CN1944642A - Ahylysantinfarctase 36KD single-stranded haemocoagulase and its preparing method - Google Patents

Abstract

The present invention is Agkistrodon acutus venon 36KD single-stranded hemocoagulase and its preparation process, and aims at providing single-stranded Agkistrodon acutus venon hemocoagulase with high potency and low toxicity. SDS-PAGE electrophoresis shows that the hemocoagulase has molecular weight of 36KD+/-2KD, single color zone, and at least 90 % homogeneity with the N end sequence of the single-stranded amino acid shown in SEQ ID No. 1. The preparation process includes dissolving Agkistrodon acutus venon in Tris-HCl buffering solution (pH8.0) and dialysis; adding the supernatant to Metal Sepharose F.F affinity chromatographic column and collecting the penetrating peak component; adding the penetrating peak component to DEAE-Sepharose F.F ion exchange column and collecting active peak component; separating in Superdex 75 column and Sephacryl S-100 column; desalting in Sephadex G-25 column; sterilizing and freeze drying.

Description

Ahylysantinfarctase 36KD strand hemocoagulase and preparation method thereof

Affiliated technical field

The present invention relates to the method for enzyme and separation thereof, enzyme purification, particularly the method for hemocoagulase and preparation thereof is obtained in separation and purification from ahylysantinfarctase (Agkistrodon acutus).

Background technology

K1obusizky in 1936 and konig isolate a kind of zymoprotein that can shorten the clotting time from willow leaf adder snake venom, be named as Coagulase, have many scholars to confirm to have this kind of enzyme in the different pallas pit vipers of Crotalinae in succession.Since the sixties, raising and biological chemistry along with the protein purification isolation technique, pharmacological further investigation, determined the hemocoagulase immense value of hemostat application clinically, wherein, reptilase (Reptilase) is Switzerland Solco Basle Ltd is about the preparation of 31KD strand hemocoagulase with the molecular weight that extracts in Brazilian spearhead Pallas pit viper (Bothrope jararaca) snake venom a hemostatic agent, its composition is except the class hemocoagulase, also contain class hemocoagulase kinases, this kinases activates preceding hemocoagulase and is hemocoagulase, impel Fibrinogen to change scleroproein into, form the Blood clotting identical with the class hemocoagulase.(" enzyme hemostasis injection reptilase " Solco Basle Ltd character) this hemostatic agent determined curative effect, but, do not have Brazilian spearhead pallas pit viper to distribute in China.In order to obtain a hemocoagulase with domestic with being born in the year of snake kind, domestic scholars has been carried out unremitting effort.For example, utilization is distributed in agkistrodon acutus (Agkistrodon acutus) the snake venom thrombin-like enzyme development of each province, China south China, application number 02138630.7 patent of invention prospectus " sTLE " and the continuous mutually zymoplasm component of isolating of application number 03140154.6 patent of invention prospectus " the agkistrodon acutus reptilase is as the medicine of treatment hemorrhagic diseases " with styptic activity, their-terminal amino acid sequences there are differences, and all form by two subunits of A, B, it is about 120U/mg than vigor.Because the difficult point that exists on the separating and purifying technology of ahylysantinfarctase strand hemocoagulase has the pertinent literature of actual reference significance few.

Before this, the present application people separates from the agkistrodon acutus snake venom and has obtained highly purified fiber eliminating enzyme, and obtains army's scientific-technical progress second prize in 2000.

Summary of the invention

The objective of the invention is to utilize the agkistrodon acutus resource of China's abundant, from this snake venom separation and purification go out that a kind of high yield, height are tired, height ratio vigor, hypotoxicity, VIGGNECDTNEEHFL.

The feature that hemocoagulase of the present invention has is through the SDS-PAGE electrophoresis, and its molecular weight is the single colour band of 36KD ± 2KD, and has 90% homogeny at least with the amino acid whose N-terminal sequence of the strand shown in the SEQ IDNO1.

Described this hemocoagulase iso-electric point is 6.59, and HPLC measures and is simple spike, and color atlas calculates by area normalization method, and purity is greater than 98%, and enzyme is 2000U/mg-3000U/mg albumen than living.

