KR100215647B1 - Novel fibrinolytic enzymes and purification method thereof - Google Patents

Abstract

The present invention relates to a novel thrombolytic enzyme isolated from Tabanus fulvus and a purification method thereof. The novel thrombolytic enzymes of the present invention, tabanase I and II, are serine protease and have a molecular weight, respectively. It is 22,000 Daltons and 27,000 Daltons, showing much better fibrinolysis than Lumbrokinase and stable over a wide range of pH.

Description

Novel thrombolytic enzymes and purification methods thereof

The present invention relates to a novel thrombolytic enzyme and a purification method thereof, and more particularly to a novel thrombolytic enzyme isolated from Tabanus fulvus and purified method thereof.

Thrombotic diseases such as myocardial infarction, stroke, and venous thrombosis are the largest causes of human death. In the United States and Europe, where myocardial infarction is the first cause, many new drugs have been developed to improve the prevention and treatment of these diseases. Research efforts are being made. Stroke has been recorded as a major cause of death along with cancer in Korea, China, and Japan. Recently, in Korea, thrombotic diseases such as myocardial infarction and venous thrombosis have increased rapidly along with westernization of lifestyles such as eating habits. National interests and measures for prevention and treatment are desired.

Currently, anticoagulants, antiplatelet agents and thrombolytic agents are used for the prevention and treatment of thrombotic diseases. Anticoagulants and antiplatelet agents can prevent the spread of blood clots, but they do not dissolve the formed clots. Fortunately, tissue-type plasminogen activator (t-PA), a urokinase-type plasminogen activator (t-PA) capable of breaking down fibrin, a major component of recent thrombi, to dissolve blood clots already formed. Type plasminogen activator (uPA) and streptokinase have been developed to clarify the prospects for the treatment of thrombotic diseases, and in particular, genetic engineering mass production methods of t-PA and u-PA have been devised for the treatment of thrombotic diseases. Has made a breakthrough electricity. In Korea, expensive recombinant t-PA is currently imported and used for the treatment of thrombotic diseases. U-PA is separated and purified from human urine by biochemical methods and used for treatment.

In the East, many natural products and herbal medicines are used for the treatment of thrombotic diseases in synonyms and folk remedies. However, scientific and scientific studies on the effects of these drugs are not available. Efforts to establish separate purification technologies and develop new drugs through such research are needed. In fact, some of the natural products currently in use are thought to contain anti-thrombotic components, and therefore, are expected to be developed into new drugs. Currently, many countries are investing enormous research funds and efforts in the development of therapeutic agents for thrombotic diseases. If successful, they will contribute to the health of human beings as well as the economy of developing countries. In light of these considerations, research and development of thrombotic disease therapeutics targeting natural products used in synonymology and folk medicine are considered to be very advantageous in terms of the success of new drug development and the efficiency and economic efficiency of investment research. Treatment should be implemented and urgently needed in the Asian region with rich experience.

Thus from the present inventors have anti-thrombotic effect is recognized and the mangchung (Tabanus fulvus) herbal medicines have been used in efforts result, thrombolytic agents from example to retrieve and remove the fibrinolytic components in the 38 kinds of natural products in donguihak and remedies Successfully isolated and purified novel thrombolytic enzymes to complete the present invention.

It is an object of the present invention to provide novel thrombolytic enzymes.

Another object of the present invention is to provide a method for separating and purifying the novel thrombolytic enzyme from natural products.

1 is a diagram schematically showing a method for separating and purifying tabanase from an insect,

Figure 2 shows the fibrinolytic ability of the fraction separated from the insect extract by Sephacryl S-200 gel filtration chromatography,

Figure 3 shows the fiber soluble capacity of the fraction separated by ion exchange chromatography fractions with high fiber soluble in Figure 2,

Figure 4 shows the cellulose soluble capacity of the fraction separated by high hydrophobic affinity column (Toyo Pearl HW) chromatography in Figure 3,

FIG. 5 shows the result of fibrin autographing three kinds of fractions having high fibrinolytic ability in the fraction of FIG.

