• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
  • 法律状态
  • 优先权
    • 专利摘要:
    A process for the preparation of thrombin-like proteolytic enzymes from snake venoms or fractions thereof comprising contacting the snake venom or fraction thereof with heparin insolubilized by reaction with a water-insoluble carrier in order to bind the thrombin-like enzyme to the insolubilized heparin, removing the undesirable accompanying substances of the snake venom and splitting the thrombin-like enzyme from the insolubilized heparin.
    公开号:SU946389A3
    申请号:SU772510853
    申请日:1977-08-15
    公开日:1982-07-23
    发明作者:Ф.Штоккер Курт
    申请人:Пентафарм Аг (Фирма)；
    IPC主号:
    专利说明:

    39 Insoluble heparin can be obtained by reacting heparin in a perceptible way through its amino groups with reactive groups of a polymeric water-insoluble carrier substance, thus linking it covalently with the carrier. Insoluble heparin can, for example, be obtained by the proposed method by binding the heparin to the agarose using the cyanogen bromide bead, binding to the putrescine carbonose and the typhosphene method or by linking with the carbamide diimide by the carbodiimide method. those. putrescine and epsilonamino-caproylcellulose. Insoluble heparin can also be obtained by reacting heparin with cross-linked cyanogen bromide agarose in the form of balls or beads of standard size. Treatment of the starting material with insoluble heparin {i.e. Binding of a thrombin-like enzyme (serpent of another and with heparin) and cleavage of the enzyme from insoluble heparin can be carried out in portions by mixing the starting material dissolved in water, an aqueous buffer solution, an aqueous solution of electrolyte Bé. a buffer action or, in extreme cases, with a small buffer action, or containing both a buffer and a neutral salt, such as sodium chloride, together with a related adsorbent (i.e., insoluble heparin) followed by filtration, or, preferably, by chromatography chemical relatedness on the column. Suitable electrolytes are inorganic and organic salts, for example, sodium chloride, ammonium chloride, magnesium sulfate, ammonium sulfate: mono, sodium acetate, calcium chloride, ammonium bicarbonate, ammonium formate, triethylamine hydrochloride; or organic acids, such as acetic acid, tartaric or citric acid; or bases, such as ammonium hydroxide, trimethylamine, triethylamine. In chromatography on chemical kinship, insoluble heparin is poured into a column, whose diameter corresponds to about one-tenth of its pysops, and equilibrated with the same aqueous medium in which the starting material is dissolved. At the exit from the column, it is advisable to install a photometric flow meter, measuring and automatically recording the optical density of the eluates at an appropriate wavelength range (280 or 2S nm). Zljaty, preferably, divided into fractions through an automatic collector of fractions and collected. Chromatography on chemical affinity are as follows. The raw material (crude snake d or its fraction) is dissolved in water, an aqueous buffer solution, or an aqueous electrolyte solution, such as sodium chloride solution, or an aqueous solution containing both a buffer and a neutral salt, such as sodium chloride. The solution is filtered until clear or centrifuged and then poured into a column. The column is washed with the same aqueous medium as the one in which the starting material is dissolved until the substance that absorbs UV rays can be detected in the eluate. The column is then washed with an aqueous aluent, for example, a buffer solution or electrolyte solution or a solution containing both a buffer and a neutral salt, for example sodium chloride, whose concentration exceeds that of the aqueous medium in which the starting material is dissolved, in order to remove the serpentine and thrombin-like enzymes from insoluble heparin. . Wash until a UV absorbing substance is eluted. It is then washed again with a buffer solution, an electrolyte solution or a solution containing both a buffer and a neutral salt, such as sodium chloride, of a concentration sufficient to remove substances without a residue from the column that are more strongly bound to insoluble heparin than thrombin-like enzymes, that after deposition, the adsorbent can be regenerated again by kinship and used for the new process. Suitable buffer solutions are aqueous solutions of salts of inorganic or organic bases with inorganic or organic acids, or salts of amphoteric substances that develop a buffering effect in the range of pH 4-10. In particular, non-toxic substances or their mixtures that do not contain aromatic groups and therefore do not absorb light with a wavelength of 280 or nm and which thus do not interfere with the UV photometric control of the chromatography process, in particular, glycine / sodium hydroxide buffers are suitable. , acetic acid, sodium hydroxide, citric acid / sodium hydroxide, triethanolamine hydrochloric acid, lysine / hydrochloric acid, glycylglycine / hydrochloric acid, sodium phosphate, as well as vacuum-volatile buffer systems like buffers based on fo ammonium ammonium, ammonium acetate, ethylene diamine tetraacetate.
