Composition of individual proteolytic ferments
FIELD: chemistry, biochemistry.
SUBSTANCE: invention relates to biotechnology, namely to obtaining composition of individual proteolytic ferments and can be applied in medicine, cosmetology. In contents of composition are included not less than five proteases with molecular weight from 23 to 36 kilodalton and with -N-end amino acid successions: I V G G T E V T P G E I P Y Q L S L Q D -; I V G G T E V T P G E I P Y Q L S F Q D -; I V G G Q E A S P G S W P X Q V G L F F -; I V G G S E A T S G Q F P Y Q X S F Q D -; I V G G Q E A T P H T W V H Q V A L F I -; I V G G Q E A T P H T X V H Q V A L F I -; A M D X T A Y X D Y D E I Q A X L K G L -; A F D X T N Y N T F E E I N S I L D G V -; A A I L G D E Y L X S G G V V P Y V F G - Obtained composition is applied for treatment of purulent-necrotic and cicatricial changes of tissues in contents of pharmacological composition or elimination of purulent-necrotic and cicatricial changes of tissues in contents of cosmetic composition.
EFFECT: obtaining possibility to hydrolyse protein substrates to individual amino acids, obtained substance is highly efficient and does not cause allergic reactions when applied for a long time
6 cl, 3 tbl, 4 ex
The invention relates to biotechnology, namely, to obtain the composition of individual enzymes with a wide range of proteolytic activity, which makes the composition is able to hydrolyze protein substrates down to individual amino acids.
In the composition include proteolytic enzymes (endo - and ectopeptidases), which clearly individualized, including N-terminal sequence and molecular weight, and is characterized by high activity against many peptide and protein substrates, including collagen types.
Description of the INVENTION
Proteolytic enzymes perform many physiological functions, from digestion of the protein to the more specific, such as activation of imagenow and the complement system, are involved in the clotting process, protein and lysis of fibrin clots, the maturation of hormones and bioactive peptides from a protein precursor and the transport of proteins across cell membranes. Comparison of amino acid sequences, three-dimensional structures and mechanisms of enzymatic reactions allows you to split proteolytic enzymes into several groups.
Classification of proteolytic enzymes, based on their mechanism of catalysis, allows you to select 4 classes: serine protease; metalloprotease; thiol (qi is tinavie) and acidic (aspartic) protease. These proteases differ in sensitivity to various inhibitors, for example, serine proteases inhibited by phenylmethylsulfonyl (PMSF) and diisopropylfluorophosphate (DFP); metalloprotease - chelating agents such as EDTA, EGTA, phenanthrolin; cysteine protease - iodoacetamide and heavy metals and aspartic - pepstatin. Serine proteases are usually slightly alkaline optimum pH, metalloprotease - neutral, and cysteine and aspartic protease - sour.
The rate of hydrolysis of specific peptide bonds by most proteases depends, as a rule, from the substrate specificity of the enzyme, the spatial availability of this peptide bond (especially if the substrate is native but not denatured protein) and does not depend on the size of the molecules of the substrate (the length of polypeptide chain). Substrate specificity is the ability of the enzyme with the highest speed to hydrolyze peptide bonds between specific amino acid residues.
Typical representatives of the most numerous class of serine protease is a digestive protease mammals, which have similar amino acid sequence and spatial structure, is able to hydrolyze substrates to small fragments and distinguishing is to be the type of cleaved peptide bond. So, chymotrypsin hydrolyzes peptide bonds involving amino acids with aromatic side chains, trypsin - positively charged, elastase - amino acid residues of glycine, to a lesser extent - leucine. The observed specificity is due to differences in the structure of the active sites of proteolytic enzymes.
Proteolytic enzymes with narrow substrate specificity, are used in molecular biology as a tool for the study of the primary structure of proteins and peptides, and the protease are convenient objects for studying protein structure and mechanisms of their functioning. Many proteases, such as trypsin, chymotrypsin, papain, and others are active beginning of many drugs used in cosmetology and veterinary medicine.
Known serine protease isolated from the digestive organs of invertebrates and fishes (Zwilling R., et al., 1975, FEBS Lett. 60, 247-9; Gran G.A., et al., 1981, Methods Enzymol 80, 722-734; Reeck G.R. and H. Neurath. 1972, Biochem. 11: 503-510, O.A.Klimova, et al., 1990, BBRC, v.166, N3, 1411-1420).
