JPH0429350B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel proteolytic enzyme extracted from earthworms such as Lumbricus rubellus. More specifically, the present invention provides four types of novel white amorphous powders obtained by purifying earthworm extracts and characterized by exhibiting fibrinolytic activity (hereinafter abbreviated as fibrinolytic activity). Protease, namely protease F--1-HM-27, F-I-
1-HM-54, F-I-2-HM-15 and F-
-Relates to HM-64. The enzyme of the present invention can be extracted, for example, by adding an aqueous solvent to earthworms and holding the mixture at an appropriate temperature for an appropriate period of time, and then extracting the extract as it is or after maintaining it at an appropriate temperature for an appropriate period of time, concentrating or drying it. adsorbent,
It can be obtained by treatment with a polar organic solvent, salting out, ultrafiltration, ion exchange chromatography or hydrophobic chromatography, or a combination thereof. In other words, these six new enzymes are earthworm-derived proteases;
Both have fibrinolytic activity. Upon separation, these enzymes are distributed into separate fractions as shown in FIGS. 21 and 22, and are distinguished by these fractions. Al activity and substrate specificity: Enzymes of the present invention, such as protease F--1-
HM-27 is identified by measuring the physical and chemical properties shown below. A) Activity and substrate specificity F--1-HM-27 has fibrinolytic activity against fibrin clots, plasminogen activation activity, and casein, p-tosyl-L-
Arginine methyl ester hydrochloride (TAMe)
It is called. ), N-a-p-tosyl-L-lysine methyl ester hydrochloride (hereinafter referred to as TLMe), L
-Pyroglutamyl-glycyl-L-arginine-
p-Nitroanilide hydrochloride (usually called Test Team S-2444 chromogenic substrate, a product of Daiichi Kagaku Yakuhin Kogyo Co., Ltd., hereinafter simply referred to as S-2444) and H-
D-valyl-L-leucyl-L-lysine-p-nitroanilide dihydrochloride (usually tested by Test Team S-
It is called 2251 chromogenic substrate and is a product of Daiichi Kagaku Yakuhin Kogyo Co., Ltd. Hereinafter, it will be simply referred to as S-2251. ).
However, N-benzoyl-L-alanine methyl ester (hereinafter referred to as BAMe) and N-benzoyl-L-tyrosine ethyl ester (hereinafter referred to as BTEe)
It is called. ) had almost no effect. This fibrinolytic activity was determined by the method of T.Astrup [Arch.Biophy., 40 , 346
(1952)]. That is, fibrinogen was dissolved in 0.17M borate buffer (PH7.8) containing 0.01M sodium chloride so that the coagulable protein was 0.15%, then 10ml was poured into a sterile shear dish with a diameter of 80mm, and a thrombin solution (20μ/ ml), mix, cover and leave at room temperature for 1 hour (standard fibrin plate). Appropriately diluted test enzyme solution (in the case of powdered enzyme, 1mg/
After preparing a solution of 0.03 ml (appropriately diluted enzyme aqueous solution), drop it vertically onto a standard fibrin plate, sandwich a filter paper between the lids, leave it for 10 minutes, and then place it in a thermostat at 37°C for 18 hours. Make it react. The fibrinolytic activity (fibrinolytic activity) of this fibrin mass was expressed by measuring the long axis and short axis of the dissolved portion formed on a standard fibrin plate, and multiplying the product (mm ² ) by the dilution factor (in the case of an aqueous solution). mm ² /ml for powder, mm ² /ml for powder
(Activity units expressed in mg). In addition, the plasminogen activation activity was 10μ for plasminogen (manufactured by Sigma) at 5μ/ml;
Mix 20μ of the test enzyme aqueous solution and 30μ of 0.17M borate buffer (PH7.8) containing 0.01M sodium chloride.
