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    • 专利摘要:
    The present invention relates to novel serine proteases, their preparation and use. More specifically, the present invention relates to novel serine proteases having thrombolytic solubility, a method for separating and purifying them from the trademarked herb of Tenodera sinensis , and the extract of the trademarked herb, and the serine proteases of the present invention as an active ingredient. The pharmaceutical composition may be usefully used as a therapeutic agent and health supplement for thrombosis related diseases.
    公开号:KR20000000902A
    申请号:KR1019980020843
    申请日:1998-06-05
    公开日:2000-01-15
    发明作者:김영식；장일무；한범수；조소연
    申请人:김영식；
    IPC主号:
    专利说明:

    Thrombolytic Serine Protease Isolated and Purified from the Herb
    [10] The present invention relates to a novel serine protease having a thrombolytic solubility, a method for separating and purifying it from the trademarked herb of Tenodera sinensis , and an extract of the trademarked herb comprising the serine protease.
    [11] In recent years, the mortality rate due to circulatory diseases such as hypertension, heart disease, and stroke, called adult diseases, is increasing gradually in Korea as well as in developed countries in the West. Cardiovascular disease, a type of blood circulation disease, is reported to be one of the most common diseases in modern people. Specifically, seizure-induced cerebrovascular diseases such as coronary artery thrombosis and stroke, which cause myocardial infarction, are reported. Disease (cerebrovascular thrombosis) and venous thrombosis that causes pulmonary embolism (Guytion, AC, Textbook of Medical Physiology, 7th Eds. , WB Saunders, Philadelphia, pp .76-86, 1986 ).
    [12] NIH reports that stroke is the third leading cause of death among adults in the United States, and is the largest contributor to disability.
    [13] In general, blood circulates in the blood vessels but quickly clots once exposed. When blood vessels are damaged, bleeding occurs. If the damaged blood vessels are large blood vessels such as arteries, surgery should prevent bleeding, but small injuries naturally hemostasis in the blood vessels. The inner surface of blood vessels is surrounded by endothelial cells, and these endothelial cells act to inhibit blood coagulation in normal cases and to act as a quick hemostasis in case of injury.
    [14] The reason why vascular endothelial cells have antithrombosis is that the surface is negatively charged like the surface of platelets, and in these cells strong anticoagulant factors such as heparan sulfate or thrombomodulin, etc. Is secreted. At this time, heparan sulfate promotes the activity of antithrombin III, and thrombomoduline inhibits the enzymatic activity of thrombin and activates protein C to prevent blood coagulation.
    [15] In addition, when blood vessels are damaged by any factor and bleeding occurs, a physiological defense mechanism triggers a hemostatic response in the blood vessels, thereby inhibiting blood loss. First, the flow of blood is suppressed and then platelets circulating in the blood adhere to each other. Done. When platelets adhere to agglomerate, a plug is formed at the site of damage, and thrombin is exploded through a blood coagulation pathway by the action of endothelial cells and platelets around the damaged blood vessel. In vivo physiological conditions, hemostasis necessarily occurs only around damaged blood vessels and do not affect other areas.
    [16] When the hemostasis process is completed and regeneration of vascular tissue is performed, the fibrin polymer formed through this process must be dissolved. Fibrin polymers are dissolved by a substance called plasmin, which is produced by activating plasminogen in the thrombolytic system. As such, plasmin acts as an enzyme that dissolves fibrin, but also has an activity of degrading fibrinogen, which is important for blood coagulation. When excessive amounts of free plasmin are present, blood coagulation is suppressed as a whole and thus a great risk exists to the living body. To overcome this problem, there is an inhibitor of plasmin in the blood and plasmin so that plasminogen activation by plasminogen activator occurs only in the place where fibrin clot is formed. The plasminogen activator inhibitor (PAI) is precisely regulated.
    [17] When blood clots are generated in the cerebrovascular system due to bioreaction mechanisms, thrombosis occurs. If the blood vessels are blocked by blood clots, heart failure or heart failure will occur, and many studies have been conducted to treat thrombus. Specifically, plasminogen activators such as streptokinase and urokinase are known to be useful for removing blood clots and intravenous injections of these have been used for the past 30 years to activate the fibrinolytic system. Although these two drugs have already been demonstrated in many clinical cases that are indeed effective for thrombolytic dissolution, on the other hand, side effects such as systemic hemorrhage occur during treatment due to lack of specificity for thrombosis.