Similar N-terminal amino acid sequences of these enzymes can be classified as a serine protease. Study of substrate specificity indicates that, in contrast to digestive proteases mammals, they are able to hydrolyze the broader the spectrum of substrates, for example, different types of collagens, which, due to the specific amino acid sequence and spatial structure, resistant to most proteases and available for hydrolysis only specific enzymes - collagenases.
True collagenase (tissue and microbial origin)belonging to the class of metalloprotease, split native collagen at one point one of the three polypeptide chains of the main structural element of native collagen - tropocollagen.
Unlike true collagenases serine protease invertebrates break down all three polypeptide chains tropocollagen, and the resulting peptides are subjected to further hydrolysis is down to individual amino acids, which are either included in the processes of anabolism, or quickly removed from the body without causing intoxication. Obviously, the ability of serine proteases invertebrates and fish to hydrolyze a wide range of substrates associated with the structural features of not only their active centers, but also the structure of the substrate binding sites - secondary interaction of the substrate with the substrate binding site significantly contribute to the increase of catalytic activity. Thus, the activity of these proteases by hydrolysis of long polypeptides above, and short synthetically the substrates WEEE, VTEE and Ac(Ala)3pNA is much lower than that of trypsin, chymotrypsin and elastase, respectively.
However, in some cases, the task is total proteolysis of different protein substrates, down to individual amino acids, so that the resulting amino acids could be used in the processes of anabolism as building blocks.
Known enzyme preparation of Collazo (Collasum), which is a mixture of two isoenzymes of serine collagenolytic protease a and C (RF Patent No. 2121503 from 1996.09.05, IPC: C12N 9/48; 9/64)with necroticism activity and fibrinolytic and thrombolytic properties. However, the mixture of these two proteases are not able to completely hydrolyze many polypeptide substrates, such as collagen fibers only partially hydrolyzed, and wounds containing partially damaged collagen fibers to be cleaned using collaz problematic.
In U.S. patent No. 4.801.451 and 4.963.491 declared a mixture of Exo - and endopeptidase isolated from Antarctic krill, and use the mixture to form a purified solution, and in patent No. 4801451 claimed the use of these enzymes for cleansing of necrotic tissue from a wound.
However, in these patents claimed untreated and poorly characterized substances. The authors call them "multifunctionally and proteolytic enzymes", that, by definition, means nespecificnomu and the unknown mechanism of action.
Closest to the claimed compositions, the mixture of serine proteases derived from fish (Atlantic cod), RF patent for the invention №2264824. This patent pending pharmaceutically and/or cosmetically active compositions containing as the active component trypsine and chymotrypsin obtained from cod, namely Atlantic cod. There are three main isoforms of trypsin in the Atlantic cod, which is already purified and characterized. They were called trypsin I, II and III (Asgeirsson et al., Eur. J. Biochem. 180:85-94, 1989). Trypsine cod have N-terminal sequence I-V-G-G-Y-Q/E-C-E/T-K/R-H-S-Q-A-H-Q-V-S-L-N-S, whereas trypsine mammals such as bovine trypsin, have N-terminal sequence I-V-G-G-Y-T-C-G-A-N-T-V-P-Y-Q-V-S-L-N-S. All three isoforms of trypsin cod have similar molecular weight of about 24 kDa.
However, it is known that serine proteases are endopeptidases and hydrolyzing polypeptide substrates to the state of small peptides, but not to individual amino acids, in addition, they are not able to hydrolyze a number of substrates, such as native collagen, which reduces the possibility of further use of such enzyme composition.
The objective of the invention is to provide a composition consisting of proteol the political enzymes which has the activity, stability in a wide temperature and pH ranges and resistance to autolysis and is intended for wide use in diagnostic, therapeutic purposes, in cosmetology and pharmacology, and biotechnology.
As a result of the research of the authors of the digestive organs of aquatic organisms were obtained from the natural complexes of proteolytic enzymes (proteases) with molecular masses 100-11 kilodaltons. Using a combination of chromatic methods of proteolytic complexes were isolated and purified to a homogeneous state of individual components with molecular masses 23-26 kilodaltons with the following N-terminal amino acid sequences:
I V G G a T E V T P G E I P Y Q L S L Q D -
I V G G a T E V T P G E I P Y Q L S F Q D -
I V G G Q E A S P G S W P X Q V G L F F -
I V G G S E A T S G Q F P Y Q X S F Q D -
I V G G Q E A T P H T W V H Q V A L F I -
I V G G Q E A T P H T X V H Q V A L F I -
A M D T A X Y X D Y D E I Q A X L K G L -
A F D X T N Y N T F E E I N S I L D G V -
A A I L G D E Y L X S G G V V P Y V F G -
Based on the complexes of the protease and/or individual components with molecular masses 23-36 kilodaltons new proteolytic composition in comparison with the previously known to have higher levels of enzymatic activity and degree of hydrolysis in relation to many peptide and protein substrates (including collagens of different types).
