RU2129438C1 - Method of preparing ovoinhibitor - Google Patents

Abstract

FIELD: biotechnology and biochemistry. SUBSTANCE: object of invention can find use as ingredient of therapeutic preparations, food additives, and element of diet therapy. Native fraction of chicken egg is isolated with distilled water, purified on water-equilibrated hydrophobic sorbent polychrome I. Active protein is eluted with distilled water after which inhibitor is subjected to rechromatography on ion-exchange sorbent. Molecular weight of inhibitor is 50120 Da and inhibitory activity with respect to trypsin 26.7 inh.un. EFFECT: increased yield.

Description

 The invention relates to medicine and the food industry, biotechnology and biochemistry and relates to a method for producing an ovoinhibitor of proteinases, which can be used as an ingredient in medicines, food additives, and therapeutic nutritional elements.

 The development of uncontrolled proteolysis is the cause of serious disorders in the body, therefore, proteinase inhibitor proteins are being studied intensively, which can be used in replacement therapy in the treatment of diseases accompanied by excessive activation of the body's proteolytic systems (inflammatory processes, pancreatitis, malignant neoplasms). In addition, proteolytic enzyme inhibitors are used to stabilize protein medications and limit proteolysis in the food industry.

 Protein proteinase inhibitors are isolated from various sources of plant and animal origin.

 A known method of an inhibitor of the Bauman-Birk type (BBI) from soybean culture, comprising the following stages: removing insoluble substances from soybeans, defatted with hexane and subjected to extraction with an aqueous solution of acid, in order to obtain soluble protein substances with a concentration of not less than 50%; adding acetone to soluble substances to obtain a crude inhibitor concentrate; dilution of the concentrate with water; removing insoluble substances from the diluted inhibitor concentrate and adding acetone to it in order to obtain a water-soluble and acetone insoluble Bauman-Birk inhibitor concentrate; drying the resulting concentrate [1]. The molecular weight of the soybean inhibitor is 8000 Da.

 The possibility of using a soybean inhibitor in the treatment of malignant neoplasms is being studied [2].

 A known method of producing a pancreatic inhibitor of the Kunitz type from the organs of cattle [3].

 The essence of the method is that from the waste products of insulin by ion exchange chromatography, chemical purification, salting out, gel chromatography and sedimentation, a high-yield substance BPTI, called ingiprol, is isolated in high yield. The molecular weight of a Kunitz-type inhibitor is 6500 Da.

 Kunitz-type pancreatic inhibitor is the active principle of drugs widely used in clinical practice, such as trasilol, grodox, contracal and aprotinin. However, a significant drawback of this inhibitor is its use in high doses due to the rapid removal from the bloodstream after intravenous injection, which is apparently due to the molecular weight of BPTI. Therefore, it is highly relevant to develop methods for the isolation of higher molecular weight proteinase inhibitors from other biological objects.

 Closest to the claimed method according to the technical nature and the achieved result is a method for producing an ovoinhibitor from egg protein, including the deposition of an inhibitor with trichloroacetic acid with acetone, followed by dialysis and lyophilization of the precipitate; the dry precipitate is dissolved in a small volume of water, the pH of the solution is adjusted to 4.0 and applied to a column with KM cellulose [4]. Chromatography is carried out in a stepwise gradient: (1) a solution of 0.1 M ammonium acetate buffer (pH 4.0), which elutes the fraction containing mainly ovomucoid; (2) 0.1 M ammonium acetate buffer (pH 4.9), which elutes the fraction containing an ovomukoid and a small amount of an ovoinhibitor, and (3) 0.1 M ammonium acetate buffer (pH 5.0) plus 1 M NaCl, which elutes the bulk of the inhibitor. After dialysis and lyophilization, the third fraction was dissolved in 0.005 M Tris-HCl buffer (pH 7.6) and dialyzed against the same buffer for 12 hours. The dialyzed product is centrifuged to remove insoluble material and applied to a column of DEAE-cellulose (2.3 x 45 cm) equilibrated with 0.005 M Tris-HCl buffer (pH 7.6). Chromatography is carried out by a linear gradient of 0.005-0.2 M Tris-HCl buffer (pH 7.6).

 The active fractions are combined, desalted on Sephadex G-25 and lyophilized. An ovoinhibitor with a molecular weight of 48,000 ± 2 Da is obtained.

 The disadvantages of the prototype method include its duration (9 stages are performed within about 9 days) laboriousness (requires multiple dialysis and lyophilization of the product). The output of the ovoinhibitor is about 1.5% of the amount of the original egg protein.

 The objective of the invention is to obtain a highly active ovoinhibitor in a simpler way with high yield.

 The problem is solved in that in the method for producing an ovoinhibitor, which provides for the isolation of the active fraction from chicken egg protein and its chromatographic purification, the isolation is carried out with distilled water, and purification is carried out on polychrome I with elution of the active protein with water and rechromatography of the inhibitor on an ion-exchange sorbent.