    [18] In addition, in 1987, a tissue-type plasminogen activator (tPA) was developed by genetic engineering methods. tPA is a protein present in trace amounts in vivo (plasma or organ) that activates plasminogen as a plasmin, which causes plasmin to break down fibrin, which facilitates blood flow. Because of its high selectivity to blood clots, it was initially recognized as an ideal thrombolytic agent, and clinical reports continue to be effective for stroke as well as for myocardial infarction. However, as a result of the clinical use, the half-life of the blood is very short, about 6 minutes, which is inconvenient to use, and there are still problems such as the side effects of streptokinase or urokinase administration. In addition, the above-described streptokinase, urokinase and tPA (tissue-type plasminogen activator) have some problems for clinical use. Specifically, the drug is not only a high risk of side effects, very expensive, and is not oral administration except eurokinase.
    [19] Therefore, in recent years, attempts have been actively made to provide better thrombolytic agents that overcome the problems of these conventional thrombolytic agents. In addition to thrombolytic agents which can be administered only by vascular injection, blood is directly orally administered or combined with vascular injection. Attention has been drawn to agents that can increase the thrombolytic capacity in the blood. For example, the first is to develop new mutant activators with strong thrombolytic activity, and the second is to develop new thrombolytic agents derived from animals, plants, or microorganisms having better thrombolytic ability from natural substances.
    [20] Efforts to develop new medicines from natural products have long been tried in our country as well as in developed countries. Thousands of attempts have been made for this purpose because thrombolytic substances present in various organisms in nature can be developed as new thrombolytic agents using a wide range of screening techniques, and thrombolytic agents of a different concept from the plasminogen activators described above, Unlike tPA, which activates plasminogen as a plasmin and causes plasmin to break down fibrin, thereby facilitating blood flow, proteolytic enzymes that directly break down fibrin are detected from natural products such as plants and plants. Its research is active. Directly decomposing fibrin may be beneficial to patients with acute thrombosis compared to conventional urokinase or tPA. Recently, studies of proteolytic enzymes that directly decompose fibrin have been actively conducted, and mollusks such as earthworms (Nakajima) et al., Biosci. Biotech. Biochem ., 57 1, 726-1730, 1993 ), snake venom (Guan et al., Arch. Biochem. Biophys., 289 , 197-207, 1991 ; Hahn et al., J. Biochem ., 119 , 835-843, 1996 ) or microorganisms ( Appl. Environ. Microbiol . 62, 2482-2488, 1996 ). It is done. In addition, leeches (Markward, Thrombosis Haemostasis , 141-152, 66 , 1991 ); Bats (Cartwright, Blood , 43 , 317-326, 1974 ); Vampire bedbugs (Noeske et al., J. Biol. Chem ., 269 , 5050-5053, 1994 ); And anticoagulant or thrombolytic enzyme proteins are isolated and purified from most aversive animals, such as Duoly (Friedly, Science 271 , 1800-1801, 1996 ) and are in clinical trials or preclinical stages for commercialization.
    [21] Insects are the largest species of life on the planet, and because of their excellent environmental stress, they live in a wide range of locations around the world. Since insects can adapt and survive successfully under various environmental conditions, there is a lot of movement to develop new biological materials from insect resources. It is noteworthy that the traditional use of insects as a medicinal herb in Korea also appears in the medical textbook. .
    [22] Insects often have substances that can dissolve blood clots, a type of defense system. For example, the manufacturing of scarab larvae dry (slugs) has traditionally been used for thrombosis, and it has been reported in laboratory rats that the extracts have the effect of treating blood clots (Ahn, Kyu-Seok, Korean Journal of Oriental Medicine) , 11, 92-101, 1990 ). In addition, hementin (Markward, Thrombosis Haemostasis , 66 , 141-152, 1991 ) isolated from leeches and a fibrinolytic enzyme (Kim, isolated from centipedes) at the Merck Institute in the United States in 1989. KY et al., Thromb. Haemost., 69 , 839, 1993 ], have been reported as new thrombolytic agents. In addition, bloodsucking bed bugs [Noeske et al., J. Biol. Chem . 269 , 5050-5053, 1994 ]. Anticoagulant or thrombolytic enzyme proteins have been isolated and purified from insects and are in clinical trials or preclinical stages for commercialization.
    [23] Therefore, the present inventors continued to develop new types of thrombolytic agents using insects, and firstly separated and purified various proteins that directly degrade fibrin from the trademark herb of Tenodera sinensis , a new serine. The present invention was completed by identifying the protease and investigating its physical properties.
    [24] It is an object of the present invention to provide a new serine protease with a thrombolytic solubility, a method for separating and purifying it from the trademark herb, and a thrombi soluble trademark herb extract.