Below are examples of the compositions with the above components.
The components of the composition with molecular weight 100-37 and 22-11, kdalton identified by electrophoresis in polyacrylamide gel (SDS page) under denaturing conditions (in the presence of sodium dodecyl sulfate); N-terminal amino acid sequence was not determined due to low content of components in the composition. The percentage of components in the composition was determined by staining electrophoregram in SDS page followed by scanning of the gel. To do this, at the end of electophoresis gel were fixed in 5%solution of trichloroacetic acid, stained with a solution of Coomassie R-250 according to standard methods (Osterman L.A. research Methods proteins and nucleic acids: Electrophoresis and ultracentrifugation (practical manual). M.: Nauka, 1981, 288 C.) and held densitometric analysis using system gel documentation Biometra, Germany (cat. No. 035-300), which provides computer information read by the optical density pre-stained gel, analysis and storage of results.
|Examples of compositions on the basis of individual proteolytic farms is now|
|The molecular weight of individual proteolytic enzyme component compositions, kdalton||N-terminal amino acid sequence||The content of the component in the composition, %|
|36||I V G G a T E V T P G E I P Y Q L S L Q D -||6,0||11,0||13,0|
|35-II||I V G G a T E V T P G E I P Y Q L S F Q D -||22,0||20,0||34,0|
|28||I V G G Q E A S P G S W P X Q V G L F F -||13,0||11,0||8,0|
|25-I||I V G G S E A T S G Q F P Y Q X S F Q D -||to 12.0||9,0||-|
|2-II||I V G G Q E A T P H T W V H Q V A L F I -||6,0||6,0||-|
|25-III||I V G G Q E A T P H T X V H Q V A L F I -||14,0||14,0||5,0|
|32||A M D T A X Y X D Y D E I Q A X L K G L -||6,0||4,0||40,0|
|35-I||A F D X T N Y N T F E E I N S I L D G V -||4,0||4,0|
|23||A A I L G D E Y L X S G G V V P Y V F G -||15,0||20,0|
Suggested songs of proteolytic enzymes in the research process showed higher enzymatic activity and the absence of allergic reactions with long-term use. The advantages of the claimed composition (in particular, the depth of proteolysis) are confirmed by the results of a comparative trial the deposits compositions with different content components (examples 1 and 3 of table 1) when exposed to collagen type I (table 2). Testing of the composition of example 2 of table 1 gave results similar to the results of the composition of example 1, therefore in table 2 are not included.
In a plastic tube type "Eppendorf" cut cap was placed 200 μl of soluble collagen from the skin of cattle (Sigma # 8919), were dialyzed against distilled water for 12 hours at +40°C, was added 300 μl of 0.05 M solution of TES with 0.36 mM CaCl2, pH 7.5, was mixed and incubated at 37°C for 15 min, then was added 10 μl of a solution of the composition of proteolytic enzymes. Aliquots of the reaction mixture of 50 µl were taken by an automatic pipette through the intervals specified in the table, and transferred into plastic tubes type "Eppendorf", containing 10 μl of 50% (W/V) solution of trichloroacetic acid, frozen at -70°C for 1 hour, thawed and centrifuged at 5000 g for 5 minutes. Supernatant carefully and thoroughly removed, precipitation was dissolved in 10 μl of a 2%aqueous solution of sodium dodecyl sulfate was added to each sample and 20 μl of buffer solution to prepare samples, and then analyzed the samples by the method of polyacrylamide gel electrophoresis according to the method of Laemmli (Nature(1970) 277, 680).
The gel was stained with 0.2% solution of Coomassie R-250 in 25% solution of isopropanol and 10% acetic acid, washed with 7% solution of acetic acid, and dried.
Electropores the MMU analyzed, and the percentage of fragments was estimated using densitometry stained gel (HOWTO MUK 4.1/4.2.588-96).
|The effect of the composition of proteolytic enzymes in collagen type I from the skin of cattle|
|The exposure time, min||5||10||15||30||60||90||120|
|The size of the fragments of collagen, kDa|
|Composition no. (No. of the example of Table 1)||1||3||1||3||1||3||1||3||1||3||1||3||1||3|
Differences in the dynamics of the accumulation of low molecular weight components in the products of hydrolysis of collagen type I allow, comprising a composition on the basis of individual proteolytic enzymes depending on the specific task, to adjust the speed and degree of hydrolysis of protein substrates.