After standing at 37℃ for 10 minutes, 0.03ml of this reaction solution was added.
was dropped vertically onto a plasminogen-free fibrin (Miles product) plate, reacted at 37°C for 18 hours, and the area of the dissolved portion was measured (expressed in ^mm2 ) as (X), and the same The value obtained by measuring in the same manner using 10μ of 0.17M borate buffer instead of plasminogen in the reaction system is defined as (Y), and the plasminogen activation activity (X)- Indicated by (Y). Next, the hydrolysis activity of casein was determined by the method of M. Kunitz [J.Gen.physiol., 30 291
(1947)]. i.e. 1.5
% milk casein (manufactured by Merck) in 1 ml of phosphate buffer (0.1M, PH8.0) solution and 1 ml of test enzyme aqueous solution.
was added, reacted for 30 minutes at 37°C, stopped the reaction by adding 2.0 ml of 0.4 M trichloroacetic acid aqueous solution, incubated for 15 minutes, centrifuged at 4000 rpm for 15 minutes, and collected the supernatant liquid at a wavelength of 280 nm. The absorbance at was measured. A casein solution, a trichloroacetic acid aqueous solution, and a test enzyme aqueous solution were added in this order and the same procedure was performed as a control, and the activity units were expressed in Kunitz units. Next, the hydrolytic activity of TAMe is
Methods in Enzymology
Enzymology, Volume 19, Page 41 (1970). That is, 19.7 mg of TAMe was dissolved in 50 ml of 0.1M Tris-HCl buffer (PH8.0), 3.0 ml of this TAMe solution was reacted with 0.15 ml of the test enzyme aqueous solution at 25°C, and the wavelength was measured after 1 minute.
Absorbance was measured at 247 nm. Note that the same measurement system in which purified water was used instead of the enzyme aqueous solution was used as a control. The activity unit is 1 μmol per minute.
The amount of enzyme used to hydrolyze TAMe was defined as 1 unit. Furthermore, the hydrolytic activity of TLMe is
Using TLMe instead of TAMe, the measurement wavelength
All measurements were performed using the same procedure as the TAMe activity measurement method, except that 250 nm was used. The activity unit was defined as the amount of enzyme that produced ΔA250nm of 1.0 per minute. Furthermore, the hydrolytic activity of BTEe was
Methods in Enzymology
Enzymology, Volume 19, Page 31 (1970). i.e. methanol
Dissolve 15.7 mg of BTEe in 30 ml, add purified water to make 50 ml, and then add 46.7 ml of 0.1M Tris-HCl buffer (PH8.0) to prepare 3.0 ml of BTEe solution.
and 0.15 ml of the enzyme aqueous solution were reacted at 25°C, and the absorbance at a wavelength of 256 nm was measured after 1 minute. Note that the same measurement system in which purified water was used instead of the enzyme aqueous solution was used as a control. The activity unit was defined as the amount of enzyme required to hydrolyze 1 μmol of BTEe per minute. In addition, the hydrolytic activity of BTEe is 19.7mg of BTEe.
The measurement was performed using a solution prepared by dissolving BTEe in 50 ml of 0.1M Tris-HCl buffer (PH8.0) and using the same procedure as the above method for measuring the activity of BTEe, except that the measurement wavelength was 255 nm. The activity unit was defined as the amount of enzyme that produced ΔA255nm of 1.0 per minute. The hydrolytic activity of S-2444 was reported in ``The Journal of Biological Chemistry''.
Journal of Biological Chemistry) Volume 265,
It was measured by the method described on page 2005 (1980). S-2444 was dissolved in 0.05M Tris-HCl buffer (PH7.4) containing 0.1M NaCl to a concentration of 0.5mM, 1ml of this substrate solution was mixed with 10μ of enzyme aqueous solution, and reacted at 25℃ for 1 hour. Wavelength 405nm after minutes
The increase in absorbance was measured. The enzyme unit was defined as the amount of enzyme required to hydrolyze 1 μmol of S-2444 per minute. Next, the hydrolysis activity of S-2251 is determined by the above-mentioned S-2251.
Use S-2251 instead of 2444 and adjust the substrate concentration.
All measurements were performed using the same procedure as the activity measurement method for S-2444, except that the concentration was 0.1mM. The activity unit is 1
The amount of enzyme required to hydrolyze 1 μmol of S-2251 per minute was defined as 1 unit. Table 1 shows the enzyme protease F--1 of the present invention.
This is a summary of the results obtained for various substrates using -HM-27.

