH 5 to 8, preferably at pH 6.5% subsequent separation of the solid phase with cystatin and re-suspending it into the aqueous phase, cystatin is released from binding with immobilized cysteine proteinase to aqueous solution at a pH of less than 3.5 or greater than 10.0.

Description

Process for the isolation of cystatins, natural inhibitors of cysteine proteinases, from a solution obtained from natural materials by means of immobilized cysteine proteinases

The method consists in isolating cystatin from solution by cysteine proteinase immobilized by polymerization of glutaraldehyde onto a polymer carrier based on polyethylene terephthalate in a batch arrangement at a pH of solution of 5 to 8, preferably at pH 6.5 after subsequent solid phase separation with cystatin and resuspending in the aqueous phase For example, cystatin is released from binding with immobilized cysteine proteinase to an aqueous solution at a pH of less than 3.5 or greater than 10.0.

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The invention relates to the isolation of cystatins, natural inhibitors of cysteine proteinases, from solutions prepared from eggs, egg white, tissue extracts and animal body fluids, fresh, dried or frozen, by cysteine immobilized proteinases.

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The procedures used to date for the isolation of cystatins-cystatins are natural inhibitors of proteinaceous cysteine proteinases of 10 to 15 kD, stable in solutions of pH 2 to 12, stable in aqueous solutions at temperatures above 80 G, capable of binding to both active and for inactivated cysteine proteinase derivatives, utilizing active or inactivated cysteine proteinases immobilized on polymeric carriers based on modified agarose gels, such as cyanogen bromide-activated Sepharose 4B gel, supplied by Pharmacia Sweden, or Reacti-gel from Pierce, USA, by column affinity chromatography . These gels are characterized by low mechanical resistance, do not allow pressure filtration or intensive mixing with rotating stirrers, without damaging the carrier.

The present invention is based on the isolation of cystatins by cysteine proteinases immobilized by polymerization of glutaraldehyde to polyethylene terephthalate. The resulting immobilized proteinase suspension on the rigid solid phase particles can be filtered and mixed by conventional techniques, which is preferably used for the isolation of cystatins.

An immobilized cysteine proteinase suspension is prepared by binding from solution to polyethylene terephthalate according to the procedure of U.S. Pat. No. 195,568.

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To isolate cystatins, the immobilized cystein proteinase suspension is mixed with a solution containing active cystatin. The pH of the solution is adjusted in the range of 5-8 in the resulting suspension.

After sufficient solid phase binding of cystatins is achieved, it is separated from the suspension by filtration or sedimentation, freely or in a centrifuge, and washed from the non-specifically sorbed components of the starting cystatin solution. Distilled water in at least six portions in an amount of 200 times the dry weight of the carrier has proven to be suitable for a suitable wash, by blasting, blasting, decanting or filtering.

After washing, the cystatin from the solid phase is eluted with a solution of pH less than 5.5 or greater than 10.0 and separated by filtration through a paper filter or by centrifugation or decantation. The number of possible uses of the solid phase depends on the quality of washing prior to elution and elution conditions.

Depending on the ratio of the eluate to the volume of the starting cystatin solution, a different degree of concentration of cystatin in the eluate to the starting solution can be achieved, while removing most of the ballast components from the cystatin solution. After concentration of the cystatin solution by freeze-sublimation and gel filtration on a suitable support, cysteine proteinases mobilized to polyethylene terephthalate can be obtained, cystatin preparations which exhibit homogeneity by electrophoresis in a 15% polyacrylamide gel in the presence of sodium dodecyl sulfate in the Laemmli arrangement. /U.K. Laemmli: Nature, / London /, 227 »p. 680 A97O //.

The use of a polymeric carrier, polyethylene terephthalate, with attached cysteine proteinases for the isolation of cystatins of the present invention therefore presents a novel approach to the problem of cystatin isolation and its main advantages lie in the high mechanical strength of the proteinase mobilized particles, ensuring good filterability and suspension, in easy separation of the solid phase by sedimentation, the possibility of recycling which reduces the disadvantage of the batch process compared to the ko242 391 ion arrangement, thus lower yield, further higher affinity step execution speed, which is manifested especially in processing large volumes of low cystatin solution, low import price and low cost polymeric carrier for proteinase immobilization. A disadvantage of the present invention is the need to use larger volumes of wash solutions to ensure good purity of the resulting cystatin preparation, but of low cost and good availability.

An essential advantage of the present invention is the possibility of using a proteinase immobilized to polyethylene terephthalate for the isolation of cystatins even from solutions which are not free from colloidal particles which are unusable for column affinity, as well as solutions of high viscosity (e.g. 3x concentrated white).

The yields of cystatin column chromatography on affinity carriers are reported by the authors in the range of 20 to 40%. Using the present invention, the yield in the particular case of egg white is known to have an approximate cystatin content of intact material of about 10% in the first cycle, but can be increased by recycling, despite the hen egg white being a cheap raw material accessible in large quantities generated as waste in the production of mayonnaise.

Example 1

A suspension containing 1.25 g of polyethylene terephthalate in 25 ml of saline was placed in a thermostat after addition of 12.5 ml of a 25% glutaraldehyde solution and mixed, and the polymer was activated at 37 ° C for 14 hours. The polymer was separated by filtration on a Buchner funnel through a paper filter and transferred without washing to a papain solution containing 300 mg papain in 30 ml 0.1 M phosphate buffer containing 0.002 M ethylenediamine tetraacetic acid (hereinafter EDTA), pH 6.3. 3.3 ml of a 1% glutaraldehyde solution was added to the suspension under stirring with a magnetic stirrer. After three hours of stirring the suspension at 22 ° C, the papain-bound polymer was separated by filtration on a Buchner funnel through a paper filter and washed successively for 2 dm? distilled water on filter. The immobilized papain thus prepared was

4,242,391 were stored in 1 N NaCl solution acidified with HCl to pH 3.0 at 4 ° C for several months without loss of binding capacity.