Hepatocytes from the liver of an adult and the embryo (5-8 weeks) were obtained by the standard method. The precipitate was collected and resuspendable 1%albumen hydrolysate. For counting living cells selected 1 ml of cell suspension, the content of intact cells was determined by staining with 0.2%solution Trypanosoma blue with subsequent microscopy.
|The effect of the composition of proteolytic enzymes in human hepatocytes.|
|P is Chen, used for isolation of hepatocytes||Proteolytic enzyme used for disaggregation of liver tissue (concentration in solution)|
|Trypsin (Sigma, T 7409), with 0.1% p-p||Collagenase (Sigma, C 9407), 0.05% p-p||The composition of individual proteolytic enzymes 0.01%-p|
|The liver of an adult||The total yield of hepatocytes, %||19,02||36,43||19,28||44,22||54,31||58,45|
|The content of live hepatocytes, %||54,11||41,37||70,51||47,22||88,29||70,14|
|The liver of the embryo (5-8 weeks)||The total yield of hepatocytes, %||24,3338,41||29,52||36,29||55,90||56,46|
|The content of live hepatocytes, %||58,62||52,49||76,11||52,12||92,22||77,72|
Identified advantages of the new composition of proteolytic enzymes allowed to use it in medicine for the correction, treatment and prevention of pathological (hypertrophic and keloid) scars on the skin, adhesions, as well as for the treatment of wounds of various origins (wounds, burns, frostbite, ulcers).
To accelerate the healing of burns due to the effective removal of the scab known application of the ointment, the composition of the active principle which is the drug elastase and polymer based on acrylic acid (U.S. patent No. 4.276.281). The elastase was obtained from the pancreas of mammals (specifically horses). It is a serine protease with a molecular weight of 25 900 daltons, consisting of 240 amino acid residues, with the known amino acid sequence (Shotten, D.M., Hartley, .S. Biochem J. 131, 643, 1973). The use of elastase based on its high efficiency for the removal of the scab and other macromolecular debris (remnants of damaged cells, part of the but or fully denatured collagen and so on) from the surface of the affected skin burns mammals, thereby expediting cleanup of affected surface and its preparation for granulation.
The main drawback of the application of elastase as is its low activity in relation to a number of polypeptide substrates, including collagens of different types, and shallow hydrolysis of polypeptide substrates (large fragments)that have to be removed from the affected surfaces by other means (mechanical or chemical), further injuring the patient.
In addition, if you frequently applied to the skin component such as elastase, the latter can cause some diseases due to its toxic and allergic action. In this regard, this remedy on the basis of elastase requires restrictions on duration of use, and for some users who are prone to allergies, it may be impossible.
The closest drug to the claimed tool is proposed in the invention "Method of treatment of diseases accompanied by formation of pus and/or necrotic tissue by the RF patent №2149644, IPC: A61K 38/48, the priority of 28.11.1997, in which the active principle used complex collagenolytic proteases with molecular weight of 10 to 40 kilodaltons, having proteolytic activity and obtained from pediatricain the x bodies of aquatic organisms. The disadvantage of this tool is that the main biologically active component is a compound containing as a minor component enzymes with non-specific activity, resulting in the damaging effect of the complex of proteases in relation to living cells is significant. In addition, the complex structure of the protease is strongly dependent on the physiological cycle of aquatic organisms, from which this complex was obtained. The content components of the complex and, consequently, its properties vary widely, which makes the use of the drug.
Medicinal products containing as active principle the proposed composition of proteolytic enzymes, consisting of individual enzymes with a high degree of purification, allows to reduce the concentration of the composition when applied due to higher enzymatic activity and does not cause allergic reactions with long-term use. In addition, the composition can be optimized (including standardized) in relation to the object of influence, which increases efficiency and reduces the side effects from the application.
Examples of the application of the composition of individual proteolytic enzymes.
Example No. 1.
Patient K., 41, sought advice from the about about scarring on the face, formed about a year ago during the self-treatment of acne urine therapy in the form of applications own urine. Developed as the result bolesno-necrotic form of erysipelas.
Application of known drugs for the treatment of scarring of the skin was ineffective.