[Table] However, ND in the table is an abbreviation of Not Detected and means almost undetected (hereinafter the same meaning). B) Optimal PH and stable PH range: Protease F- using fibrin mass as a substrate. The optimum pH for the fibrin-degrading action of -1-HM-27 was around 8 as shown in FIG. Furthermore, the stable pH range using fibrin mass as a substrate was approximately stable within the pH range of 5 to 12, as shown in FIG. PH stability was determined by measuring the residual activity of protease F--1-HM-27 after standing at 37°C for 60 minutes. C) Suitable temperature range for action: Using a fibrin mass with a pH of 7.8, 2
Protease F--1- when reacted for time
Figure 3 shows changes in the fibrin-degrading action of HM-27. The suitable temperature for action was within the range of 30 to 60°C, and the optimum temperature was around 50°C. D) Conditions for inactivation by various temperatures: protease F--1-HM-27 at PH7.8;
The residual fibrin-degrading activity of the fibrin clots after being incubated at various temperatures for 60 minutes is shown in Figure 4. From this, protease F--1-HM-27
was found to be completely inactivated by incubation at 70°C for 60 minutes. E) Molecular weight: 32400±2000 (measured by SDS polyacrylamide gel electrophoresis): Bovine serum albumin (molecular weight: 67000), ovalbumin (molecular weight: 43000) and chymotrypsinogen A (molecular weight: 25000) were treated with protease. F--
It was used as a standard material for determining the molecular weight of 1-HM-27. F) Ultraviolet absorption spectrum: Absorption maximum is around 280 nm and absorption minimum is around 250 nm. G) Isoelectric point: pI=3.6±0.1 H) Effect of inhibitors: The effects of various enzyme inhibitors on the fibrinolytic activity of protease F--1-HM-27 in fibrin clots were investigated. That is, 20μ of various inhibitor aqueous solutions (4mg/ml) were added to protease F--
1-Mix with 80 ml of HM-27 aqueous solution (2.5 μg/ml),
After incubating at 37°C for 10 minutes, 30μ of the reaction solution was taken and the fibrin degrading activity of the fibrin mass was measured. The results are shown in Table 2.

[Table] As is clear from this table, protease F-
-1-HM-27 is a serine reagent difluorophosphate, a protease inhibitor lima bean trypsin inhibitor, soybean trypsin inhibitor,
It is completely inhibited by antipain, leupeptin, and transilol (trade name: Bayer products), and considerably inhibited by egg white trypsin inhibitor and trans-4-(aminomethyl)cyclohexanecarboxylic acid, but epsilon- The results showed that aminocaproic acid, chymostatin and pepstatin inhibited the effect to some extent. However, the divalent cation chelator disodium ethylenediaminetetraacetate (hereinafter referred to as disodium ethylenediaminetetraacetate)
Abbreviated as EDTA. ) and the SH reagent N-ethylmaleimide did not inhibit the activity. I) Amino acid composition:

[Table] The equivalent of 0.2 mg of protease F--1-HM-27 was diluted to 0.5 ml with norleucine as an internal standard, 6N hydrochloric acid was added, and the diluted solution was hydrolyzed at 110°C for 24 hours, followed by analysis using an automatic amino acid analyzer. J) Elemental analysis values: C 48.61%, H 6.58%, N 14.75%, S
2.03% Next, a new protease F was prepared as a white amorphous powder.
For the physical and chemical properties of -I-1-HM-54, F-I-2-HM-15 and F--HM-64, please refer to the measurement method described in the section for protease F--1-HM-27. Measured using the same method as .
The physicochemical properties of these five types of proteases are listed below. A') Activity and substrate specificity: three new proteases F-I-1-HM-
54, F-I-2-HM-15 and F--HM-64
Table 4 shows the activity of the compound against various substrates.

[Table] FI-1-HM-54 has fibrinolytic activity, plasminogen activation activity, and acts on casein, TAMe, and BTEe. It has a slight effect on TLMe and S-2444, but has almost no effect on BAMe and S-2251. F-I-1-HM-15 has fibrinolytic activity against fibrin clots, plasminogen activating activity, casein, TAMe and BTEe.
