The complex nature of cationic proteins and method thereof

 

(57) Abstract:

The invention relates to medicine, in particular to drugs, protein-containing, and methods for their preparation, namely natural cationic proteins from the tissues of the thymus gland of calves - histones. The invention consists in obtaining a natural complex of cationic proteins containing histones H2A, as H2B, H3, H4 of the tissues of the thymus gland of calves, which form a stable solution of heterogeneous molecular weight conjugates of proteins formed by covalently interconnected by free amino groups of lysine histones, as well as methods for their production using a cross-linking agent, pH, temperature and time of incubation of the reaction mixture to complete the modification of the available amino groups of proteins in the product. The technical result of the present invention is a purified complex of histones from the tissues of the thymus gland of calves with stability in solutions of high ionic strength and low pH values, preserving the antibiotic activity of all classes of histones, incorporated easily diffuse through biological membranes, and method for producing such a complex. 2 S. p. f-crystals, 2 Il.

The invention otnosenia, namely, natural cationic proteins from the tissues of the thymus gland of calves - histones.

The histone - cationic (basic) proteins contained in the cell nuclei of all tissues of animals and plants. In total there are five classes of histones, which differ in content in the molecule of the basic amino acids lysine and arginine.

Classes of histones have the following designations: histone H1 (rich in lysine), histone H2B (moderately rich in lysine), histone H2A (rich in arginine and lysine), histone H3 (rich in arginine), histone H4 (rich in arginine and glycine). Histones heterogeneous and molecular weight. The highest molecular weight has a histone H1 - 21500, the next largest histones H3, H2A, H2B, H4 - 15320, 14000, 13774, 11280 Yes, respectively. Exogenous histones are antibacterial and antiviral agents of broad-spectrum, and some of them have a potentiating effect on the activity of antibacterial agents in a joint application. However, certain classes of histones vary in range and intensity of their direct antibiotic action, which limits the possibility of using each of them as pharmacological agents of broad-spectrum. the e effect on the activity of antibacterial agents, and can also act as a protein carrier to transport those medicines, which are characterized by limited permeability through cell membranes and tissue barriers, allowing you to expand the range of practical application of histones as a drug [1].

A method of obtaining complex natural cationic proteins-histones from tissue of calf thymus [2]. This known method of producing a complex of histones based on chromatographic fractionation of total histone sulfuric acid from tissue of the thymus gland of calves, including primary dissolution of the original product in an environment containing 0,02 N. HCl, 8 M urea, 1% 2-mercaptoethanol, chromatography on Biogel P-60 at a pH of 1.7, which receive four protein peak: histone H1, a mixture of histones H2A+H3, histone H2B and histone H4, primary dialysis target product against distilled water and its primary lyophilization. This method allows the separation of histone H1 from the histones H2A, H2B, H3, H4, but its disadvantages include proteolytic degradation of individual classes of histones during chromatographic separation.

The closest in technical essence to this isposal allows to obtain a highly complex nature of cationic proteins from the tissues of the thymus gland of calves contain non-degraded histones H2A, H2B, H3, H4, by preventing their proteolytic degradation in process chromatography, which is used for fractionation of the preparation of histone sulfuric acid from tissue of the thymus gland of calves that do not contain histone H1, which is associated nonhistone proteins with proteinase activity. This known method of producing a complex of histones based on the primary dissolution total histone sulfuric acid from tissue of the thymus gland of calves in HCl, the processing solution of inorganic acid (perchloric acid) to a final concentration of 0.5 M to separate soluble in perchloric acid of the histone H1 from insoluble histones H2A, H2B, H3, H4, the precipitate was perchlorates of histones by centrifugation, secondary dissolution and chromatographic separation, you get: traces of histone H1, histone H2B, a mixture of histones H2A+H3 and histone H4, then protein fractions are combined and conduct initial dialysis target product against 0,01-0,02 N. HCl and its primary lyophilization. In solutions of low ionic strength (physiological sodium chloride solution or 0.01 M sodium phosphate) and in the environment of the organism histones H2A, H2B, H3, H4 interact and exist in veterinarinary histones much more than between homologous and non-covalent interactions (ionic bonds, hydrogen bonds, hydrophobic interactions) between heterologous histones is peculiar: a strong protein-protein interactions are formed between pairs of histones H2A-H2B, H3-H4, H2B-H4 and weak links between pairs of H2A-H3, H2B-H3, H2A-H4 [4]. However, non-covalent interaction between histones in solutions of high ionic strength and low pH values is violated, and in such solutions the histone proteins exist as monomers [5].