The patient was scheduled outpatient treatment at the Department of balneology and physiotherapy of the Military medical Academy and spent 3 course electrophoresis on 10 sessions with a composition of proteolytic enzymes on the scars of the skin.
Breaks between courses was 1 month.
As a result, the scars were almost invisible, decreased their density and intensity of staining compared to the original state.
Example No. 2.
Patient K., 25 years old, came about scars, forehead and eyelids. From the anamnesis it is known that 3 months ago was in a car accident in which he sustained multiple injuries of soft tissues of the face with shards of glass, the Wound of the left upper eyelid sutured. Patient concern: the presence of itching in the area of scars, the presence of palpable small encapsulated foreign bodies under the skin disfigurement.
Completed 15 sessions of the composition of proteolytic enzymes. As a result of the treatment came the destruction of the capsules and the yield of fragments is flowed out. Just came out more than 20 pieces. Subsequently, the patient was performed chemical peels average power using trichloroacetic acid. The end result of treatment was cleansing facial from small encapsulated foreign bodies, correction of scars and restore the aesthetic appearance of the face of the patient.
Example No. 3.
Patient P., 21 years. Asked about cicatricial ectropion of left upper eyelid. The injury was 2.5 months ago during a car accident. Of the complaints noted an increasing degree of ectropion, conjunctivitis (due to drying of the eyeball). Completed 10 sessions of 10 sessions phonophoresis with "composition of proteolytic enzymes". In addition, 3 times fulfilled injection drug Collalysin".
As a result within 2 months of treatment, eyelid was better to fall and almost merges.
Example No. 4.
Palatka H., 42, asked about the long-term unhealed wounds on the anterior surface of the thorax on the left. 2 months ago the operation was performed is the removal of the breast on Halstead. Subsequently there was a marginal necrosis of the skin flap. For local treatment used ointment "Levocin" and drug "Curiosin".
At survey: granulating wound oblong shape with a size of 3.5×6 see the bottom of the wound issue is Leno pale pink granulation, coatings fibrin. Marginal epithelization sluggish.
On the wound surface during the week, put a bandage with a composition of proteolytic enzymes, followed by a bandage with ointment "Levocin". Wound healing was completed by the end of the 10-th day.
1. The composition of individual proteolytic enzymes with a molecular mass of from 11 to 100 kDa, composed of not less than five protease with a molecular mass of from 23 to 36 kDa N-terminal amino acid sequences from the following list:
IVGGQEASPGS WPXQVGLFF -
2. The composition of individual proteolytic enzymes for hydrolysis of proteins to individual amino acids, in the following composition:
100-37 KD - 0,6%
36 KD IVGGTEVTPGEIPYQLSLQD - 6%
35-II KD IVGGTEVTPGEIPYQLSFQD - 22%
28 KD IVGGQEASPGSWPXQVGLFP - 13%
25-I KD IVGGSEATSGQFPYQXSFQD - 12%
25-II KD IVGGQEATPHTWVHQVALFI - 6%
25-KD III VGGQEATPHTXVHQVALFI - 14%
32 KD AMDXTAYXDYDEIQAXLKGL - 6%
35-I KD AFDXTNYNTFEEINSILDGV - 4%
23 KD AAILGDEYLXSGGVVPYVFG - 15%
22-11 KD of 1.4%
3. The composition of individual proteolytic enzymes for hydrolysis of proteins to individual amino acids, in the following composition:
36 KD IVGGTEVTPGEIPYQLSLQD - 11%
35-II KD IVGGTEVTPGEIPYQLSFQD - 20%
28 KD IVGGQEASPGSWPXQVGLFF - 11%
25-I KD IVGGSEATSGQFPYQXSFQD - 9%
25-II KD IVGGQEATPHTWVHQVALFI - 6%
25-KD III IVGGQEATPHTXVHQVALFI - 14%
32 KD AMDXTAYXDDEIQAXLKGL - 4%
35-I KD AFDXTNYNTFEEINSILDGV - 4%
23 KD AAILGDEYLXSGGVVPYVFG - 20%
22-11 KD of 0.7%
4. The composition of proteolytic enzymes according to any one of claims 1 to 3, characterized in that it is able to hydrolyze native and fully or partially denatured proteins, including collagens of different types to individual amino acids.
5. Pharmaceutical composition for treatment of purulent-necrotic and scarring of tissue in a patient, comprising the composition according to any one of claims 1 to 3 in an effective amount and a pharmaceutically acceptable carrier.