    [1] Figure 1a is a chromatographic separation of various serine proteases (MEF-1, MEF-2, MEF-3) having thrombolytic action from the trademark herb using a Bio-Gel P-60 column It shows the result of refinement,
    [2] FIG. 1B shows the result of separating and purifying the active fraction obtained in the process of FIG. 1A by chromatography using a DEAE-Affi-Gel Blue column.
    [3] Figure 2a shows the result of performing SDS-polyacrylamide gel electrophoresis (SDS-PAGE) to purify the serine protease (MEF-1) of the present invention,
    [4] Figure 2b shows the results of performing SDS-polyacrylamide gel electrophoresis by purifying serine proteases (MEF-1, MEF-2, MEF-3) of the present invention,
    [5] Figure 3a shows the result of incubating the serine protease (MEF-1) with fibrinogen and then taking samples by time and performing SDS-polyacrylamide gel electrophoresis,
    [6] Figure 3b shows the result of culturing the serine protease (MEF-2) with fibrinogen and then taking samples by time and performing SDS-polyacrylamide gel electrophoresis,
    [7] Figure 3c shows the result of performing the SDS-polyacrylamide gel electrophoresis after incubating the serine protease (MEF-3) with fibrinogen and then taking samples at different times.
    [8] Figure 4 is a culture of the serine protease (MEF-1) with an existing enzyme inhibitor and then confirmed the activity of the protease with azocaine produced.
    [9] Figure 5 shows the result of quantifying the D-dimer produced after incubating the serine protease (MEF-1) with fibrin.
    [25] In order to achieve the above object, the present invention provides new serine protease (thrombotic soluble).
    [26] Specifically, the present invention
    [27] (1) Molecular weight 31,500 Daltons, isoelectric point 6.1, sensitive to chymostatin, sensitive to metal ions of Cu 2+ and Mn 2+ , optimal in temperature range of 20-40 ° C. and pH 5.0-8.0 Serine protease (MEF-1), indicating the activity of
    [28] (2) A serine protease (MEF-2) having a molecular weight of 32,900 Daltons, sensitive to TLCK and soybean trypsin inhibitors and exhibiting optimal activity in the temperature range of 10-40 ° C. and pH 5.0-10.0. ),
    [29] (3) Serine protease (MEF-3) having a molecular weight of 35,600 Daltons, sensitive to chymostatin and exhibiting optimal activity in the temperature range of 10-50 ° C. and in the range of pH 5.0-10.0.
    [30] The serine protease (MEF-1) of the present invention comprises the amino acid sequence of SEQ ID NO: 1 at the amino terminus, and the serine protease (MEF-2) comprises the amino acid sequence of SEQ ID NO: 2 at the amino terminus.
    [31] In addition, the present invention elutes the supernatant with tris-hydrochloric acid buffer solution, centrifuged, the supernatant is precipitated by ammonium chloride, and then the precipitate fraction is dissolved and centrifuged again, the supernatant is concentrated and purified by gel chromatography. It provides a method for preparing thrombolytic serine proteases consisting of a process.
    [32] The present invention also provides a thrombi soluble trademark herb extract obtained by eluting the trademark herb with a buffer solution.
    [33] In addition, the present invention provides a pharmaceutical composition for treating a thrombosis-related disease comprising the serine proteases as an active ingredient. The pharmaceutical composition may be administered orally and used to treat coronary artery disease and cerebrovascular disease.
    [34] The present invention also provides a health supplement comprising an active ingredient as part or all of the thrombolytic enzyme proteins.
    [35] Hereinafter, the present invention will be described in detail.
    [36] The present invention isolates a new serine protease with a thrombolytic action from the extract of the trademark herb, an alveolar of a wart.
    [37] In the present invention, first, in order to obtain a trademark herb extract having thrombogenic solubility, the trademark herb powder is eluted with Tris-HCl buffer at low temperature, centrifuged to separate the supernatant, and the supernatant is precipitated with ammonium lactate. Using the supernatant and ammonium lactate precipitate, the activity of the thrombolytic enzyme is measured according to the method for measuring the activity of the thrombolytic enzyme to identify the thrombolytic enzyme present in the extract of the trademark (see Table 1 ).
    [38] Specifically, the activity of the enzyme is measured by the fibrin plate method, that is, the dissolution area on the fibrin plate, and the like, and at this time, plasmin, a well-known thrombolytic agent, is used as a control.
    [39] In the present invention, in order to obtain the enzyme proteins having the thrombus solubility, the ammonium lactate precipitate fraction is dissolved and centrifuged again, the supernatant is separated, and the separated supernatant is concentrated to perform gel chromatography and the like to repeat the thrombus solubility. Fine thrombolytic enzyme proteins are purified.