Problem to be solved and the technical result of the present invention is a purified complex of histones from the tissues of the thymus gland of calves with stability in solutions of high ionic strength and low pH values, preserving the antibiotic activity of all classes of histones, incorporated easily diffuse through biological membranes, and the receipt of such a complex of histones is associated a single inventive concept with the creation of the method of its production.

Target product under specific conditions is a complex chemically cross-stitched proteins formed Kovalenko interconnected histones from a number of H2A, H2B, H3, H4 with sustainable education is more than 70 kDa, respectively. This protein complex has no histone H1 and nonhistone proteins that are associated with it, and no degradation products of separate classes of histones and histones are the complexes in solutions of a wide range of ionic strength and pH due to covalent binding of interacting proteins, it is achieved that the product obtained after the initial lyophilization, dissolved in the solution medium ionic strength and exercise kavalee binding proteins cross-linking agent without precipitation of proteins from solution at pH the temperature and time of incubation of the reaction mixture to complete the modification of the available amino groups of proteins in the product with the formation of a stable solution of conjugates of proteins with a molecular mass of not more than 30 kDa and not more than 70 kDa, and the reaction of covalent binding stops regenerating agent, followed by repeated dialysis target product against a buffer solution of low ionic strength, it again lyophilizer.

In the private case complex chemically cross-stitched natural cationic proteins from tissue of calf thymus contains stable in solution are heterogeneous in molecular weight conjugates of proteins formed by covalently with whom. Valentino interconnected histones from a number of H2A, H2B, H3, and H4 form dimers and oligomers with a molecular mass of 25-30 kDa and 65-70 kDa, respectively.

In the private case, the method of producing complex natural cationic proteins from the tissues of the thymus gland of calves is as follows: the protein product after repeated lyophilization was dissolved in a solution pH of 7.6-7.8 to a final concentration of protein in solution is not more than 0.4% and the resulting solution was added a bifunctional crosslinking reagent, which is used as glutaric aldehyde in a concentration of not more than 0,510-2mol/l, and conducting the reaction of covalent bonding at a temperature of not more than 23oC, the incubation time of 30 minutes, and repeated dialysis of the target product is carried out at a temperature of not more than 10oC in a period of time not more than 24 hours against 0.01 M phosphate buffer solution pH 7.0.

Due to the high efficiency of interaction with amino groups of proteins bifunctional crosslinking agent is glutaric aldehyde is widely used in the synthesis of covalent conjugates, while the reaction does not require prior modification of proteins. Chemical linking using glutaraldehyde, Aldi using this cross-linking reagent. In each case it is necessary to select conditions maximum binding proteins, which is controlled by the combination of several parameters and depends on the concentration of glutaraldehyde in the incubation medium and the quantity of reagent per unit mass of the protein, the pH of the incubation medium, temperature and time of incubation, in which the reaction product remains in solution, and thus all the available amino groups of proteins modified.

During experiments it was determined that the molar content available to update the amino groups in the preparation containing histones H2A, H2B, H3, H4, 6,7 0,110-4mol/g (the number of free amino groups is determined in reactions with 2,4,6-trinitrobenzenesulfonic (6)), and the effective concentration of glutaraldehyde equimolarly the amount available for the modification of amino groups in the product and is 0.3-0,510-2mol/l When the concentration of the protein in solution of 0.2-0.4% of the reaction product is stable in solution, and modified all of the amino groups of proteins. By increasing the concentration of protein in solution at twice the aggregation of the conjugates of proteins and a gel is formed, and by increasing the concentration of protein and glutaraldehyde twice is formed of ocado is revealed, the linking may be carried out at a pH of 7.6 to 7.8, temperature 21-23oC for 15-30 minutes, and under these conditions there is a complete modification of amino groups of proteins and the reaction product is in solution. The reaction of covalent binding of proteins to stop using a reducing reagent, followed by dialysis of the target product against 0.01 M phosphate buffer pH 7.0 at a temperature of 8-10oC for 18-24 hours and lyophilizers. Estimation of molecular weight and charge of the molecules in the composition of the synthesized target product containing conjugates of histones from a number of H2A, H2B, H3, H4, carried out on the basis of the results of its electrophoretic analysis in polyacrylamide gel in different systems division.

Example 1.