It acts on It has a slight effect on TLMe and S-2444, but has almost no effect on BAMe and S-2251. F--HM-64 has fibrinolytic activity on fibrin clots, has plasminogen activating activity, acts on casein, and has a slight effect on TAMe. It has a slight effect on TLMe and S-2444, and a very slight effect on S-2251, but
It has little effect on BAMe and BTEe. As shown above, these three new proteases F-I-1-HM-54, F-I-2-HM-
The substrate specificities of 15 and F--HM-64 differ slightly in their activity. The terms used to express the strength or weakness of the hydrolyzing activity of each of the three new proteases on each substrate act when the enzyme unit (μ/mg) is 1 or more. Similarly, when it is in the range of 0.1 or more and less than 1, it has a slight effect. Similarly, it has a slight effect in the range of 0.01 or more and less than 0.1. Similarly, in the range of 0.001 or more and less than 0.01, it has a very slight effect. Similarly, when it is less than 0.001 and hardly detected, it is indicated as ND in Tables 1 and 4, and is expressed as having almost no effect. Such activity expressions of substrate specificity have the same meaning in Table 1. B') Optimal PH and stable PH range: 3 new proteases F-I-1-HM-
The optimum PH and stable PH range of 54, F-I-HM-15 and F--HM-64 are shown in FIGS. 5 to 10, respectively. That is, protease F-I
-1-HM-54 (Figure 5) and F-I-2-HM
The optimal pH of protease F-1-HM-64 (Fig. 6) is around 8 to 10, respectively.
The optimum pH for (Figure) was around 7-8. and protease F--1-HM-54
(Figure 8) and F-I-2-HM-15 (Figure 9).
The PH stability range was almost stable between PH4 and 12, and the PH stable range of protease F--HM-64 (Figure 10) was almost stable between PH5 and 12. C') Suitable temperature range for action: 3 types of proteases F-I-1-HM-54, F
-I-2-HM-15 and F--HM-64 are shown in FIG. 11 to FIG. 13, respectively. That is, protease F-I-1-
3 of HM-54 (Figure 11), F-I-2-HM-15 (Figure 12) and F--HM-64 (Figure 13)
The optimum temperature for action of all of these enzymes was in the range of 30 to 60°C, and the optimum temperature was about 50 to 60°C. D') Conditions for inactivation by various temperatures: 5 new proteases F-I-1-HM-
54, F-I-2-HM-15 and F--HM-64
Figures 14 to 16 show the residual fibrin-degrading activity of the fibrin clots after incubation at various temperatures at pH 7.8 for 60 minutes. Protease FI-1-HM-54 (Figure 14), FI-2
It can be seen that both -HM-15 (Fig. 15) and F--HM-64 (Fig. 16) are completely inactivated by incubation at 70°C for 60 minutes. Note that the white circles in Figures 1 and 5 to 7 represent phosphate buffer solutions,
Black circles represent Tris-glycine buffer, white circles in Figures 2 and 8 to 10 represent acetate buffer,
Black circles indicate phosphate buffer and triangles indicate Tris-glycine buffer. E') Molecular weight: Molecular weight was measured by SDS polyacrylamide gel electrophoresis. The results were as follows. F-I-1-HM-54 27500±2000 F-I-2-HM-15 27000±2000 F--HM-64 27800±2000 F') Ultraviolet absorption spectrum: Protease F-I-1-HM-54 ,F--
The absorption maxima of 2-HM-15 and F--HM-64 are both around 280 nm, and the absorption minima are both
It was around 250nm. G') Isoelectric point: The isoelectric point of protease F-I-1-HM-54 is
pI=4.0±0.1; Protease F-I-2-HM-
The isoelectric point of 15 is pI = 3.9 ± 0.1; protease F-
The isoelectric point of -HM-64 was pI=3.8±0.1; H') Effect of inhibitor: In the measurement method for F--1-HM-27, an aqueous solution of F--1-HM-27 (2.5 μg/
ml) instead of protease F-I-2-HM-
54 aqueous solution (12.5 μg/ml); Protease F-I-
2-HM-15 aqueous solution (12.5 μg/ml); Protease F--HM-64 aqueous solution (25 μg/ml) using 80 μ of each enzyme aqueous solution, and otherwise performing the same procedure to prepare Protease F-I-1. -HM-
54, F-I-2-HM-15 and F--HM-64
The effects of various inhibitors on The results are shown in Table 5. As is clear from this, protease F--1-HM-54 is
It is completely inhibited by lima bean trypsin inhibitor, difluorophosphate and N-ethylmaleimide, significantly inhibited by leupeptin and soybean trypsin inhibitor, and to some extent inhibited by chymostatin, t-AMCHA and egg white trypsin inhibitor. ,EDTA,
It was not inhibited at all by pepstatin, antipain, trasylol, and epsilon-aminocaproic acid. Protease F-I-2-HM-15