    [40] It is preferable to use DEAE-Affi-Gel Blue chromatography, which is not commonly used in the purification process of the present invention, which simultaneously uses the principles of ion exchange, size exclusion and adsorption. The pigment contained in the extract can be removed.
    [41] Through the above procedure, the enzyme proteins of the present invention having high thrombolytic solubility having a molecular weight of 31,500 (MEF-1), 32,900 (MEF-2), 35,600 (MEF-3) Dalton and an isoelectric point of 6.1 (MEF-1) are obtained. . (See FIGS . 2A and 2B )
    [42] In order to investigate the properties of the enzyme proteins, the change in enzyme activity was measured by fibrin plate method while keeping it warm. As shown in Table 1 , the enzyme protein isolated and purified from the trademark herb extract shows excellent thrombolytic activity.
    [43] In addition, the enzyme proteins exhibit optimal thrombolytic activity, respectively, in the temperature range of 10-40 ° C. and in the range of pH 5.0-10.0.
    [44] Specifically, the MEF-1 enzyme protein has a molecular weight of 31,500 Daltons, has an isoelectric point of 6.1, and exhibits optimum activity in a temperature range of 20 to 40 ° C. and a pH of 5.0 to 8.0. MEF-2 exhibits optimal activity in the temperature range of 10-40 ° C. and pH 5.0-10.0, and MEF-3 exhibits optimal activity in the temperature range of 10-50 ° C. and pH 5.0-10.0. For MEF-1 the amino terminus comprises the amino acid sequence of SEQ ID NO: 1 (Ala-Asp-Val-Val-Gln-Gly-Asp-Ala-Pro-Ser-), and for MEF-2 the amino terminus is Amino acid sequence (Ile-Val-Gly-Gly-Glu-Glu-Ala-Val-Ala-Gly-Asp-Phe Pro-X-Ile-Val-Ser-Leu-Gln-Glu: X is an unidentified amino acid) Include.
    [45] In addition, the present invention compared the properties of the enzyme protein of the existing enzyme inhibitor. Namely, inhibitors include benzamidine, ethylenediaminetetraacetic acid (EDTA), ethyleneglycol-bis (beta-aminoethyether) -N, N, N, N, -tetraacetic acid], cysteine, phenylmethylsulfonyl fluoride; Hereinafter, the enzyme proteins of the present invention have been tested for activity using β-mercaptoethanol, β-mercaptoethanol, trypsin inhibitor and aprotinin. (serine protease) lineage was found.
    [46] In addition, to determine what form the serine proteases of the present invention, elastanal (elastanal); Np-tosyl-L-phenylalanine chloromethyl ketone (Np-tosyl-L-phenylalanine chloromethylketone; hereafter abbreviated as "TPCK"); Np-tosyl-L-lysine chloromethyl ketone (Np-tosyl-L-lysine chloromethylketone; hereafter abbreviated as "TLCK"); And testing with inhibitors of chymostatin revealed that MEF-1 and MEF-3 of the serine proteases of the present invention are sensitive to chymostatin, which is a protease found in existing mammals or microorganisms. It was confirmed that the enzyme is different from the property. And MEF-2 was inhibited by TLCK and soybean trypsin inhibitor (see Fig. 4 , Table 3 and Table 4 ).
    [47] Serine proteases of the present invention can be isolated and purified from the trademark herb, but since the amount is extremely small, the gene of the enzyme can be obtained and expressed in a suitable host organism including Escherichia coli, thereby producing a large amount of protease that is thrombolytic.
    [48] The present invention provides pharmaceutical compositions and dietary supplements for the treatment of thrombosis-related diseases comprising enzyme proteins having the above characteristics as active ingredients. Here, the thrombosis-related diseases include coronary artery disease and cerebrovascular disease.
    [49] The pharmaceutical composition may be administered orally or parenterally, and an effective dosage of enzyme proteins is preferably 10,000 to 25,000 U per day.
    [50] In the pharmaceutical use of the enzyme proteins of the present invention can be produced and produced by a method known in the pharmaceutical field, it can be used in the formulation of tablets, capsules, powders, solutions, suspensions, injections, etc. using an appropriate excipient. have.
    [51] Hereinafter, an Example demonstrates this invention in detail.
    [52] The following examples illustrate the invention and are not intended to limit the scope of the invention.