Preparations of total histone sulfuric acid from tissue of the thymus calves receive a known manner [7] . Sample total histone sulfuric acid from tissue of the thymus gland of calves in the amount of 100 mg was dissolved in 20 ml of 0.01 N. HCl. To the protein solution was added perchloric acid to a final concentration of 0.5 M. the Solution centrifuged. At this stage, separate soluble in 0.5 M perchloric acid histone H1. The precipitated perchlorate histone containing histones H2A, H2B, H3, H4, washed with acetone and vyaspeeth, containing histones H2A, H2B, H3, H4, dissolved in 5 ml of a solution of the following composition: 0,015 N. HCl, 005 M sodium chloride, pH of 1.7. Protein solution contribute to column length 94 cm and 2.5 cm in diameter, filled with gel acrylics P-30. Chromatography was performed at room temperature. The elution of protein from the column conducting the initial solution. Check out the protein with the column carried out spectrophotometrically at a wavelength of 230 nm and on the basis of these data to build a profile of elution of the protein from the column. Get four protein peak: peak 1 - traces of histone H1, peak 2 is a mixture of histones H2A+H3, peak 3 - histone H2B, peak 4 - histone H4. Protein fractions corresponding to the peaks 2-4, unite and deleteroute against 0,01 N. HCl atoC and lyophilizers. Sample preparation after the initial lyophilization containing histones H2A, H2B, H3, H4, dissolved in 3 ml of 0.1 M phosphate buffer pH of 7.9 to final concentration of protein in solution 4 mg in 1 ml of the protein solution with constant stirring on a magnetic stirrer is added dropwise a solution of glutaraldehyde (original solution previously diluted 10-fold) to a final concentration of 0,510-2mol/l and in 2 minutes using 0.1 M NaOH the pH of the reaction mixture was adjusted to 7.8 (adding glutaraldehyde solution pH CH the AI on a magnetic stirrer and after 30 minutes of exposure reaction is stopped by the addition to the incubation mixture a reducing reagent, then the reaction mixture is twice deleteroute at 10oC against 300 ml of 0.01 M phosphate buffer pH 7.0, consistently for 3 and 15 hours and target product lyophilizer.

To determine the molecular weight of covalent conjugates of histones carry out electrophoretic separation of the target product in 18% polyacrylamide gel in the presence of 0.1% sodium dodecyl sulfate [8]. According to electrophoretic separation in polyacrylamide gel target product is heterogeneous in molecular weight (Fig. 1). In Fig. 1 shows experimental data on the electrophoretic separation of covalent conjugates of histones in 18% polyacrylamide gel in the presence of 0.1% sodium dodecyl sulfate, the time separation of 3 hours and 30 minutes, the amount of protein made in one well of the gel 30 mg, where indicated: (A) preparation of total histone; (B) is a drug containing histones H2A, H2B, H3, H4; (C) the preparation of covalent conjugates of histones; 1-3 - protein zone. When comparing electrophoregram separation covalent conjugates of histones with the original drug containing histones H2A, H2B, H3, H4, and total histone was used as control samples, it is seen that the preparations of conjugates of the Rfall factions of histones. According to this observation is the molecular mass of the obtained conjugates histone determined from a calibration curve constructed with consideration of the sum of marker proteins (albumin, pepsin, Unkasa, cytochrome C, insulin), is in the range of 25-30 kDa and 65-70 kDa when a weight ratio of at least 10:1, respectively (according to the densitometry of protein peaks). The absence in samples of conjugates "free" fractions of histones suggests that they are involved in the complex, i.e., covalently linked to each other via glutaric aldehyde with the formation of dimers and oligomers of histones. It should be noted that electrophoregram in this system electrophoretic separation of proteins has a minor protein zone 3 with a value of Rfclose to the value of Rfhistone H2B. Based on the available data difficult to establish what fraction of histones present in zone 3. It can be assumed that some of the molecules of histone H2B is not involved in the formation of covalent conjugates of histones, however, we cannot exclude that area 3 is homologous dimers of histone H4.

The content in the composition of the covalent conjugates of the histone protein molecules that act in a 15% polyacrylamide gel in the system acetic acid - urea [9].

According to electrophoretic separation in this system, the target product is heterogeneous in charge (Fig. 2). In Fig. 2 shows experimental data on the electrophoretic separation of covalent conjugates of histones in 15% polyacrylamide gel in the acidic buffer system in the presence of 2.5 M urea, the time separation of 3 hours and 30 minutes, the amount of protein introduced on 1 tube of gel, 30 μg (A) and 100 μg (B) where indicated: (A) preparation of total histone; (B) - the preparation of covalent conjugates of histones; 1-3 - protein zone. A comparative analysis of electrophoregram drug covalent conjugates of histones with the preparation of total histone taken as the control sample, it is seen that the target product contains conjugates of histones with low, medium and high electrophoretic mobility, which can be divided into three protein zone. In the preparation of conjugates of histone there are no "free" fraction of histones, which in this division take polyacrylamide gel position with a certain value of Rf. Conjugates of histones zone 1 low electrophoretic mobility and occupy a position in polyacrylates, conjugates of proteins minor zone 2 major and zone 3 is characterized by medium and high electrophoretic mobility in polyacrylamide gel. Thus the conjugates of histones zone 2 have a value of Rfclose to the value of Rfhistone H1 and histone conjugates zone 3 have a value of Rfhigher than the value of Rfof histone H4. The presence of the target product major protein zone 3 indicates that it also has a covalent conjugates of histones with high positive charge on the surface of the molecule.