was completely inhibited by lima bean trypsin inhibitor, difluorophosphate and N-ethylmaleimide, significantly inhibited by egg white trypsin inhibitor and leupeptin, antipain, soybean trypsin inhibitor, pepstatin, epsilon-aminocaproic acid, chymostatin. , EDTA and t-AMCHA to some extent, but not at all by trasylol. Protease F--HM-64 was completely inhibited by lima bean trypsin inhibitor, difluorophosphate, N-ethylmaleimide and soybean trypsin inhibitor, and by egg white trypsin inhibitor, trasylol, pepstatin, t-AMCHA and chymostatin. and to some extent by antipain and epsilon-aminocaproic acid;
It was not inhibited at all by EDTA and leupeptin. In addition, complete inhibition means that the relative activity value in Table 5 is 0, considerable inhibition means that the relative activity value is less than 50%, and some inhibition means that the relative activity value is less than 50%. is 50% or more, and the relative value is 100 to indicate that there is no inhibition at all. This expression has the same meaning in the case of Table 2 above.

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[Table] J') Elemental analysis values: 1) Elemental analysis values of protease F-I-1-HM-54: C 48.93% H 6.65% N 15.95% S 1.34% 2) Protease F-I-2-HM- Elemental analysis values of 15: C 46.15% H 6.64% N 16.02% S 2.05% 3) Elemental analysis values of Protease F--HM-64: C 48.23% H 6.53% N 15.93% S 1.43% Novel protease of the present invention F--1-HM
-27, F-I-1-HM-54, F-I-2-HM
It was found that the four enzymes, -15 and F--HM-64, all have excellent fibrin decomposition activity for fibrin clots, as described above, and also have plasminogen activating activity. In other words, the four new proteases of the present invention are stable new enzymes that were extracted and isolated from earthworms for the first time by the present inventors, and are expected to have the following clinical effects through their excellent fibrinolytic activity. be done. Fibrin, which is generally converted from fibrinogen by enzymes, is one of the important causes of thrombosis and embolism. The four new proteases of the present invention can be used to treat peripheral arteriovenous thrombosis, pulmonary embolism, coronary artery occlusion, myocardial infarction, cerebrovascular occlusion, etc. due to the above-mentioned active action.
It is expected to be effective in preventing and treating retinal arteriovenous thrombosis, vitreous hemorrhage, anterior chamber hemorrhage, etc. In addition, it is expected to have a combined effect against cancer when used in combination with anticancer drugs, and is also expected to be effective as an anticoagulant during blood transfusion, to prevent embolization of suture lines in vascular surgery, and to maintain long-term function of arteriovenous shunts in hemodialysis. be done. Next, methods for producing, separating, and purifying various novel proteases of the present invention will be explained in detail in Examples. Example 1 (1) Add 0.9% sodium chloride aqueous solution containing 0.1% sodium benzoate to 1 kg of freeze-dried earthworm powder, stir at 30°C for 72 hours to extract, filter, and reduce the residual amount to 0.1% sodium benzoate. The extract was washed with a 0.9% aqueous sodium chloride solution containing 0.9% sodium benzoate, and the extract and washing solution were combined to obtain a clear extract (fibrinolytic activity against fibrin clots: 450 mm ² /ml at 10-fold dilution) 13. This extract was ultraconcentrated to a liquid volume of 0.71, and 0.71 of ethanol was added to it to precipitate. Ethanol was added to the filtrate after fractionation so that the final concentration of ethanol was 80%, and the resulting precipitate was After further washing with ethanol, vacuum drying and dry powder 42g
(The fibrin-degrading activity of this product against the fibrin mass was 1322 mm ² /mg.) The powder was dissolved in 1000 ml of purified water, and treated with DEAE-Cellulofine (product of Chitsuso Corporation) column chromatography. and, as shown in FIG. 17, F-I-1 containing a novel protease,
Four fractions, F-I-2, F- and F-, were obtained. (2) After salting out each fraction at 0.6 saturated ammonium sulfate, the resulting precipitate was added to a small amount of 10mM phosphate buffer (PH8.0).