    [53] I. Isolation and Purification of Thrombolytic Enzyme Proteins
    [54] Example 1 Isolation and Purification of Thrombolytic Enzyme Protein
    [55] The supernatant was collected in a mixed solution of 50 mM Tris-HCl buffer and 0.1 M aqueous sodium chloride at pH 7.4, eluted at 4 ° C. for 1 hour, and centrifuged at 8,000 rpm for 30 minutes to collect the supernatant. The supernatant was then precipitated by treatment with 60-90% ammonium lactate, and the precipitate fraction was dissolved in the mixed solution (pH 7.4) of the 50 mM Tris-HCl buffer solution and 0.1 M sodium chloride solution and centrifuged again at 10,000 rpm for 30 minutes. Isolate and collect the supernatant. The supernatant was concentrated by ultrafiltration and equilibrated with a mixed solution of 50 mM Tris-HCl buffer solution and 0.1 M sodium chloride solution (pH 7.4) (Bio-Gel P-60 column; 7.8 × Gel chromatography was performed under conditions of a flow rate of 11.0 mL / hour using 3 cm). (Step A)
    [56] At this time, the absorbance of each fraction was measured at 280 nm, the fractions having high thrombus lytic activity were collected by fibrin plate method, concentrated by ultrafiltration, and then equilibrated with the concentrate (DEAE epi-gel blue column (flow rate: 11.3 ml / hour). , 7.8 × 3 cm). (Step B)
    [57] The measurement of the thrombolytic action by the fibrin plate method was performed using a 0.8% fibrinogen solution dissolved in 0.2 M dispersion buffer (0.05 M Na 2 B 4 O 7 10H 2 O: 0.2 MH 3 BO 4 -0.05 M NaCl = 2: 8). The filtrate was poured into a petri dish and 15 ml, and 0.5 ml of 10 U / ml of thrombin was mixed and incubated at 37 ° C. for 30 minutes. 0.1, 0.5, and 1 µg of the concentrate and plasmin 0.001, 0.005, 0.01, 0.02, 0.04, 0.06, 0.08 and 0.1 U were added dropwise and incubated at 37 ° C. for 18 hours, and then the dissolved diameter was measured. In the chromatography process, each fraction was collected while varying the concentration of the sodium chloride solution to 0.01, 0.2, 0.4, 1.0, and 2.0M in a mixed solution of 50 mM Tris-HCl buffer solution and sodium chloride solution, respectively, to absorb absorbance (A 280 ) and to dissolve blood clots. Activity was measured to collect fractions with high absorbance and thrombolytic activity. These fractions were subjected to SDS-polyacrylamide gel electrophoresis to collect purified fractions having one molecular weight for various analysis. Protein quantification was carried out by adding 200 μl of Bio-Rad Protein Assay staining concentrate by Bradford method, mixing with 5-10 μl of the purified fraction, and measuring absorbance at 595 nm. The results for are shown in FIG. 1 .
    [58] II. Identification of Enzyme Proteins
    [59] Example 2 Confirmation of Molecular Weight and Amino Acid Sequence of Thrombolytic Soluble Enzyme Protein
    [60] 500 μl (20 μg) of the enzyme protein obtained in Example 1 was lyophilized at −70 ° C., dissolved in 20 μl of water to make 1 μg / μL, and then 10 μg of SDS-PAGE was performed thereafter, followed by separation therefrom. The protein was confirmed without drying the gel. As a result, the initial sequence of the amino terminus of the purified protein was confirmed. (See Sequence Listing )
    [61] On the other hand, in order to measure the molecular weight and isoelectric point of the purified protein was subjected to SDS-PAGE and isoelectric point according to the above method. As a result, the molecular weights of the enzyme proteins (MEF-1, MEF-2, MEF-3) of the present invention are 31,500 (MEF-1), 32,900 (MEF-2), 35,600 (MEF-3) Dalton, respectively, and the isoelectric point is 6.1. It was confirmed that it is (MEF-1). (See FIGS . 2A and 2B )
    [62] <Example 3> Activity measurement of thrombolytic enzyme protein
    [63] As a substrate, 1 ml of azocasein was dissolved in 0.2 M buffer at a concentration of 2 mg / ml, and 0.5 and 1 µg of the enzyme proteins were added thereto and left at 37 ° C for 1 hour. Then, the absorbance of the supernatant obtained by mixing 1 ml of 5% trichloroacetic acid (TCA) in 250 μl of this sample and centrifuging at 12,000 rpm for 5 minutes was measured at 340 nm to obtain the activity of the enzyme protein obtained in Example 1. Was measured. As a result, the activity of the purified enzyme protein was 335.8 U / mg (MEF-1), 56.1 U / mg (MEF-2) and 153.1 U / mg (MEF-3), respectively (see Table 1 ).