6. Cosmetic composition for elimination of purulent-necrotic and scar skin defects, including composition according to any one of claims 1 to 3 in an effective amount and a cosmetically acceptable carrier.
SUBSTANCE: for separating carboxypeptidase B from pig pancreas extraction, precipitation by ammonium sulphate and purification are carried out in buffer solutions, which contain 0.001-0.005 M of zinc chloride. Purification is carried out by double chromatography on Q-sepharose, and during chromatography process elution of 0.07-0.08 M NaCl in 0.005 M tris HCI buffer, pH 7.6, is used.
EFFECT: invention ensures simplification of method of carboxypeptidase B obtaining preserving high specific activity of final medication and carboxypeptidase B stability.
FIELD: biotechnology, pharmacology, medicine.
SUBSTANCE: DNA sequence encoding of new kind of human serine protease (DPRP-2), is cloned and characterized, wherein said DPRP-2 is cognate to dipeptidyl oligopeptidase IV and hase gomology wherewith of 39 % determined using amino acid sequence. By using of recombinant DNA technologe recombinant form of DPRP-2 is obtained and kinetic properties of new enzyme (substrate specifity, Km values, activity inhibitors, etc.) are investigated.
EFFECT: new means for screening and development of new therapeutic agents for treatment of various deceases in human.
4 cl, 7 dwg, 7 ex
FIELD: biotechnology, biochemistry.
SUBSTANCE: invention represents enzyme that catalyzes reaction for formation of peptide from carboxyl component and amine component. Also, invention relates to strains of microorganism Empedobacter and genus Sphingobacterium that produce this enzyme, and to a method for preparing dipeptides from carboxyl component and amine component using this enzyme. Invention provides synthesis of peptide without carrying out the complex method of synthesis.
EFFECT: improved and easy method of synthesis, reduced cost, high yield of peptide.
11 cl, 18 tbl, 27 ex
SUBSTANCE: disclosed is new enzyme useful as catalyst of peptide synthesis reaction from carboxyl component and amino component. Said enzyme is produced by bacterial host cell cultivation transformed with DNA encoding peptide-forming enzyme, followed by accumulation of target enzyme. Obtained culture is blended with carboxyl component and amino component to produce dipeptide.
EFFECT: simplified method with increased yield.
38 cl, 4 dwg, 18 tbl, 39 ex
FIELD: biotechnology, veterinary, food processing industry.
SUBSTANCE: claimed method includes homogenization of crab hepatopancreas in 0.1 M ammonium acetate, containing 0.1-1 mM of calcium acetate at pH 6.4-7.9 followed by homogenate centrifugation. Ammonium sulfate is added to obtained supernatant up to 60-70 % saturation followed by centrifugation. Obtained enzyme complex in dissolved in distilled water in which 0.1-1 mM of calcium acetate is added to produce molecular solution followed by purification and concentration thereof. Enzymes are desalted on polyfiber membrane with pore size of 100 kDa. Supernatant containing collagenase inhibitor is washed with distilled water, heated up to 60-100°C and precipitate is separated by centrifugation. Enzyme solution is lyophilized.
EFFECT: new method for collagenase inhibitor production.
3 cl, 1 dwg, 1 tbl, 2 ex
SUBSTANCE: invention relates to new staphylokinase derivatives which represent expression products in heterogeneous system of mutant genes obtained by local gene PCR-mutagenesis ← wild type enzyme and differ from respective native staphylokinase form in one or more amino acid substitutions between 104 and 113 amino acid residues that leads to formation of RGD or KGD sequence in said site. Obtained recombinant staphylokinase derivatives (RGD/KGD-Sak), as well as variants thereof having deletions of 1-16 amino acids from NH2-terminal combine properties of thrombolitic and antocoagulating agents and are characterized by decreased polymerization ability and immunogenicity in contrast to wild type enzyme.
EFFECT: new effective agents for thrombosis preventing and treatment.
25 cl, 3 dwg, 3 tbl, 2 ex
FIELD: multienzymatic preparation from sea animal raw materials, useful in biotechnology, medicine, cosmetology, veterinary, agriculture, etc.
SUBSTANCE: claimed preparation is obtained by homogenizing of hepatopancreas of commercial crab species in buffer solutions; solid contaminant removing by homogenate centrifugation and lipid flocculation with chitosan, wherein homogenate is additionally purified by centrifugation. Ultrafiltration of enzyme preparation solution is carried out on membrane permeable for substances of molecular mass 15 kDa or less and further on membrane non-permeable for substances of molecular mass 50 kDa or more. Solution having enzymatic activity is lyophilized. Multienzymatic preparation contains collagenolytic and metal-dependent proteinases having molecular mass less than 30 kDa, rybonucleases. Deoxyribonucleases, phosphodiesterases, phosphatases, amylases, lipases, and β-glucanases.