After gel filtration using Sephacryl S-200, desalting and concentration using Ultrafiltration, and freeze-drying, 0.209 g of F-I-1 fraction and 0.42 g of F-I-2 fraction were obtained. g,
0.879 g of purified protease from F-fraction and 1.070 g of F-fraction were obtained. The fibrinolytic activity of each of these fractions on fibrin clots was determined by F-I-1.
15200mm ² /mg, F-I-2 is 12000mm ² /mg, F-
was 9290 mm ² /mg and F- was 17620 mm ² /mg. Example 2 After obtaining F-I-1 fraction, F-I-2 fraction, F-fraction, and F-fraction by the same method as in Example 1-(1), F-I-1 fraction was obtained. Each of the fractions and F-I-2 fractions was passed through a DEAE-cellulofine column equilibrated with 10mM phosphate buffer (PH8.0) to adsorb the active fraction, and then treated with the same buffer. The active fraction was eluted with a concentration gradient of 0 to 100 mM sodium chloride, the obtained active fraction was collected, and the purified product was purified by polyacrylamide gel electrophoresis by gel filtration with Sephadex G-75.
I-1-HM-54 is 0.07g and F-I-2-HM
-15 obtained 0.06g. The F-fraction was passed through a Toyo Pearl HW-55 (Toyo Soda product) column equilibrated at a saturation concentration of ammonium sulfate of 0.3 to adsorb the active fraction.
Elution is performed with a concentration gradient of ammonium sulfate 0.3 to 0.1 saturation, the active fraction is collected and desalted, and this is passed through a hexyl-Sepharose column equilibrated with 10mM phosphate buffer (PH6.0) to determine the activity. After the fraction is adsorbed, the active fraction is eluted with the same buffer using a concentration gradient of 0 to 15mM of sodium chloride. F- of refined products
-HM-64 obtained 0.1g. The F-fraction was passed through an egg white trypsin inhibitor (Sigma product) Sepharose affinity carrier column equilibrated with 20mM phosphate buffer (PH8.0), and after adsorbing the active fraction, 1M The same buffer containing salt and 0.1M
After washing with acetate buffer (PH5.0), acetate buffer (PH5.0) containing 0.5M arginine and 1M NaCl.
The active fraction was eluted to obtain the F-fraction. The F-
The fractions were further passed through a Toyopearl HW-55 column equilibrated with a 0.3 saturated solution of ammonium sulfate to adsorb the active fraction, and eluted with a concentration gradient of 0.3 saturated to 0.1 saturated ammonium sulfate to perform polyacrylamide gel electrophoresis. Single purified product F--1-HM-
27 obtained 0.07g and other fractions 0.06g (No. 18
figure).

[Brief explanation of drawings]

Figures 1 to 4 show protease F-- in terms of fibrinolytic activity against fibrin clots.
1-Graphs showing the optimum PH, PH stability, suitable temperature for action, and temperature stability of HM-27, Figures 5 and 8
11 and 14 are graphs respectively showing the optimal pH, pH stability, optimal temperature for action, and temperature stability of the novel protease F-I-1-HM-54 in terms of fibrinolytic activity against fibrin clots. 6th
Figures 9, 12, and 15 show the optimal PH, PH stability, optimal temperature for action, and temperature stability of the novel protease F-I-2-HM-15 in terms of fibrinolytic activity against fibrin clots. The graphs shown in Figures 7, 10, 13 and 16 respectively show the optimal PH and PH of the novel protease F--HM-64 in terms of fibrin degrading activity against fibrin clots.
Graphs showing stability, suitable temperature for action, and temperature stability, respectively. Figure 17 shows the results of the present invention in terms of fibrin degrading activity on fibrin masses obtained by subjecting earthworm extract to alcohol precipitation treatment and then DEAE-cellulofine chromatography treatment. Figure 18 shows the fractionation pattern of the novel protease after the earthworm extract was subjected to alcohol precipitation.