Example 2. Obtaining a product containing histones H2A, H2B, H3, H4, spend the same way as in example 1, except that chromatography of histones the elution of protein from the column, gel filled acrylics P-30, carried out with a solution that does not contain sodium chloride. Get two protein peak: peak 1 - traces of histone H1, peak 2 is a mixture of histones H2A+H2B+H3+H4. The reaction of covalent binding of histones spend the same way as in example 1, except that the protein solution after its activation with glutaraldehyde add glycine (2 mg/ml of reaction medium) and conducting the reaction of covalent binding, as described above. The presence of amino acids in the incubation medium new product, containing conjugates of histones, assessed the results of electrophoretic analysis of the target product in the two systems polyacrylamide gel, identical when used in the reaction of covalent binding of histones from a number of H2A, H2B, H3, H4, obtained by chromatographic separation on acrilex P-30.

Sources of information

1. Patent N 2045278 of the Russian Federation. The means of potentiating the anti-TB activity of isoniazid/ Ashmarin I. P., Perelman, A. E., Gorukhina O. A., Wisniewski B. I., Gaber, N. E., The Vavilin, GI, BI N 28. - 1995. - S. 127.

2. Von Holt C., Brandt W. F. Fractionation of histones on molecular sieve matrices// In Methods in cell Biology. - 1977. - Vol. 16. - P. 205-225. Acad. Press, New York.

3. Patent N 1319352 A1 of the Russian Federation. The purification method of the preparation of histone H4 from the tissue of the thymus/ Gorukhina O. A., mühlberg A. A., Crieve M. A., Tishkin I.e. 1985.

4. D'anna J. A., Isenberg I. A histone cross-complexing pattern// Biochemistry. - 1974. - Vol. 13, N 24. - P. 4992-4997.

5. Dobretsov, E. , Gorukhina O. A., borschevskaya T. A. Status of the polar groups and the aggregation of histones in aqueous solutions// Biochemistry. - 1969. - T. 34, vol. 4. - S. 806-809.

6. Sarantonis E. G., Diamandis E. P., M. Karayannis I. Kinetic study of the reaction between trinitrobenzenesulfonic acid and amino acids with a trinitrobenzenesulfonate ion-selective electrode// Anal. Biochem. - 1986. - Vol. 115, N 1. - P. 129-134.

7. Patent N 843915 of the Russian Federation. The method of obtaining sums K., Osborn M. The reliability of molecular weight determination by dodecylsulfate polyacrylamide gel electrophoresis// J. Biol. Chem. - 1969. - Vol. 224, N 16. - P. 4406-4412.

9. Panyim, S., Chalkley R. High resolution acrylamide gel electrophoresis of histones// Arch. Biochem. Biophys. - 1969. - Vol. 130, N 1-2. - P. 337-346.

1. Complex natural cationic proteins containing histones H2A, as H2B, H3, H4, of the tissues of the thymus gland of calves, characterized in that the chemically cross-stitched protein complex formed in solution heterogeneous in charge and molecular weight conjugates of histones, covalently linked by free amino groups of lysine histones from a number of H2A, as H2B, H3, H4 with the formation of dimers and oligomers with mol. m is not more than 30 kDa and 70 kDa, respectively, at a weight ratio of 10:1, and the molar content of free amino groups in the original drug is 6,70,110-4mol/g

2. The method of producing complex natural cationic proteins by primary dilution total histone sulfuric acid from tissue of the thymus gland of calves and deposition of the resulting solution of histones H2A, as H2B, H3, H4 solution of inorganic acid and subsequent secondary precipitate dissolution and chromatographic separation of the target product, the subsequent primary dialysis and primary lyophilization, characterized in that the product poluchayuca protein cross-linking agent without precipitation of proteins from solution at pH, the temperature and time of incubation of the reaction mixture to complete the modification of the available amino groups of proteins in the product with the formation of a stable solution of conjugates of proteins, mol. m is not more than 30 kDa and not more than 70 kDa, and as a crosslinking agent using a bifunctional reagent, which choose glutaric aldehyde in a concentration of not more than 0,510-2mol/l when the concentration of the protein in solution is not more than 0.4%, and the reaction of covalent binding is carried out at a pH of 7.6 to 7.8, temperature 23oC, the incubation time is not more than 30 min, and the reaction of covalent binding stops regenerating agent, followed by repeated dialysis target product against a buffer solution of low ionic strength, and the dialysis is carried out at a temperature of not more than 10oWith in a period of time not more than 24 hours against 0.01 M buffer solution of pH 7.0 and target product again lyophilizer.