    Buffers based on sodium bicarbonate and ammonium bicarbonate are suitable for use in the discontinuous method, however, they are unsuitable for chromatographic operations due to the release of CO. Thrombin-like enzymes from various serpentine poisons have a different affinity for insoluble heparin bound by insoluble filler. Accordingly, the composition of buffers of biological origin should be coordinated with the secreted enzymes.
    The lower the concentration of the buffer or electrolyte solution or the buffer neutral salt solution, the better the enzyme binds to insoluble heparin.
    However, at low concentrations the maximum is the non-specific ability of heparin to bind proteins, concentrations must be chosen such that the thrombin-like enzyme binds, while the rest of the proteins pass through the column without delay. For each individual and optimal concentration conditions can be determined by simple preliminary experiments.
    Binding of all studied thrombin-like enzymes from snake venoms occurs at concentrations of buffer solutions or electrolyte solutions or solutions of a buffer-neutral salt within the range of 0.01-0.5 and pH -10 molar impurity depending on the type of species.
    For cleavage of thrombin-like enzymes from snake pools associated with insoluble heparin, buffer solutions or electrolyte solutions or buffer-neutral salt solutions are used which have a higher concentration than the solution used to bind the enzyme to insoluble heparin. Preferably, the same plant is used. the thief buffer, as for binding the enzyme to insoluble heparin, however, increases its concentration by adding a strongly dissociating salt of neutral character, preferably sodium chloride, so that the bound enzyme is cleaved from insoluble heparin.
    0 Since some serpentine dahs contain other substances that are related to the insoluble, heparin, the concentration used for the elution of the solution is preferably adjusted to a value at which the thrombin-like enzyme is cleaved off, while other substances with stronger kinship remain associated with insoluble heparin. .
    Cleavage of all thrombin-like enzymes associated with insoluble heparin from serpentine poisons is carried out preferably at concentrations ranging from 0.1-2 moles and at a pH range of t-IO.
    It is advisable to remove all components of the poison associated with insoluble heparin with a relationship to heparin greater than that of thrombin-like enzymes using buffer solutions or electrolyte solutions or solutions of buffer / neutral salt, with a molarity of 0, and a pH range of -10. Preferably, the concentration of the first buffer solution used to bind the enzyme, and insoluble heparin, is adjusted by the addition of a neutral salt, preferably ...
    but sodium chloride, to the desired value. The adsorbent is transferred to the working state by washing with the first buffer solution used to bind the enzyme with insoluble heparin.