Figure 6 shows the fibrinolytic capacity of the fraction separated from the fraction 2 of Figure 4 by Sephadex G-75 gel filtration chromatography,

Figure 7 shows the fibrinolytic capacity of the fraction obtained by separating fraction 3 of Figure 4 by Sephadex G-75 gel filtration chromatography,

FIG. 8 shows the results of electrophoresis (SDS-PAGE) and fibrin autographing the fractions having high fibrinolytic ability in the chromatograms of FIGS. 6 and 7,

FIG. 9 shows the results obtained by comparing fibrinolytic activity of tabanase I and II with lumbrokinase fraction F-III-2 on a fibrin plate. FIG.

Figure 10 shows the results of the investigation of the effect of pH and temperature on the activity of tabanase I,

Figure 11 shows the results of the investigation of the effect of pH and temperature on the activity of tabanase II,

Figure 12 shows the stability of the fibrinolytic ability with various fibrinolytic enzymes over time.

In accordance with the above object, the present invention provides thrombolytic enzymes tabanase I and II isolated and purified from Tabanus fulvus .

According to the above another object, in the present invention, by adding ammonium sulfate to the physiological saline extract of the insects to obtain a precipitate; The precipitate is suspended in a buffer solution and then dialyzed in the same buffer solution; The dialysed solution is filtered through an anion exchange resin column; A novel method for separating thrombolytic enzymes is provided that includes concentrating an eluted protein and then performing gel filtration chromatography, anion exchange column chromatography, hydrophobic affinity column chromatography, and gel filtration chromatography. .

Hereinafter, the present invention will be described in detail.

The thrombolytic enzymes of the present invention are two kinds of serine-based proteases that are separated from Tabanus fulvus and named tabanase I and II, respectively, by the present inventors, and have a molecular weight of 22,000 daltons and Banase II is 27,000 Daltons. Their fibrinolytic inactivity is over 30,000 eurokinase international units per mg, much higher than the international unit of 4,000 eurokinase of lumbrokinase, the currently used thrombolytic enzyme. The enzymes are stable over a wide pH range of pH 4.0 to 11.0 and the proper pH for the activity of the enzyme is 7.0 to 9.0.

The amino terminal sequence of the thrombolytic enzymes of the present invention is N-Asp-Ile-Gly-Asn-Arg-Pro-Ile-Asp-Ile-Gln-Gln-Phe-Arg-Thr-Trp- C and N-Asn-Val-Gly-Trp-His-Arg-Ile-Asp-Ser-Gln-C for tabanase II, and to date no peptide with such N-terminal sequence is known.

The thrombolytic enzymes of the present invention can be obtained through several steps of separation and purification from nematodes, but can also be prepared by synthesizing the same amino acid sequence by conventional chemical synthesis after confirming the amino acid sequence of the separated enzymes.

According to the present invention, a method for separating and purifying a thrombolytic enzyme from a nematode is as follows.

The worms are crushed, and then saline is added and the extract obtained by shaking overnight is centrifuged and the supernatant is filtered. Ammonium sulfate was added to the filtrate and saturated to 60%, and the resulting precipitate was suspended in a small amount of buffer (0.01 mol tris hydrochloric acid, 0.01 mol sodium chloride, pH 8.0), and then dialyzed in the same buffer.

The dialyzed protein solution is placed in an anion exchange column, preferably DE-52 anion exchange column, previously equilibrated with the same buffer, washed with the same buffer until no more protein is present, and then buffered with 0.6 molar sodium chloride. The protein is eluted at once in solution. The protein eluate is concentrated, and then the protein is separated by gel filtration column chromatography, wherein Sephacryl S-200 is preferably used as the column. The eluted fractions were measured for absorbance at 280 nm, and the fibrinolytic ability of each fraction was measured using a fibrin plate.

The fractions of the fibrinolytic ability identified above are collected to separate proteins by anion exchange column chromatography. It is preferable to use a DE-52 column, and the concentration gradient elution using 0.1 mol sodium chloride buffer and 0.6 mol sodium chloride buffer is performed to elute the protein, and then the absorbance and fibrinolytic ability of the eluted fractions are measured, respectively.