EFFECT: enhanced assortment of multienzymatic preparations.
2 cl, 4 ex
FIELD: biotechnology, preparative biochemistry, enzymes.
SUBSTANCE: invention relates to a method for preparing the highly purified and highly active enzyme preparation collagenase. Method for preparing the collagenase preparation involves homogenization of hepatopancreas tissue of industrial species crabs in buffer aqueous solutions, isolation of collagenase solution, its purification by successive two-stage ultrafiltration on membranes providing cutting off inert substances and lyophilic drying of the preparation. Ultrafiltration is carried out on poly-fibrous membranes with size of pores 15 kDa and concentrate prepared at the 1-st stage of ultrafiltration is diluted with water in the ratio = 1:(5-10), and concentrate prepared at the 2-d stage of ultrafiltration is subjected for additional successive microfiltration on membranes with size of pore 100 kDa and ultrafiltration on membranes with size of pores 15 kDa. Homogenization of hepatopancreas of industrial species crabs is carried out in acetate-ammonium buffer containing calcium acetate at pH 6.4-6.7 followed by precipitation of collagenolytic enzymes with ammonium sulfate at 60-70% degree of saturation. Invention provides increasing the total collagenolytic activity, specific activity of the preparation and its purity. The collagenase preparation can be used in medicine, veterinary science, biochemistry and food industry and for research aims also.
EFFECT: improved preparing method.
2 cl, 2 ex
SUBSTANCE: raw material obtained out of gray substance homogenate should be supplemented with ionol (2.6-di-tret-butyl-4-methylphenol) at quantity necessary to prepare 0.0001 M final solution. Addition of this substance to raw material enables to keep its high activity being equal to 12.5-16.0 sec and enables to prolong its storage terms at (-40 ± 2) C up to 10 wk before carrying out sublimation and prevent the decrease of activity during sublimation process.
EFFECT: higher efficiency.
1 ex, 2 tbl
SUBSTANCE: invention concerns medicine and pharmacology, namely pharmaceutical injection compositions for treatment of diseases with immunodeficiency signs, described as containing Tilorone and hydrophilic solvent in certain ratio.
EFFECT: invention provides extended application of common active substance Tilorone, reconstitution of local immunity and biocoenosis of skin and mucous membranes in treatment of pyodestructive processes of skin and mucous membranes, especially in deep lesion focus, reduction of secondary necrosis of affected tissues, relief of inflammatory reactions, maintenance of local anaesthetic effect, acceleration of healing.
4 cl, 5 ex, 5 tbl
SUBSTANCE: ointment contains an admixture of sulphated mucopolysaccharides, methyl cellulose, chitosan, vinyl butyl acetate, Nipagin and water at a certain parity of components.
EFFECT: rising of therapeutic effect at the expense of the expressed regenerating action, reduction of terms of treatment, wound repair without formation of a vulnerary cicatrix.
SUBSTANCE: invention concerns medicine, namely, to surgical stomatology and can be used at treatment of septic wounds of maxillofacial area. For this purpose the 4.16% cycloferon immunomodulator solution, prepared by its dilution with the 0.9% sodium chloride solution is injected into an exudative phase of a septic wound process against complex medicamental treatment in a wound on turundas. In case of the expressed exudation, the cycloferon solution is diluted with the 10% sodium chloride hypertonic solution. Dressings are performed daily. In a proliferative phase of a wound process cycloferon is injected into a wound a kind of 5% liniment, thus dressings are performed in a day. The way allows raising efficiency of complex treatment of patients with the given pathology without system influence on immune system at the expense of local cycloferon immunocorrection taking into account wound process phases.
EFFECT: increase of efficiency of complex treatment of patients with the given pathology without system influence on immune system at the expense of local cycloferon immunocorrection taking into account wound process phases.
3 tbl, 3 ex
SUBSTANCE: invention concerns medicine. The way of a uniform covering of foam or a dressing material with the antimicrobial polymer containing such agents, as silver, and foam or a dressing material received in this way is described. Such foam or a dressing material is especially effective in a combination with vacuum therapy of a wound.
EFFECT: development of a way of a uniform covering of foam or a dressing material with antimicrobial polymer.