    The relationship of insoluble heparin to thrombin-like enzymes from snake vents at low temperature is maximal and decreases with increasing temperature. This phenomenon can be used to initiate the binding of the enzyme to insoluble heparin at a low temperature, for example, in a column cooled with ice water, as well as removing the enzyme by increasing the temperature in the cooling or heating jacket of the column, e.g. one single buffer solution of constant concentration and constant pH. The enzyme is cleaved from insoluble heparin at a constant concentration of the aqueous medium on the buffer and / or neutral salt by raising or lowering the pH above or, respectively, below that in which the enzyme binds to insoluble heparin. The pH of each enzyme is determined by simple preliminary experiments. The content of the thrombin-like enzyme from the serpentine and in the eluates obtained by chromatography on a column is determined by experiment on fluxing, preferably on fibrinogen. As an experience in coagulation, it is suitable, for example, to determine the coagulation time in seconds after adding 0.2 ml of the diluted eluate to 0.2 ml of 0.4-fibrinogen (cattle) at pH 7, k and 37 C. Simple determining the clotting time is suitable for plotting the activity profile in the form of elution curves. Using the same technique with a standard thrombin preparation or using a standard preparation for a given thrombin-like enzyme from a snake and a calibration activity curve, it can be obtained from the coagulation time in quantitative data (National Institute of Heat units (NIH) or Ancrod units or Batroxobin units ). The content of the thrombin-like enzyme can also be determined photometrically using a synthetic chromogenic thrombin substrate, for example Tos-Gly-Pro Arg-pNA.tiC in enzyme units. One unit (U) in this case is designated the amount of enzyme that cleaves under the conditions of the experiment for 1 minute and 1 m mole of the substrate. Unwanted buffering agents and salts are then completely or partially dialyzed from the collected active fractions. Simultaneous desalting and concentration of the collected active fractions is carried out by ultrafiltration, for example using a Diafto-Membran membrane and M-2. The need for desalting and the concentration of the fraction containing the thrombin-like serpentine enzyme depends on the type of buffer used, the amount of substance treated by chromatography, and the purpose of the product. When using non-toxic buffer systems, such as glycine / NaOH / NaC, for the preparation of thrombin-like enzyme preparations for experimental and / or therapeutic defibrogenation to be able to produce the eluate from the collected active fractions by known methods isotonic sterile, not containing. pyrogen solution, only partial desalination is required or it is not required at all. Partial desalting is also sufficient when the thrombin-like snake enzyme contained in the eluate and need to be rechromatographed for further purification, in which case it is sufficient to bring the concentration of the buffer or salt of the eluate to a lower value for chemical bonding. the enzyme was able to re-associate with insoluble heparin. Example1. 9g heparin sodium specific biological activity 1b2 international units per mg is dissolved in 1 liter of O, 1 molar sodium bicarbonate buffer containing 0.5 mol / l of sodium chloride and the pH of which is 8.3 ° C. CNBr Sepharose B, which was previously allowed to swell and be purified in 0.001 N hydrochloric acid. The mixture is stirred for 2 hours. At the end of the reaction, the mixture is filtered on suction glass of type G-3, and the filtered product, sepharose-heparin, is washed five times with notry bicarbonate buffer of the indicated composition, each containing 300 ml. To saturate the reactively capable CNBr Sepharose-Heparin groups, 2 hours.
    mix together with 1 l of 0.5% ethanoliin in sodium bicarbonate buffer of the indicated composition. Sepharose-heparin is then collected again on a glass of suction and washed three times with 300 ml of sodium bicarbonate buffer. After that, the Sepharose-Heparin until then is washed with 0.1 M sodium acetate buffer, pH 0, until the pH of the filtrate is adjusted to 4.0. Finally, the insoluble heparin-is washed with several portions of O, 1-molar glycine / NaOH buffer, pH 8.5; is filled into a column with a diameter of 2 mm and is precipitated with the same glycine-based buffer. In sodium hydroxide. The column is then readjusted to a downward chromatography, column height is 29 cm, thereby the content of insoluble heparin is approximately 153 ml. The exit from the column is connected via a photometric flow meter set up to measure the wavelength range of 280 nm and equipped with an automatic recorder, to a collection of fractions set to collect fractions of 350 drops each.
    30 g yesa snakes of Bothrops atrox are dissolved in BOO ml of distilled water. The pH of the solution is adjusted to 3.0 with 1N hydrogen chloride and after one hour of the incubation process, to 1N sodium hydroxide. The resulting flaked product is separated by centrifugation and discarded. To the residue, add a solution of 15 g of sodium salicylate in 300 ml of distilled water. The pH of the solution was adjusted to 3.0 with 1N hydrogen chloride. After 60 minutes, the precipitate formed is removed by centrifugation, dissolved in 200 ml of a 0.1 molar glycine / sodium hydroxide-based buffer at pH 8.5 and washed until this with an ultrafilter buffer, while adding ferric chloride (III) the x-valent sample of the filtrate will not stain purple, i.e. will no longer contain salicylic acid.