The preparation method of ahylysantinfarctase 36KD strand hemocoagulase of the present invention may further comprise the steps:

(1) take by weighing after the agkistrodon acutus snake venom adds Tris-HCl (PH8.0) damping fluid dissolving, the small-molecule substance and the inorganic salt of the following molecular weight of 1KD are removed in dialysis, are convenient to next step purifying;

(2) Metal Sepharose F.F affinity column on the supernatant liquor is collected and is penetrated the peak, removes most of non-Thrombin-like enzyme albumen, lipid, carbohydrate and other material, obtains the thick component of 36KD hemocoagulase and fiber eliminating enzyme;

(3) penetrate DEAE-Sepharose F.F ion exchange column on the peak, collect active peak, separate 36KD hemocoagulase and fiber eliminating enzyme;

(4) will have Superdex 75 posts and Sephacryl S-100 molecular sieve column on active the 5th peak of 36KD hemocoagulase, collect the 2nd peak, obtain the 3.6KD strand hemocoagulase of purifying;

(5) Sephadex G-25 post desalination, degerming, freeze-drying obtain purity greater than 98% 36KD strand hemocoagulase.

In preparation method of the present invention, in order to remove the small-molecule substance of the following molecular weight of 10KD, avoid influence to next step purifying, the agkistrodon acutus snake venom is done dialysed overnight after with damping fluid.

In the above content, homogeny be meant two or more aminoacid sequences relatively in the same or analogous percentage of sequence, the homogeny percentage between two seed amino acid sequences such as A sequence and the B sequence calculates by following formula:

The present invention utilizes method separation and purification from ahylysantinfarctase such as affinity chromatography to obtain hemocoagulase, disclosed from the abundant ahylysantinfarctase of Natural Resources in China and separated the homology that the hemocoagulase aminoacid sequence that obtains has height with external spearhead agkistrodon halyx pallas venom, this homology has constituted the internal condition that obtains the strand hemocoagulase of feature basically identical from ahylysantinfarctase and spearhead agkistrodon halyx pallas venom.

Biology of the present invention and pharmacodynamic analysis show that hemocoagulase is the zymoprotein of single component, have negated except the class hemocoagulase, also contain so-called class hemocoagulase kinases, and think that this is inducing of a mistake.

Hemocoagulase of the present invention has hemostasis in the body, obviously shortens the animal body intravascular coagulation time, height is tired, height ratio vigor, hypotoxicity, does not have other hematology rheological phenomena, does not have hemorrhage poison and undue toxicity.

Stable preparation process of the present invention, high yield, hemocoagulase product meet the quality standard of country to the snake-poison hemostatic medicine.

Description of drawings

Fig. 1 is a process flow sheet of the present invention.

Fig. 2 is an ahylysantinfarctase hemocoagulase reductibility SDS-PAGE electrophorogram of the present invention.

Fig. 3 is ahylysantinfarctase hemocoagulase HPLC purity testing figure of the present invention.

Embodiment

(1) affirmation of Agkistrodon acutus hemocoagulase atrox of the present invention

The aminoacid sequence of Agkistrodon acutus hemocoagulase atrox of the present invention and characteristic parameter are:

1, HPLC mensuration Agkistrodon acutus hemocoagulase atrox is single symmetrical peak, and purity is greater than 98%.

2, SDS-PAGE electrophoresis test: Agkistrodon acutus hemocoagulase atrox is a single district band, and molecular weight is 36 ± 2KD.

3, isoelectric focusing electrophoresis: the Agkistrodon acutus hemocoagulase atrox iso-electric point is 6.59.

4, the measurement result of 15 aminoacid sequences of N-terminal is: VIGGNECDTNEEHFL.

5, the external Freshman blood plasma that can condense, enzyme are 2000-3000U/mg albumen than living.

(2) preparation of ahylysantinfarctase hemocoagulase of the present invention

Embodiment one, 20060418 batches of ahylysantinfarctase 3.6KD hemocoagulase production instances

Take by weighing the snake poison lyophilized product 10g of agkistrodon acutus and add the 50ml sterile water for injection, 4 ℃ of deionized water dialysed overnight, 8000rpm, 15min, 4 ℃ centrifugal, after supernatant is regulated electricity and is directed at 3.5ms/cm with 1M Tris-HCl (PH8.0), on use the good affinity column Metal Sepharose F.F post of 0.05M Tris-HCl (PH8.0) balance in advance, 1M NH ₄Cl carries out stepwise elution, collects to penetrate the peak; Penetrate the DEAE-Sepharose post that balance is good on the peak, after 0.05M Tris-HCl (PH8.0) zero washes 1 times of column volume, do gradient elution with 0.05M Tris-HCl+1.0M NaCl (PH8.0), under detecting, ultraviolet 280nm collects wash-out 5 peaks (being the thick component of hemocoagulase), the desalination freeze-drying of active peak.Dried frozen aquatic products is dissolved in 10-20ml 0.05M Tris-HCl (PH7.5)+0.1M KCl, on use good Superdex 75 posts of 0.05MTris-HCl (PH7.5)+0.1M KCl balance in advance, above-mentioned damping fluid carries out wash-out, collects second peak; Go up the good Sephacryl S-100 post of 0.05M Tris-HCl (PH7.5)+0.1M KCl balance after the active ingredient desalination freeze-drying again, above-mentioned damping fluid carries out wash-out, collects second peak; Active peak Sephadex G-25 XK50/100 post desalination, and carry out quality examination, detected result is as follows: HPLC purity: 98.6%; SDS-PAGE reductibility electrophoresis molecular weight is 36KD, single district band; Than 2458U/mg alive, hemorrhage malicious 50U/ml mouse is subcutaneous no hemorrhage; 0.22um freeze-drying was preserved after membrane filtration removed mattress, obtained hemocoagulase 7153U altogether.