    [64] Amount of protein (mg)Azocaine resolution (U / mg)Fibrin Resolution (U / mg) Herb extract110-0.02 Ammonium Salt Precipitation Fraction89.717.20.3* Bio-Gel P-60 fractions (A phase)2.3614910 DEAE-Epi-gel-blue fraction (Phase B)0.226 (MEF-1) 0.045 (MEF-2) 0.054 (MEF-3)335.8 (MEF-1) 56.1 (MEF-2) 153.1 (MEF-3)36.9 (MEF-1) 102.4 (MEF-2) 32.7 (MEF-3) Bio-gel P-60
    [65] <Example 4> Identification of thrombolytic enzyme protein
    [66] 1 μg of each of the enzyme proteins obtained in Example 1 and 10 mM of benzamidine (10 μl) as an inhibitor; 5 mM EDTA (5 μL); 5 mM EGTA (5 μl); 5 mM cysteine (5 μl); 0.02, 0.2 and 2 mM PMSF (2 μl); 10 mM β-mercaptoethanol (5 μl); 50 mM trypsin inhibitor (5 μL); And 50mM of aprotinin (3.25μL) was taken, and 50mM tris- hydrochloric acid (pH 7.5) was added so that each solution was 100μL and left for 1 hour. 1 ml of azocaine (concentration: 2 mg / ml) was added thereto, and left at 37 ° C. for 1-2 hours. Then, 250 µl of the solution was added, 5% TCA (1 ml) was added, and centrifuged at 12,000 rpm for 5 minutes. The absorbance of the supernatant obtained by separation was measured at 340 nm. (See Figure 4 , Table 2 and Table 3 )
    [67] As a result, in the inhibitor, MEF-1 showed 11% activity at 0.02 mM PMSF and MEF-2 and MEF-3 showed 1% and 3%, respectively. I found out that.
    [68] On the other hand, the serine protease of the present invention in order to determine which form of elastase, chymotrypsin or trypsin (elastonal) of 10, 50 and 100 μM; 100 μM TPCK; TLCK of 10, 50 and 100 μM; And 10, 50 and 100 μM of chymostatin inhibitors were tested in the same manner as described above to measure the absorbance.
    [69] As a result, in the MEF-1 and MEF-3 of the present invention, serine proteases were not inhibited in elastanal, TPCK and TLCK, but the activity was reduced by 90% or more even at the lowest concentration of 10 μM in chymostatin. The serine protease of 1 was found to be a new enzyme whose properties differ from the serine proteases found in existing mammals and microorganisms. In the case of MEF-2, at least 100% of the soybean trypsin inhibitor was reduced by 90% or more, and at 100μM of TLCK, the activity was reduced by more than 65%, thus the MEF-2 of the present invention was trypsin. It was confirmed that the protease belongs to.
    [70] InhibitorConcentration (mM)Relative activity (%) 100 EDTA585.8 EGTA590.6 Cysteine590.8 2-mercaptoethanol1086.4 PMSF21.37 Chymotrypsin0.184.1 Elastanal0.187.1 TPCK0.1114.4 TLCK0.10.20.533.65.223.34 Soybean Trypsin Inhibitor50 * 100 *26.15.85 Aprotinin50 * 100 * 250 *24.60.420.27 Benzaamidine10205030.52.030.98
    [71] InhibitorConcentration (mM)Relative activity (%) 100 EDTA5103.1 EGTA5103.4 Cysteine598.2 2-mercaptoethanol10110.1 PMSF22.6 Chymotrypsin0.15.2 Elastanal0.1106.7 TPCK0.1110.4 TLCK0.1110.9 Soybean Trypsin Inhibitor50 *106.8 Aprotinin50 *109.2 Benzaamidine10111.1
    [72] * Concentration: μM
    [73] <Example 5> Investigation of the Effect of Metal Ions on Thrombolytic Serine Protease
    [74] In the present invention, the effect of metal ions on the protease (MEF-1) was investigated by the following method. That is, CaCl 2 , MgCl 2 , BaCl 2 , CuCl 2 , ZnCl 2 and MnCl 2 are dissolved in water, respectively, and 5 mM of cations (Ca 2+ , Mg 2+ , Ba 2+ , Zn 2+ , Cu 2+ and Mn 2). + ), 5 μl each of them were mixed with 1 μg of serine protease, and 50 μM tris-hydrochloric acid buffer solution (pH 7.5) was added to make 100 μl and left at 37 ° C. for 1-2 hours. 1 ml of 2 mg / ml azocaine was added and left at 37 ° C. for 2 hours. Then, 250 μl of the solution was added, 5% TCA (1 mL) was added, followed by centrifugation at 12,000 rpm for 5 minutes, and the absorbance of the supernatant was measured at 340 nm.