    About 30 ml of concentrate remaining on the filter is treated with 0.1 molar glycine / sodium hydroxide buffer with a pH value of 8.5 to achieve a volume of
    50 ml. The content of batroxobin units (B) per ml is then the product is loaded into a prepared Sepharose-heparin column. By rinsing 750 ml of O, 1 molar glycine / sodium hydroxide-based buffer with a pH value of 8.5 (buffer 1), the ballast materials are eluted. Then the column is eluted
    0-, 1-molar buffer based on glycine-sodium hydroxide with a pH value of 8.5 (buffer P) containing 0.1 mol / l of sodium chloride, until 1120 ml of liquid passes, while again removing Ballast substances are eluted. After that, the thrombin-like enzyme is captured in the form of two absorbing UV rays coagulating fibrinogen
    zones by elution using an O, 1-molar glycine / sodium hydroxide-based buffer with a pH value of 8.5 (buffer III) containing 0.25 mol / l of sodium chloride, up to the amount of flowing liquid -. 900 million I
    Finally, by elution with 600 ml of glycine / sodium hydroxide-based buffer at pH 8.5 (buffer IV) containing 1 mol / l sodium chloride, the substances remaining bound to separose-heparin are removed.
    The thrombin-like enzyme (ba.trak sobin) is contained in 800 ml of eluate at a concentration of 100 batraxobine units per ml. The yield in terms of the applied 112,000 batraxobine units (B) is 71, the Eluate is concentrated on an ultrafilter (Diaflomembran UM-2 Ami con) in 80 ml, after which it serves as a batroxobin concentrate to obtain a batraxobin content of 22 units per milliliter for therapeutic defibrogenation.
    Example2. 0.1 g of an Agkistrodon contortrix snake and a snake are dissolved in 1.5 ml of aqueous 6.1 mol of glycine / sodium hydroxide-based buffer at pH 6.0. Solution
    权利要求:
    Claims (2)
    [1]
    loaded into a column (mm, 16 ml) from heparin-sepharose. The column is dusted with the same buffer until the washing liquid at 280 nm shows no absorption of light. The column is then rinsed with 0.1 M glycine buffer pH 6.0; containing 0.75 mol of sodium chloride per liter, until any amount of UV absorbing product is removed from the column. This eluate containing ballast products is selected. Then, a thrombin-like enzyme was isolated by elution using an O molar glycine buffer with a pH of 6.0 and containing 1.0 mol of sodium chloride per liter. Get 22 ml of the eluate with coagulating action on the fibrin gene. According to the measurement on the synthetic chromogenic substrate (benzoyl-Pro-Pye-Agde-and-nitroanilide), the activity in total is b99b milli-units. The total percentage of active eluate is 6,
    [2]
    2. The thrombin-like enzyme from the snake Agkistrodon con tort gix purified in this way, upon electrophoresis on polyacryl amide gel in the presence of sodium dodecyl sulfate, shows a single zone; when tested on an unheated fibrin plate, the absence of fibrinolytic activity is detected. It is then concentrated on an ultrafilter (DiafJomembran UM-2, Am icon) to a volume of 1-2 ml. 150 mg of dactose is added to the concentrate, then diluted with distilled water to 30 ml, poured into 1, O ml and lyophilized. 30 vials with an enzyme content of 200 milli-units each, in the form of a stable, immediately dissolved solution of the product. In this form, the preparation is suitable for the study of fibrin-to-fibrin transformation under physiological and pathological conditions. Froze 0.1 g of an Agkistrodon rhodostoma snake and a snake are dissolved in 1.5 ml of an aqueous 0.01 molar glycine buffer with a pH value of 6.0. The solution is loaded into a column (17x70 mm, 16 ug) of heparin-Sepharose precipitated with the same buffer. Then it is washed with the same buffer until using a photometer with a flow meter in the eluate can no longer detect the UV-absorbing product. The column is then eluted to remove ballast material 0,010 molar glycine buffer with a pH value of 6.0 and containing 0.25 mol of sodium chloride per liter. 