Fibrin-degrading activity, S-2444-degrading activity, and S-2251-degrading activity for the F-fraction obtained by DEAE-cellulofine chromatography treatment, and the fibrin clot obtained by Toyopearl HW-55 column chromatography treatment. The novel protease of the present invention F--1-HM-
27 is a graph showing fractionation patterns of 27 and other fractions.

Claims (1)

[Claims] 1. A white amorphous powdered protease F--1-HM-27 having the following physical and chemical properties. A) Activity and substrate specificity: Fibrin degrading action on fibrin clots, plasminogen activation action, casein, p-tosyl-L-arginine methyl ester hydrochloride, N-
a-p-tosyl-L-lysine methyl ester hydrochloride, L-pyroglutamyl-glycyl-L-arginine-p-nitroanilide hydrochloride and H-D-valyl-L-leucyl-L-lysine-p-nitroanilide・It has a strong effect on dihydrochloride, but
It has no effect on N-benzoyl-L-alanine methyl ester and N-benzoyl-L-tyrosine ethyl ester. B) Optimal PH and stable PH range Optimum PH for fibrin decomposition action when using fibrin mass as a substrate: around 8, stable PH range 5
~12. C) Suitable temperature range for action: Suitable temperature for action in fibrin decomposition reaction against fibrin lumps at PH7.8: 30-60°C, optimal temperature approximately 50°C
℃. D) Inactivation conditions: Complete inactivation by keeping at 70°C for 60 minutes. E) Molecular weight: 32400±2000 F) Ultraviolet absorption spectrum: Maximum absorption near 280nm and minimum absorption near 250nm. G) Isoelectric point: pI=3.6±0.1 H) Effect of inhibitor: The fibrin degrading effect on fibrin clots is
Completely inhibited by lima bean trypsin inhibitor, difluorophosphate, soybean trypsin inhibitor, antipain, leupeptin and trasylol, considerably inhibited by egg white trypsin inhibitor and trans-4-(aminomethyl)cyclohexanecarboxylic acid, ε-aminocaproic acid , chymostatin, and pepstatin, but not substantially inhibited by ethylenediaminetetraacetic acid disodium salt and N-ethylmaleimide. I) Amino acid composition: [Table] J) Elemental analysis values: C 48.61%, H 6.58%, N 14.75%, S
2.03%. 2 White amorphous powdered protease F-I-1-HM-54 having the following physical and chemical properties. A) Activity and substrate specificity: fibrin degrading action on fibrin clots, plasminogen activation action, casein, p-tosyl-L-arginine methyl ester hydrochloride and N
-Has a strong effect on benzoyl-L-tyrosine ethyl ester, but N-a-p-tosyl-
L-lysine methyl ester hydrochloride and L-pyroglutamyl-L-glycyl-L-arginine-p-
It has a slight effect on nitroanilide hydrochloride, and N-
Benzoyl-L-alanine methyl ester and H
-D-valyl-L-leucyl-L-lysine-p-
It has little effect on nitroanilide dihydrochloride. B) Optimal PH and stable PH range Optimum PH for fibrin decomposition action when using fibrin clot as a substrate: approximately 8 to 10, stable PH range 4
~12. C) Suitable temperature range for action: Suitable temperature for action in fibrin decomposition reaction against fibrin lumps at PH7.8: 30-60°C, optimal temperature approximately 50°C
℃~60℃. D) Inactivation conditions: Complete inactivation by keeping at 70°C for 60 minutes. E) Molecular weight: 27500±2000. F) Ultraviolet absorption spectrum: Shows absorption maximum near 280 nm and absorption minimum near 250 nm. G) Isoelectric point: pI=4.0±0.1. H) Effect of inhibitors: The fibrin degrading effect on fibrin clots is
Completely inhibited by lima bean trypsin inhibitor, difluorophosphate and N-ethylmaleimide, significantly inhibited by leupeptin and soybean trypsin inhibitor, chymostatin, trans-4-(aminomethyl)cyclohexanecarboxylic acid and egg white trypsin inhibitor. It is inhibited to some extent by disodium ethylenediaminetetraacetate, pepstatin, antipain, trandiol, and ε-aminocaproic acid. I) Amino acid composition: [Table] [Table] J) Elemental analysis values: C 48.93%, H 6.65%, N 15.95%, S