    [75] As a result, the serine protease (MEF-1) of the present invention had almost the same level of activity as compared to the control group without the addition of metal ions, but the activity was 60-60 when Cu 2+ and Mn 2+ were added. 80% decrease.
    [76] <Example 6> Investigation of Temperature Stability of Proteases
    [77] In the present invention, the following experiment was performed to investigate the temperature stability of serine proteases. That is, 50 μl of tris-hydrochloric acid buffer (pH 7.5) is added to 1 μg of each serine protease to make 100 μl, and then 10 ° C., 20 ° C., 30 ° C., 40 ° C., 50 ° C., 60 ° C., 70 ° C. and 80 ° C. Each sample was left for 15 minutes at &lt; RTI ID = 0.0 &gt; C, &lt; / RTI &gt; Then, 250 μl of the solution was added, 5% TCA (1 mL) was added, followed by centrifugation at 12,000 rpm for 5 minutes, and the absorbance of the supernatant was measured at 340 nm. At this time, the optimal activity temperature of the serine protease (MEF-1) of the present invention was 37 ℃, the activity was sharply reduced above 50 ℃. In the case of the serine protease (MEF-2) of the present invention, the optimum activity temperature was 10 ° C., and its activity rapidly decreased above 50 ° C. In the case of the serine protease (MEF-3) of the present invention, the optimum activity temperature was 30 ° C., and its activity rapidly decreased at 60 ° C. or higher. As a result, it was confirmed that the serine proteases of the present invention exhibited suitable activity in the range of 10-50 ° C.
    [78] <Example 7> PH stability investigation of protease
    [79] In the present invention, the following experiment was performed to investigate the pH stability of serine proteases. First, a citric acid buffer solution having a pH of 3.0, 4.0, and 5.0, respectively, having 0.1 M of citric acid and 0.1 M of sodium citrate having the composition of Table 1 was prepared. In addition, a pH of 7.0, 8.0 and 9.0 were prepared with 50 mM Tris-HCl buffer, respectively, and a buffer of pH 10.0 and 11.0 was prepared with 50 mM CAPS [3- (cyclohexylamino) -1-propanesulfonic acid]. It was. Then, 1 μg (4.5 μl) of each of the serine proteases of the present invention lyophilized by dialysis was mixed with 95.5 μl of the buffer solution prepared above, and left at 25 ° C. for 2 hours. To this was added 1 ml of 2 mg / ml azocasein, which was left at 37 ° C. for 30 minutes, and left at 37 ° C. for 1 hour. Then, 250 μl of the solution was added, 5% TCA (1 mL) was added, followed by centrifugation at 12,000 rpm for 5 minutes, and the absorbance of the supernatant was measured at 340 nm.
    [80] Citric Acid (0.1M)Sodium Citrate (0.1M) pH 3.046.53.5 pH 4.033.017.0 pH 5.020.529.5 pH 6.09.541.5 Unit: ml
    [81] As a result, the MEF-1 protease of the present invention showed little change in activity at pH 4.0 or higher, and was observed to exhibit optimal activity in the range of pH 5.0 to 8.0. MEF-2 protease exhibits optimal activity in the temperature range of 10-40 ° C. and in the range of pH 5.0-10.0. MEF-3 protease exhibits optimal activity in the temperature range of 10-50 ° C. and in the range of pH 5.0-10.0.
    [82] III. Thrombolytic Solubility of Proteases
    [83] Example 8 Measurement of Thrombolytic Activity of Protease Against Fibrinogen
    [84] In order to measure the thrombolytic action of fibrinogen of the proteases of the present invention, the following experiment was conducted. That is, 5 μg of the protease of the present invention was added to 200 μl of bovine fibrinogen (1 mg / ml) dissolved in 50 mM Tris-HCl buffer solution, respectively, for 30 seconds, 1 minute, 3 minutes, 5 minutes, 10 minutes, 20 minutes, Incubated for 30 minutes, 1 hour, 3 hours 5 hours and 24 hours. Then, 2 × SDS polyacrylamide gel developing buffer solution was added at a ratio of 1: 1 and boiled at 100 ° C. for 4 minutes, followed by 15 μl of electrophoresis. At the same time, the fibrinogen solution not treated with the protease was incubated for 30 minutes in a buffer solution. (See Figures 3A , 3B and 3C )
    [85] As a result, when the proteases and the fibrinogen were incubated, it was found that the α, β and γ chains of the fibrinogen were degraded within several minutes so that the proteases of the present invention acted directly on the fibrinogen.