120 ml of this solution are obtained with a content of 27 units of Anorod per milliliter. This product is shown by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate in a single zone. The proposed method permits, on a column, for chromatography with a volume of only 20 ml, to cleave more than 4 g of crude or an equivalent amount of a previously purified fraction of the enzyme, and thrombin-like enzymes of high purity are formed. This corresponds to approximately 40 times the performance of ion exchange chromatography on cellulose exchangers, approximately 00 times the performance of gel chromatography, and approximately 25 times the performance of chromatics, tography on chemical relatedness on p-aminobenz-amidine-succinyl-diamino-dipylaminoagarose. This strong ability to form insoluble heparin bonds not only improves the cleavage performance of a relatively small chromatography device, but also ensures that the active fractions of the eluate contain the enzyme in significantly higher concentrations compared to ion exchange gel chromatography, which significantly reduces the labor intensity of concentration operations and desalting eluates. According to the proposed method, thrombin-like enzymes from snake venoms are obtained for a small number of operations that are easy to carry out, are largely automated and are well recorded. The use of toxic eluents is not necessary. DETAILED DESCRIPTION OF THE INVENTION A method for producing thrombin-like enzymes by chromatography of snake cattle, characterized in that, in order to simplify the process, snake d in aqueous solution or in pH buffer C-10 with a content of 0.05-0.5 mol per liter of sodium chloride. treated with heparin, immobilizM9 + b3891,
    via amino groups on inert 1. Holteman and Weiss. Extraction of the carrier. And isolate the desired product. and Isolation of Thrombin ike Enzy Sources of information, mes. J. BioE. Chem. 1976, 252,
    taken into account in the examination of j. 1663-1669.
    类似技术:
    公开号 | 公开日 | 专利标题
    Wuepper et al.1972|Plasma prekallikrein: isolation, characterization, and mechanism of activation
    SU946389A3|1982-07-23|Method of producing trombine like enzymes
    US4849403A|1989-07-18|Protein C activator, methods of preparation and use thereof
    Revak et al.1974|Structural changes accompanying enzymatic activation of human Hageman factor
    Winn-Deen et al.1988|Development of a direct assay for alpha-amylase.
    Seegers et al.1958|Further studies on the purification of thrombin
    Rosenberg et al.1970|Multiple bovine thrombin components
    Polakoski et al.1973|Boar Acrosin: I. PURIFICATION AND PRELIMINARY CHARACTERIZATION OF A PROTEINASE FROM BOAR SPERM ACROSOMES
    US5118614A|1992-06-02|Concentrates of coagulation factors ii, vii, ix and x, method of their preparation and use
    US4381346A|1983-04-26|Isolation of plasminogen activators useful as therapeutic and diagnostic agents
    Furie et al.1976|[16] Coagulant protein of Russell's viper venom
    KR20010049988A|2001-06-15|Process for the preparation in pure form of the protease activating blood clotting factor VII, its proenzyme or a mixture of both proteins by means of ion-exchange chromatography
    Bach et al.1984|Immunoaffinity purification of bovine factor VII
    EP0040238B1|1984-07-11|Plasminogen activators useful as therapeutic and diagnostic agents and their isolation
    Miller-Andersson et al.1980|Preparation and stability of a highly purified human thrombin standard