19 cl, 5 dwg
SUBSTANCE: invention concerns a chemico-pharmaceutical industry, namely to creation of a composition for treatment of atopic dermatitises, allergic conditions of a skin and blackhead. The composition contains: terpenes of Ginkgo biloba; b) phloroglucinol reagents in the pure state or their admixture, extracted from Humulus lupulus, from species of Hypericum and species of Mirtus; c) a lipophilic extract from Zanthoxylum bungeanum or Echinacea angustifolia, for treatment of atopic dermatitis, allergic conditions of a skin and blackhead.
EFFECT: development of a composition which shows fast favourable influence and possesses antiinflammatory, antibacterial, antimicotic and antipruritic action.
22 cl, 5 ex
SUBSTANCE: invention refers to medicine, namely to dermatology and can be used in treatment of patients suffering from hydradenitis. It is ensured by application of a gauze tampon plentifully impregnated with ethanol 50% on the region of formed infiltration. The tampon is applied for 6-12 hours within one day with multiple impregnations as drying. The therapeutic course is 24-48 hours.
EFFECT: method allows preventing further development of infiltration into purulent process ensured by prolonged exposure to dissolved alcohol at the initial stage.
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention concerns medicine and can be used for treatment of damages of a skin or dermal wounds. For this purpose a copolymer-1 is administered locally in a dose effective for this purpose. The pharmaceutical composition for local introduction, containing a copolymer-1 is also offered.
EFFECT: maintenance of fast closing of wounds, acceleration of formation of cicatrix.
25 cl, 27 ex, 43 dwg, 10 tbl
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention concerns a chemicopharmaceutical industry, and also to medicine and veterinary science and represents a pharmaceutical injection composition for treatment of the purulent-destructive processes, characterised that it contains Tilorone and a hydrophylic dissolvent, where as a hydrophylic dissolvent it contains the substances chosen from a number: the emulsion of perfluoroorganic bonds, an isotonic solution of sodium chloride or their admixture, and in addition contains Novocainum; thereat, the components in the composition are in a certain mass parity.
EFFECT: tilorone application expansion, restoration of local immunity and a biocenosis of a skin and mucosas at treatment of purulent-destructive processes of skin and mucosas, especially at a deep locating of the centres of a lesion, depression of a secondary necrosis of tissues on the amased sites, cupping of inflammatory reactions, maintenance of local anesthetic effect, healing acceleration.
3 cl, 1 ex, 2 tbl
SUBSTANCE: invention relates to medicine, namely to dermatology, and can be used for trophic ulcer treatment. For this purpose applications of carbonic acid snow, formed in shape of cylinder with diametre 0.5-0.7 cm are performed. Base of obtained cylinder is put on ulcer periphery in turn in 4 points. Application exposure in each point constitutes 1.5-2 minutes.
EFFECT: acceleration of trophic ulcer healing process without formation of course scars due to launching cell regeneration mechanism because of water crystallisation in cells resulting from their sharp freezing and their further destruction resulting from slow thawing.
1 ex, 1 dwg
SUBSTANCE: persistent wound and trophic ulcer healing in the patients suffering from pancreatic diabetes is ensured by application of medicinal leeches on the wound surface 2-3 times a day followed by application of ointment Piyavit within preparation of the wound surface. The procedure follows until the entire ulcerous bottom is filled with granulation tissue. Then skin flap pre-cured with the ointment Piyavit is transplanted. From the first postoperative days, medicinal leeches are applied on the skin flap in number of 1-3 pieces per a session for course 8-10 days. Then the operative wound is closed with a sterile dressing applied over bandage with ointment Piyavit. The whole therapeutic course involves introduction of capsulated Piyavit in dose 300 mg 2 times a day.
EFFECT: reduced intensity of inflammatory reaction; accelerated start of wound healing with decreased area and reduced recurrence.
SUBSTANCE: material, selected from group, which consists of glass, covered with silicon dioxin, glass, covered with silicone, is used as material of internal wall of container, which includes (i) wall section and (ii) one or more closing partitions, which do not constitute part of said wall section, and which contains protein composition, which has domain with amine-end y-carboxyglutamine acid (Gla), with 9-12 residues Gla; where said material reduces inclination of said protein to be subjected to di-, oligo- and/or polymerisation.
EFFECT: improvement of water composition stability - stability of VIIa (FVlla) factor in water solutions, which is important for containing and temporary storage of liquid composition of protein, especially of protein water composition.
38 cl, 3 ex, 3 tbl