Cystatin-containing extract was prepared from bovine kidney. 800 g of frozen bovine kidneys were thawed, mechanically defatted and ground in a meat mincer, then mixed with

I 600 ml of distilled water and homogenized in a conventional kitchen mixer for 5 minutes. After standing for 10 minutes, coarse tissue debris was removed from the homogenate by centrifugation at 6,000 xg for 10 minutes and the supernatant was further centrifuged at 25,000 xg for 30 minutes, the pH of the supernatant was adjusted to 11.0 by addition of 5M NaOH and allowed to stand. after. for 2 hours. All operations were performed at 4 ° C.

After standing for 2 hours at pH 11.0, the pH of the solution was adjusted to 7.5 by the addition of 11 M HCl and the resulting precipitate was centrifuged at 6000 x g for 10 minutes. The pH of the solution was then adjusted to 2.8

II M HG1 and the solution was heated to 90 ° C in a water bath over 20 minutes. After standing at this temperature for 10 minutes, the solution was quenched under running water to 25 ° C and the pH was adjusted to 5.4 by the addition of 5 M NaOH. The resulting precipitate was centrifuged at 6000 x g for 10 minutes.

150 ml of the clarified extract thus obtained was mixed with a suspension containing 1 g of polyethylene terephthalate-mobilized papain in 50 ml of 0.1 M phosphate buffer containing 0.002 M EDTA and 0.002 M L-cysteine hydrochloride, pH 6.3 · The suspension was vigorously stirred with a magnetic stirrer. The solid was collected by filtration through a paper filter on a Buchner funnel, washed with 1 dmL of distilled water, and suspended in 150 mL of distilled water. In the suspension, the pH was adjusted to 2.8 by the addition of 11 M HCl, and after stirring at 25 ° C for 10 minutes, the solid phase was separated by filtration. The solution containing the released cystatin was concentrated by freeze-drying.

Example 2

Using the procedure of Example 1, 4 g of immobilized papain (weight of enzyme-linked support) were prepared. From 30 pieces of fresh chicken eggs B / Prague Poultry Plant n.p., plant Libuš /, 900 ml of egg white was separated and mixed with 900 ml of 0.25 M NaCl in a kitchen mixer. The resulting solution had a pH of 8.7

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5M sodium formate, pH 5.0, the pH in the white solution was adjusted to 6.2, and the resulting ovomycin precipitate was centrifuged at 6000 x g for 10 minutes »

The supernatant was mixed with a suspension containing 4 g of papyrate-bound polyethylene terephthalate in 100 ml of 0.1 M phosphate buffer with 0.002 M EDTA, pH 6.5, which did not contain L-cysteine hydrochloride. The suspension was stirred overnight with a magnetic stirrer. The solid phase was separated by centrifugation at 1000 x g and washed with 6x11 distilled water by centrifugation at 1000 x g for 1 minute.

The sediment was transferred to 200 mL of distilled water and cystatin was released by acidifying the slurry of 11M HCl to pH 1.8. After stirring for ten minutes with a magnetic stirrer at 25 ° C, the solution containing the release of cystatin was separated from the solid phase by filtration through a pleated paper filter and concentrated by freeze-drying.

Except for elution, all operations were performed at 4 ° C.

Example 3

The egg white, once used and separated from the solid phase prior to washing in Example 2, was re-mixed with the immobilized papain suspension separated after elution in Example 2 and under conditions similar to Example 2 the cysratin remaining in the white solution was coupled to the mobilized papain and eluted. bound cystatin. The yield of cystatin in the eluate was 21% lower than in Example 2.

Example 4

27.5 g of dried hen egg white (Prague poultry factory) were mixed and 80 ml of distilled water. The resulting foam was removed by centrifugation, 1000 x g for 10 minutes or standing for 1 hour. Removal of the ovomycin, binding of cystatin and washing of the suspension of mobilized cysteine-bound papain was performed as in Example 2. The pH of the suspension was adjusted to 11.5 by the addition of 5 M NaOH and after stirring at 25 ° C for 10 minutes the solid was collected by centrifugation at 1. The pH was adjusted to 7.0 by addition of HCl in the supernatant. The supernatant containing the released Cystatin was concentrated by freeze-sublimation.

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Cystatin eluates contained 5 to 10 mg of active cystatin for the procedures described in Examples 2, 5, 4 S 0.7 and 2.0 mg of cystatin for the procedure of Example 1. Cystatin levels were determined by titration with a cathepsin H 2 O solution. known content of proteinesis, using 4-nitroanilide L-phenylalanine as substrate.

Claims (1)

SUBJECT OF THE INVENTION Method for isolating cystatins, natural inhibitors of cysteine proteinases, from solutions obtained from natural materials by means of immobilized cysteine proteinases, characterized in that cystatin from solution binds to cysteine proteinases immobilized by polymerization of glutaraldehyde to a polymer carrier based on polyethylene terephthalate in a batch arrangement at pH 5-8. with benefit at pH 6.5, after subsequent separation of the solid phase with cystatin and resuspension in the aqueous phase, cystatin is released from the immobilized cysteine proteinase binding to an aqueous solution at a pH of less than 3.5 or greater than 10.0.