Fractions with high fibrinolytic ability were collected and made into a 30% saturated ammonium sulfate solution, followed by chromatography using a hydrophobic column, preferably Toyopearl HW-55. The protein was eluted using a concentration gradient of 30% to 10% of ammonium sulfate, and then the absorbance and fiber solubility of the eluted fractions were measured.

In order to confirm the fibrinolytic ability and molecular weight of the fraction showing high fibrinolytic ability, fibrin autography was performed to confirm the fraction of fibrinolytic components present in the nematode, and then concentrated, respectively, followed by Sephadex paper (Sephadex). Pure purification can be achieved by performing two gel filtration chromatography each with G) -75.

Hereinafter, the present invention will be described in more detail by the following examples. The following examples are given for the purpose of illustrating the invention and do not limit the scope of the invention.

Example 1 Separation and Purification of Thrombolytic Components Existing in a Nematode

300 g of worms were pulverized, and 10 times of saline was added thereto, followed by shaking overnight at 4 ° C. All procedures below were performed at 4 ° C unless otherwise noted. The shake was centrifuged at 5,000 rpm for 15 minutes and the supernatant was separated and filtered. Ammonium sulfate was added to the filtrate and saturated to 60%, and the solution was centrifuged at 12,000 rpm for 30 minutes. The resulting precipitate was suspended in a small amount of buffer (0.01 mol tris hydrochloric acid, 0.01 mol sodium chloride, pH 8.0) and then dialyzed in the same buffer.

The dialyzed protein solution was placed in a DE-52 anion exchange column previously equilibrated with the same buffer, washed with the same buffer until no more protein was present, and then the protein was temporarily removed with a buffer containing 0.6 mol sodium chloride. Eluted. Protein solution was concentrated using Amicon, and then protein was separated by gel filtration using Sephacryl S-200. The eluted fractions were measured for absorbance at 280 nm, and the fibrous solubility of each fraction was measured using a fibrous plate (see FIG. 2).

The fractions of the fibrinolytic ability confirmed above were collected to separate proteins using a DE-52 anion exchange column. The protein was eluted by forming a concentration gradient with 0.1 mol sodium chloride buffer and 0.6 mol sodium chloride buffer, and the absorbance and fibrinolytic ability of the eluted fractions were measured, respectively, and the results are shown in FIG. 3.

Fractions showing high fibrinolytic ability in FIG. 3 were collected and made into a 30% saturated ammonium sulfate solution, followed by chromatography using a hydrophobic column, Toyopearl (Toyopearl HW-55). The protein was eluted using a concentration gradient of 30% to 10% saturation concentration of ammonium sulfate, and the absorbance and fibrinolytic ability of the eluted fractions were measured, respectively, and the results are shown in FIG. 4.

Fibrin autography was performed in order to confirm their fibrinolytic ability and molecular weight since three fractions showed high fibrinolytic ability in FIG. 4, and the results are shown in FIG. 5. From the results of FIG. 5, fractions 2 and 3 were found to be fibrinolytic components present in the nematode.

Fractions 2 and 3 were each concentrated, followed by two gel filtration chromatography using Sephadex G-75, respectively. Absorbance and fibrinolytic ability of the eluted fractions were measured and shown in FIGS. 6 and 7, respectively. As a result, fractions showing high fibrinolytic ability were collected and subjected to polyacrylamide gel electrophoresis (SDS-PAGE) and fibrin autographing (see FIG. 8). In FIG. 8, two kinds of fibrinolytic components were identified, and the results of analyzing the sequence of the amino terminal ends of Fraction 2 and Fraction 3 were as follows.

N-Asp-Ile-Gly-Asn-Arg-Pro-Ile-Asp-Ile-Gln-Gln-Phe-Arg-Thr-Trp-C

N-Asn-Val-Gly-Trp-His-Arg-Ile-Asp-Ser-Gln-C

Searching the amino acid sequences in the database revealed that these two substances were novel proteins, and we named them Tabanase I and II, respectively. The process of separating the tabanase I, II from the nematode can be summarized as shown in Figure 1, the total volume of the sample, the amount of protein in the sample and the recovery rate of the protein is shown in Table 1 below.