    Kerr1981|[6] The second component of human complement
    USRE32271E|1986-10-28|Isolation of plasminogen activators useful as therapeutic and diagnostic agents
    Heberlein et al.1968|Canine plasminogen: purification and a demonstration of multimolecular forms
    PT95193A|1991-05-22|METHOD FOR ACTIVITY OF PROTEIN C
    Somer et al.1970|Coagulation factor IX in normal and haemophilia B plasma
    JPH09183794A|1997-07-15|Preparation and recovery of protein
    Kurachi et al.1981|[17] Human factor XI |
    Mannhalter1987|Biochemical and functional properties of factor XI and prekallikrein
    Váradi et al.1976|Purification and properties of human factor IXa
    CA1151542A|1983-08-09|Process for preparing the third componentof the complement from human blood plasma
    同族专利:
    公开号 | 公开日
    ATA590477A|1980-08-15|
    NL7708515A|1978-02-21|
    SE433619B|1984-06-04|
    IL52607D0|1977-10-31|
    CH626917A5|1981-12-15|
    DE2734427B2|1979-08-09|
    US4137127A|1979-01-30|
    NO772862L|1978-02-20|
    CA1078732A|1980-06-03|
    NL186018C|1990-09-03|
    IL52607A|1980-02-29|
    FR2362155A1|1978-03-17|
    IT1083927B|1985-05-25|
    DK364077A|1978-02-18|
    JPS5710718B2|1982-02-27|
    NL186018B|1990-04-02|
    FR2362155B1|1980-06-06|
    NO147526B|1983-01-17|
    LU77957A1|1977-12-14|
    AU2763377A|1979-02-08|
    DE2734427A1|1978-02-23|
    BE857876A|1977-12-16|
    SE7709241L|1978-02-18|
    DE2734427C3|1980-04-17|
    NO147526C|1983-04-27|
    GB1591333A|1981-06-17|
    DK147408B|1984-07-23|
    JPS5324095A|1978-03-06|
    ES461549A1|1978-06-01|
    AU507161B2|1980-02-07|
    DK147408C|1985-02-04|
    AT361616B|1981-03-25|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    CH586233A5|1971-01-18|1977-03-31|Pentapharm Ag|
    SE420321B|1973-09-03|1981-09-28|Fargal Pharmasint Lab Biochim|CHROMATOGRAPHIC EXTRACTION AND INSULATION OF ENZYMATIC COMPONENT FROM ORMAN POISON|DE3512910A1|1985-04-11|1986-10-16|Behringwerke Ag, 3550 Marburg|METHOD FOR CLEANING PLASMINOGEN ACTIVATORS|
    US4849403A|1985-05-29|1989-07-18|Pentapharm Ag|Protein C activator, methods of preparation and use thereof|
    US5100668A|1988-06-14|1992-03-31|Massachusetts Institute Of Technology|Controlled release systems containing heparin and growth factors|
    US5196193A|1989-10-31|1993-03-23|Ophidian Pharmaceuticals, Inc.|Antivenoms and methods for making antivenoms|
    ES2131504T3|1990-02-20|1999-08-01|Baxter Int|VIRICALLY SAFE PURIFIED HUMAN THROMBINE.|
    EP0585504A1|1992-09-04|1994-03-09|Pentapharm A.G.|Phospholipid dependant prothrombin activator and its use in lupus anticoagulant testing|
    DE19607210A1|1996-02-26|1997-08-28|Knoll Ag|Process for the purification of thrombin-like proteases from snake venoms|
    IL130728A|1997-01-08|2002-11-10|Squibb Bristol Myers Co|Centrifuge apparatus with temperature control means|
    US6132598A|1997-01-08|2000-10-17|Bristol-Myers Squibb Company|Centrifuge apparatus with temperature control means|
    WO2001048486A1|1999-12-24|2001-07-05|International Reagents Corporation|Means of stabilizing compositions and reagents|
    RU2362582C2|2004-06-24|2009-07-27|Тобиси Фармасьютикал Ко., Лтд.|Preparation containing batroxobin, for inhibition of local invasion of malignant tumours|
    CA2623950C|2005-09-30|2016-01-26|Tobishi Pharmaceutical Co., Ltd.|Activating agent of stem cells and/or progenitor cells|
    EP2217267B1|2007-12-14|2016-05-25|Tobishi Pharmaceutical Co., Ltd.|An agent for reducing a side effect of an anticancer drug|
    TWI482629B|2008-07-01|2015-05-01|Tobishi Pharmaceutical Co|A therapeutic agent for lower urinary tract diseases and an improving agent for lower urinary tract symptoms|
    法律状态:
    优先权:
    申请号 | 申请日 | 专利标题
    CH1049376A|CH626917A5|1976-08-17|1976-08-17|
    [返回顶部]