 

Same patents:

The invention relates to novel conjugates of affinity ligand - matrix containing the ligand associated with the matrix carrier, optionally via a spacer elements located between the matrix and ligand, and new conjugates affinity ligand - matrix, to their preparation and use for the purification of protein materials, such as, for example, immunoglobulins, insulin, factor VII, or human growth factor, or its analogs, derivatives and fragments, and their predecessors

The invention relates to new methods of chromatography, designed for the purification of crude extracts containing cyclosporine, for use in the pharmaceutical industry

The invention relates to structures of nanometer size used to construct the microscopic and macroscopic structures

The invention relates to medicine and relates to a method of evaluation of snake venom on the presence or absence of platelet aggregation (PA1), based on the specific binding with receptor purified and isolated from snake venom PA1 and its shortened standards, allocation method PA1 from snake venom, and pharmaceutical compositions based on it
The invention relates to methods for selection of individual amino acids (tyrosine and tryptophan) from their mixtures and can be used in chemical, food, microbiological industry and agriculture
The invention relates to methods for selection of individual amino acids (tyrosine and tryptophan) from their mixtures and can be used in chemical, food, microbiological industry and agriculture

-l-aspartyl-l-phenylalanine methyl ester from the aqueous solution and the device" target="_blank">

The invention relates to a method of cooling an aqueous solution of aspartame and crystallization from him aspartame, excluding mobilizatio in water crystallization system by (i) filing of a hot aqueous solution of aspartame in the means for dispersing droplets; (ii) dispersion of droplets in an immiscible with water, the fluid whose temperature is at least 20°C lower for drops there was no formation of crystallization centers while passing them through immiscible with water, liquid; (iii) cooling mentioned immiscible with water fluid with the for the drops effectively cooled to achieve an initial supersaturation-L-aspartyl-L-phenylalanine methyl ester inside the drops in the range from 1 to 6, preferably from 1.2 to 4; (iv) collecting the cooled droplets for crystallization aspartame; and (v) provide sufficient time for this

The invention relates to novel conjugates of affinity ligand - matrix containing the ligand associated with the matrix carrier, optionally via a spacer elements located between the matrix and ligand, and new conjugates affinity ligand - matrix, to their preparation and use for the purification of protein materials, such as, for example, immunoglobulins, insulin, factor VII, or human growth factor, or its analogs, derivatives and fragments, and their predecessors

The invention relates to new methods of chromatography, designed for the purification of crude extracts containing cyclosporine, for use in the pharmaceutical industry

The invention relates to structures of nanometer size used to construct the microscopic and macroscopic structures

The invention relates to medicine and relates to a method of evaluation of snake venom on the presence or absence of platelet aggregation (PA1), based on the specific binding with receptor purified and isolated from snake venom PA1 and its shortened standards, allocation method PA1 from snake venom, and pharmaceutical compositions based on it
The invention relates to methods for selection of individual amino acids (tyrosine and tryptophan) from their mixtures and can be used in chemical, food, microbiological industry and agriculture
The invention relates to methods for selection of individual amino acids (tyrosine and tryptophan) from their mixtures and can be used in chemical, food, microbiological industry and agriculture

-l-aspartyl-l-phenylalanine methyl ester from the aqueous solution and the device" target="_blank">

The invention relates to a method of cooling an aqueous solution of aspartame and crystallization from him aspartame, excluding mobilizatio in water crystallization system by (i) filing of a hot aqueous solution of aspartame in the means for dispersing droplets; (ii) dispersion of droplets in an immiscible with water, the fluid whose temperature is at least 20°C lower for drops there was no formation of crystallization centers while passing them through immiscible with water, liquid; (iii) cooling mentioned immiscible with water fluid with the for the drops effectively cooled to achieve an initial supersaturation-L-aspartyl-L-phenylalanine methyl ester inside the drops in the range from 1 to 6, preferably from 1.2 to 4; (iv) collecting the cooled droplets for crystallization aspartame; and (v) provide sufficient time for this
The invention relates to biochemical pharmacology
Up!