1.34%. 3 White amorphous powdered protease F-I-2-HM-15 having the following physical and chemical properties. A) Activity and substrate specificity: fibrin degrading action on fibrin clots, plasminogen activation action, casein, p-tosyl-L-arginine methyl ester hydrochloride and N
-Has a strong effect on benzoyl-L-tyrosine ethyl ester, but N-a-p-tosyl-
It has a slight effect on L-lysine methyl ester hydrochloride and L-pyroglutamyl-glycyl-L-arginine-p-nitroanilide hydrochloride, and has a slight effect on N-benzoyl-L-alanine methyl ester and H-D
It has almost no effect on -valyl-L-leucyl-L-lysine-p-nitroanilide dihydrochloride. B) Optimal PH and stable PH range: Optimal PH for fibrin decomposition action using fibrin mass as a substrate is about 8-10, stable PH range 4-12. C) Suitable temperature range for action: Suitable temperature for action on fibrin clots at PH7.8: 30~
60℃, optimal temperature about 50-60℃. D) Inactivation conditions: Complete inactivation by keeping at 70°C for 60 minutes. E) Molecular weight: 27000±2000. F) Ultraviolet absorption spectrum: Shows absorption maximum near 280 nm and absorption minimum near 250 nm. G) Isoelectric point: pI=3.9±0.1. H) Effect of inhibitors: The fibrin degrading effect on fibrin clots is
Completely inhibited by lima bean trypsin inhibitor, difluorophosphate and N-ethylmaleimide, significantly inhibited by egg white trypsin inhibitor and leupeptin, antipain, soybean trypsin inhibitor, pepstatin, ε-aminocaproic acid, chymostatin, ethylenediaminetetraacetic acid disodium and trans-
It is inhibited to some extent by 4-aminomethylcyclohexanecarboxylic acid, but not by transiol. I) Amino acid composition: [Table] Not available.
J) Elemental analysis values: C 46.15%, H 6.64%, N 16.02%, S
2.05%. 4 White amorphous powdered protease F--HM-64 having the following physical and chemical properties. A) Activity and substrate specificity: It has a fibrin degrading effect on fibrin clots, a plasminogen activation effect, a strong effect on casein, and an effect on p-tosyl-L-arginine methyl ester hydrochloride, but N-a-p
-tosyl-L-lysine methyl ester hydrochloride and L-pyroglutamyl-glycyl-L-arginine-p-nitroanilide hydrochloride, H-D-valyl-L-leucyl-L-lysine-p - It acts only slightly on nitroanilide dihydrochloride, and has almost no effect on N-benzoyl-L-alanine methyl ester and N-benzoyl-L-tyrosine ethyl ester. B) Optimal PH and stable PH range Optimum PH for fibrin decomposition action when using fibrin clot as a substrate: approximately 7 to 8, stable PH range 5
~12. C) Suitable temperature range for action: Suitable temperature for action in fibrin decomposition reaction against fibrin lumps at PH7.8: 30-60°C, optimal temperature approximately 50°C
℃~60℃. D) Inactivation conditions: Complete inactivation by keeping at 70°C for 60 minutes. E) Molecular weight: 27800±2000. F) Ultraviolet absorption spectrum: Shows absorption maximum near 280 nm and absorption minimum near 250 nm. G) Isoelectric point: pI=3.8±0.1 H) Effect of inhibitor: The fibrin degrading effect on fibrin clots is
Completely inhibited by lima bean trypsin inhibitor, difluorophosphate, N-ethylmaleimide, and soybean trypsin inhibitor, and significantly inhibited by egg white trypsin inhibitor, trasylol, pepstatin, chymostatin, and trans-4-(aminomethyl)cyclohexanecarboxylic acid. and is inhibited to some extent by antipain and ε-aminocaproic acid, but not by disodium ethylenediaminetetraacetate and leupeptin. I) Amino acid composition: [Table] J) Elemental analysis values: C 48.23%, H 6.53%, N 15.93%, S
1.43%.