 The essence of the method is as follows. The protein of the chicken egg is mixed with an equal volume of distilled water and the resulting solution is centrifuged. The supernatant is drained, the precipitate is mixed with water and centrifuged again. The precipitate is discarded. In the supernatant, protein is determined according to the Dowry method [5] and inhibitory activity against trypsin. The result is an aqueous solution containing protein components having high inhibitory activity against trypsin (34.5 IE). Then, proteins are purified using hydrophobic chromatography on polychrome I (polytetrafluoroethylene). The active protein is eluted with polychrome distilled water. The fractions containing the active protein are combined and freeze-dried. The eluate contains the main component with a molecular mass of 50 120 Da and two minor components with a molecular mass of 81280 and 12700 Da. The inhibitory activity against trypsin is 33.0 IE. Further purification of the inhibitor is carried out using ion exchange chromatography. Elution is carried out with 0.001 M ammonium acetate acetate buffer pH 4.5. In this case, the active protein goes immediately beyond the free volume. Trypsin-inhibiting fractions are combined and freeze-dried. The yield of purified ovoinhibitor is 47.0% of the protein introduced into the column or 19.74% in terms of the initial water-soluble chicken egg protein. The duration of the allocation is approximately 4 days.

 The proposed method for producing an ovoinhibitor from chicken egg protein is characterized in that already at the first stage of isolation, the fraction of native components with high inhibitory activity against trypsis is concentrated (34.5 IE); at the second stage (purification on polychrome I), an ovoinhibitor of only two minor proteins is released. The high capacity of the sorbent and its hydrodynamic properties make it possible to obtain preparative amounts of the substance and reduce the cleaning time. In the third stage, a pure ovoinhibitor was obtained with a high yield of 19.74% of the original chicken egg protein and high inhibitory activity against trypsin (26.7 IE).

 The possibility of implementing the proposed method is illustrated by an example.

 Example.

 To the proteins of 5 eggs (volume 170 ml, weight 170 g), an equal volume of distilled water is added, mechanically mixed until a white mucous precipitate appears, and the resulting solution is centrifuged for 15 minutes in a K-70 centrifuge (GDR) at 6000 rpm. The yellowish supernatant is drained, the precipitate is mixed with 50 ml of water and centrifuged again. The supernatants are combined and the pellet is discarded. Protein in solutions for all operations is determined by the Lowry method. The inhibitory activity against trypsin is determined using a synthetic substrate - N-benzoyl-D, L-arginine p-nitroanilide (BAPA). The calculations are based on the determination of the amounts of inhibitor required to achieve 50% inhibition in 1 minute based on 1 g of trypsin.

 The result is an aqueous solution containing 12 g of protein (according to Lowry) with a specific activity of 34.5 IE.

 Protein is applied to a column with polychrome (17x4 cm), previously balanced with water. After applying the whole supernatant, the polychrome is washed with distilled water until the unbound active protein is completely removed. The eluent (1.5 L) containing the active protein (42% of the original) is combined and freeze-dried. Protein homogeneity and its molecular weight are determined by electrophoresis in 15% PAAG with 1% sodium dodecyl sulfate in the presence of β-mercaptoethanol in the protein preparation according to the method [6]. As protein markers use a set of standards for determining the molecular weight of "Serva" (Germany).

 The eluate contains the main component with a molecular mass of 50 120 Da and two minor components with a molecular weight of 81,380 and 12,700 Da, apparently ovotransferryl and cystatin, respectively. The inhibitory activity against trypsin is 33.0 IE.

Further purification of the inhibitor is carried out using ion exchange chromatography: 1.8 g of lyophilized eluate are dissolved in 5 ml of 0.001 M ammonium acetate buffer with a pH of 4.5 and applied to a column with SP-Sephadex G-25 (35x1.7 cm), balanced same buffer. Elution is carried out with the original buffer at a rate of 7 ml / min. In this case, the active protein goes immediately beyond the free volume. Trypsin-inhibiting fractions are combined and freeze-dried. The yield of purified inhibitor (840 mg) is 47% of the protein added to the column (19.74% in terms of the original protein). The homogeneity of the drug is proved by electrophoresis in SDS page. The molecular weight is 50120 Yes. The inhibitory activity against trypsin is 26.7 IE. In 0.1 M Tris-HCl buffer (pH 8.0), the protein has a maximum adsorption at 280.9 nm with a coefficient of E $\genfrac{}{}{0ex}{}{\text{0.1}}{\text{280.9}}$ = 0.62, which corresponds to the literature data for ovoinhibitor. The absence of an excess in the region of 290–293 nm corresponding to tryptophan is also characteristic of an ovoinhibitor isolated from chicken egg protein [4].

Literature
1. US patent N 5217717, 78, 47/00; B 11B 1/00; A 23 L 1/20, 1993.

 2. Witschi H., Kennedy A.R. // Carcinogenesis, 1989. V. 10. P2275-2277.

 3. Larionova N. I., Balabushevich N. G., Gladysheva I. P., Moroz N. A., Kazanskaya N. F., Polekhina O. V., Donetsk I. A. // Questions of medical chemistry. 1994.V. 40. N 3. S. 25-31.

 4. Liu Woo-Hoe, Means Gary E., Feeney Robert E. // Biochim. Biophys. Acta. 1971. V.229. P. 176-185.

 5. Lowry O. H., Rosebrough N. J., Farr A. L., Randall J. // J. Biol. Chem. 1951. V. 193. P.265-275.

 6. Osterman L.A. // Methods for the study of proteins and nucleic acids. M. "Science". 1981.

Claims (1)

 A method for producing an ovoinhibitor, which involves the isolation of the active fraction from chicken egg protein and its chromatographic purification, characterized in that the isolation is carried out with distilled water, and purification on polychrome 1 with elution of the active protein with water and rechromatography on an ion-exchange sorbent.