Protein amount and recovery at each purification step sample Total volume (ml) Protein concentration (㎍ / ㎖) Total protein mass (mg) % Recovery Insect extract 1880 381.5 717.4 100 Anion exchange resin column (filtration method) 750 298.2 223.7 31.2 Gel filtration (sepacryl S-200) 240 324.3 77.8 10.8 Anion Exchange Resin Column (DE-52) 520 110.2 57.3 8.0 Hydrophobic Column (I) ^* 33 55.7 1.8 0.26 Hydrophobic Column (II) ^* 30 17.2 0.5 0.072 Gel Filtration (I) ^* (Sephadex G-75) 8.9 15.2 0.14 0.019 Gel Filtration (II) ^* (Sephadex G-75) 11.5 16.1 0.19 0.026 (I) ^* : Tabanase I (II) ^* : Tabanase II

Example 2

Tabanases I and II, identified as monomaterials, were used to investigate molecular weight, specific activity on fibrinolytic ability, pH and temperature effects, effects by protease inhibitors, and dependence on plasminogen.

1. Molecular Weight of Tabanase

The molecular weights of tabanases I and II are phosphorylase b (molecular weight 97,400 daltons), bovine serum albumin (molecular weight 66,200 daltons), ovalbumin (molecular weight 42,699 daltons), carbonic anhydrase ( sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) based on carbonic anhydrase (molecular weight 31,000 daltons), soybean trypsin inhibitor (molecular weight 21,500 daltons) and lysozyme (molecular weight 14,400 daltons) Determined by. The experiment was performed three times and the average value was calculated. The standard deviation was less than 500. As a result, the molecular weights of tabanase I and II were confirmed to be 22,000 and 27,000, respectively.

2. Inactivity to fibrinolytic ability

The specific activity of fibrinolytic activity was measured by standardizing the enzyme activity of urokinase (UK) using a fibrin plate. Compared to F-III-2, the most active of the lumbrokinase fractions isolated from the guin as a conventional fibrinolytic agent, the activity per mg of enzyme was 34,330 UK international units, Banase II appeared in 43,330 UK international units, and lumbrokinase in 4,000 UK international units, respectively, indicating that the cellulose solubles of tabanases I and II were much better (see FIG. 9).

3. Effect of pH and Temperature

In order to investigate the effect of pH and temperature on the fibrinolytic ability of tabanase, the tabanase was incubated at pH 3.0-12.0, 37 ° C. and 60 ° C. for 2 hours, and then the fibrinolytic ability was examined by the fibrin plate method. . FIG. 10 shows the effect of pH and temperature on fibrinolytic ability of tabanase I. FIG. As can be seen, tabanase I was found to have stable fibrin solubility in the range of pH 4.0-10.7 and a proper pH of 7.0-8.6, and the activity was maintained at 37 ° C, but all of the fibrous solubility was lost at 60 ° C. It can be seen that.

FIG. 11 shows the effect of pH and temperature on fibrinolytic ability of tabanase II. As can be seen, tabanase II was found to have stable fibrin solubility in the range of pH 4.0-10.0 and a proper pH of 7.0-9.0, and the activity was maintained at 37 ° C, but all of the fibrous solubility was lost at 60 ° C. It can be seen that.

From the above results, the fibrinolytic ability of tabanases I and II was found to be stable over a wide range with respect to pH, but sensitive to temperature.

4. Effect by protease inhibitors

The effects of protease inhibitors on tabanases are: phenylmethylsulfonyl fluoride (PMSF), disodium ethylenediaminetetraacetate (EDTA), ε-aminocaproic acid, soybean trypsin inhibitor, aprotinin ), Trans-4- (aminomethyl) cyclohexane carboxylic acid (t-AMCHA), and the like, and fibrinolytic activity was measured by a fibrin plate method. The remaining activity was measured in comparison with the fibrinolytic activity when no protease inhibitor was added and the results are summarized in Table 2. As shown in Table 2, PMSF, soybean trypsin inhibitor and aprotinin were shown to inhibit 100% of the cellulose soluble ability of tabanase, and thus, it was found that tabanase was a serine protease.