    [86] Example 9 Quantification of D-Dimer by Enzyme Immunoassay
    [87] First, 1.5 ml of human plasma (pH 7.8) calciumated by adding 0.15 M Tris-HCl buffer solution and 0.005 M calcium chloride (CaCl 2 ) aqueous solution was added thereto, and 30 μl of 2.5 M calcium chloride aqueous solution was added thereto. Fibrin was prepared by incubating for 4 hours. To this fibrin, 450 μl of buffer and 1.25 μg of protease were added and reacted at 37 ° C. for 1 hour, 3 hours, 5 hours, and 7 hours, and 5 μl of each supernatant was collected using a Biopool Kit. The amount of D-dimer was measured by enzyme immunoassay. The amount of D-dimer increases when fibrin, which is the main component of thrombi, is linked to fibrinopeptide by plasmin. (See FIG. 5 )
    [88] As a result of the experiment, the amount of D-dimer was measured to be 10.2 mg per hour, indicating that the protease of the present invention functions to break down fibrin.
    [89] As described above, the enzyme proteins of the present invention are new serine proteases that are completely different from the existing proteases, and thus can be usefully used as therapeutic agents for thrombosis-related diseases such as coronary artery and cerebrovascular diseases because of their excellent resolution to fibrin.
    [90] (Sequence list)
    [91] SEQ ID NO: 1
    [92] Length of sequence: 10
    [93] Type of sequence: amino acid
    [94] Number of chains: 1 chain
    [95] Form: Straight
    [96] Type of sequence: peptide
    [97] Ala Asp Val Val Gln Gly Asp Ala Pro Ser
    [98] (Sequence list)
    [99] SEQ ID NO: 2
    [100] Sequence length: 20
    [101] Type of sequence: amino acid
    [102] Number of chains: 1 chain
    [103] Form: Straight
    [104] Type of sequence: peptide
    [105] Ile Val Gly Gly Glu Glu Ala Val Ala Gly Asp Phe Pro Ile Val Ser Leu Gln Glu
    权利要求:
    Claims (8)
    [1" claim-type="Currently amended] New serine protease with thrombolytic solubility.
    [2" claim-type="Currently amended] 2. The method of claim 1 wherein the molecular weight is 31,500 Daltons, isoelectric point is 6.1, sensitive to chymostatin, sensitive to metal ions of Cu 2+ and Mn 2+ , temperature range of 20-40 ° C. and pH 5.0-8.0 A serine protease (MEF-1), which exhibits optimal activity in a range, and whose amino terminus has the amino acid sequence of SEQ ID NO: 1.
    [3" claim-type="Currently amended] The method of claim 1, the molecular weight is 32,900 Daltons, sensitive to TLCK and soybean trypsin inhibitor, exhibits optimum activity in the temperature range of 10-40 ℃ and pH 5.0-10.0, the amino Serine protease (MEF-2), characterized in that the terminal has the amino acid sequence of SEQ ID NO: 2.
    [4" claim-type="Currently amended] The serine protease (MEF-3) according to claim 1, characterized in that the molecular weight is 35,600 daltons, sensitive to chymostatin and exhibits optimal activity in the temperature range of 10-50 ° C. and in the range of pH 5.0-10.0.
    [5" claim-type="Currently amended] The thrombus soluble serine protease of claim 1 consisting of eluting and extracting the supernatant, centrifuging the supernatant, and then dissolving the precipitate fraction and centrifuging the supernatant again and concentrating the purified supernatant. Manufacturing Method
    [6" claim-type="Currently amended] A thrombi soluble trademark herb extract obtained by eluting the trademark candle with a buffer solution.
    [7" claim-type="Currently amended] A pharmaceutical composition for the treatment of thrombosis-related diseases including coronary artery disease and cerebrovascular disease, comprising the serine protease of claim 1 or the trademark herb extract of claim 5 as an active ingredient
    [8" claim-type="Currently amended] A dietary supplement containing part or all of the serine protease of claim 1 or the trademarked herb extract of claim 5 as an active ingredient
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    同族专利:
    公开号 | 公开日
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    法律状态:
    1998-06-05|Application filed by 김영식
    1998-06-05|Priority to KR1019980020843A
    1998-06-05|Priority claimed from KR1019980020843A
    2000-01-15|Publication of KR20000000902A
    2002-10-12|Application granted
    2002-10-12|Publication of KR100320948B1
    优先权:
    申请号 | 申请日 | 专利标题
    KR1019980020843A|KR100320948B1|1998-06-05|Thrombolytic serine protease separated and purified from a herbaceous plant and method for producing the same|
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