Effect of Protease Inhibitors Protease inhibitors Tabanase I Tabanase II Fibrinolytic capacity (mm ² ₎ Residual Activity (%) Fiber soluble capacity (mm ² ) Residual Activity (%) PMSF 0 0 0 0 EDTA 28.3 73.5 38.5 76.6 ε-aminocaproic acid 38.5 100 50.3 100 Soybean Trypsin Inhibitor 0 0 0 0 Aprotinin 0 0 0 0 t-AMCHA 31.2 81.0 38.5 76.6 Control 38.5 50.3

5. Effect of Plasminogen on Fibrin Solubility

In order to confirm the effect of plasminogen on the fibrinolytic ability of tavanase, the activity of tavanase I and II was compared with and without plasminogen in the fibrous plate, and as a result, plasminogen was added. Differences in the solubility of cellulose were not observed.

6. Stability over time of fiber soluble capacity

The specific activity of fibrinolytic ability of various fibrinolytic enzymes was investigated using a fibrin plate, and the results are shown in Table 3. As can be seen in the table below, the urokinase type plasminogen activator (u-PA) showed the highest specific activity and the lumpbrokinase (LK) showed the lowest specific activity.

Inactivity of Fibrinolytic Activity of Various Fibrinolytic Enzymes t-PA u-PA LK Tabanase I Tabanase II Inactivity (mm ² / μg) 636 949 59 723 785

The stability of fibrinolytic ability over time of various fibrinolytic enzymes was investigated at 37 ° C., and the results are shown in FIG. 12 as% of inactivity remaining after a certain time when the initial inactivation was 100. As can be seen in the figure, t-PA almost lost activity after 3 weeks, and activity was completely lost after 4 weeks. In the case of u-PA, activity was almost lost at 4 weeks and completely lost after 5 weeks. For LK, tabanase I (T1) and tabanase II (T2), the activity remained up to 20%, 23% and 10% of the original after 5 weeks, respectively.

From the above results, u-PA has a high initial activity but rapidly loses its activity over time, and thus has low stability, while LK has high stability but low activity itself, while a new thrombolytic enzyme tabanase of the present invention is present. It can be seen that it is an excellent thrombolytic enzyme with high activity and high stability.

Tavanase II, a novel thrombolytic enzyme of the present invention, is an excellent thrombolytic enzyme with high activity and high stability.

Claims (7)

A thrombolytic enzyme extracted from Tabanus fulvus , having a molecular weight of 27,000 Daltons, and an N-terminal amino acid sequence of SEQ ID NO: 2. SEQ ID NO: 2 N-Asn-Val-Gly-Trp-His-Arg-Ile-Asp-Ser-Gln-C (A) ammonium sulfate was added to the physiological saline extract of the insects to obtain a precipitate; (B) suspending the precipitate in a buffer solution and dialysis in the same buffer solution; (C) the dialysed solution is filtered through an anion exchange resin column; (D) concentrating the eluted protein, followed by gel filtration chromatography, anion exchange column chromatography, hydrophobic column chromatography, and gel filtration chromatography in sequence, wherein the thrombolytic enzyme of claim 1 from the nematode Method of purification. The method of claim 2, A method of eluting the protein with a buffer containing 0.6 M NaCl using the DE-52 anion exchange column in (c) above. The method of claim 2, Method of using Sephacryl S-200 column in the first gel filtration chromatography of (d). The method of claim 2, A method of eluting the protein by using a DE-52 column in the anion exchange column chromatography of (D) and forming a concentration gradient with 0.1 M NaCl buffer and 0.6 M NaCl buffer. The method of claim 2, A method of eluting the protein using a Toyopearl HW column in the hydrophobic column chromatography of (D) and using an ammonium sulfate concentration gradient of 30% to 10%. The method of claim 2, The second gel filtration chromatography of (d) using a Sephadex G-75 column and performing two chromatographys in succession.