Embodiment two, 20060428 batches of ahylysantinfarctase 3.6KD hemocoagulase production instances

Take by weighing the snake poison lyophilized product 10g of agkistrodon acutus and add the 50ml sterile water for injection, 4 ℃ of deionized water dialysed overnight, 8000rpm, 15min, 4 ℃ centrifugal, after supernatant is regulated electricity and is directed at 3.5ms/cm with 1M Tris-HCl (PH8.0), on use the good affinity column Metal Sepharose F.F post of 0.05M Tris-HCl (PH8.0) balance in advance, 1M NH ₄Cl carries out stepwise elution, collects to penetrate the peak; Penetrate the DEAE-Sepharose post that balance is good on the peak, after 0.05M Tris-HCl (PH8.0) zero washes 1 times of column volume, do gradient elution with 0.05M Tris-HCl+1.0M NaCl (PH8.0), under detecting, ultraviolet 280nm collects wash-out 5 peaks (being the thick component of hemocoagulase), the desalination freeze-drying of active peak.Dried frozen aquatic products is dissolved in 10-20ml 0.05M Tris-HCl (PH7.5)+0.1M KCl, on use good Superdex 75 posts of 0.05MTris-HCl (PH7.5)+0.1M KCl balance in advance, above-mentioned damping fluid carries out wash-out, collects second peak; Go up the good Sephacryl S-100 post of 0.05M Tris-HCl (PH7.5)+0.1M KCl balance after the active ingredient desalination freeze-drying again, above-mentioned damping fluid carries out wash-out, collects second peak; Active peak Sephadex G-25 post desalination, and carry out quality examination.Detected result is as follows: HPLC purity: 98.67%; SDS-PAGE reductibility electrophoresis molecular weight is 36KD, single district band; Than 2630U/mg alive, hemorrhage malicious 50U/ml mouse is subcutaneous no hemorrhage; 0.22um freeze-drying is preserved after the membrane filtration degerming, obtains hemocoagulase 9500U altogether.

Embodiment three, 20060523 batches of ahylysantinfarctase 3.6KD hemocoagulase production instances

Taking by weighing the snake poison lyophilized product 10g of agkistrodon acutus head adds 50ml and does not have mattress water for injection, 4 ℃ of deionized water dialysed overnight, 8000rpm, 15min, 4 ℃ centrifugal, after supernatant is regulated electricity and is directed at 3.3ms/cm with 1M Tris-HCl (PH8.0), on use the good Metal sepharose F.F XK50/30 post (GE Healthcare company product) of 0.05MTris-HCl (PH8.0) balance in advance, 1M NH ₄Cl carries out stepwise elution, collects to penetrate the peak; Penetrate the DEAE-sepharose post that balance is good on the peak, after 0.05M Tris-HCl (PH8.0) zero washes 1 times of column volume, do gradient elution with 0.05M Tris-HCl+1.0M NaCl (PH8.0), under detecting, ultraviolet 280nm collects wash-out 5 peaks (being the thick component of hemocoagulase), the desalination freeze-drying of active peak.Dried frozen aquatic products is dissolved in 10-20ml 0.05MTris-HCl (PH7.5)+0.1M KCl, on use the Superdex75 post that 0.05M Tris-HCl (PH7.5)+0.1M KCl balance is good in advance, above-mentioned damping fluid carries out wash-out, collects second peak; Go up the good sephacryl S-100 post of 0.05M Tris-HCl (PH7.5)+0.1M KCl balance after the active ingredient desalination freeze-drying again), above-mentioned damping fluid carries out wash-out, collects second peak; Active peak SephadexG-25 post desalination, and carry out quality examination.Detected result is as follows: HPLC purity 99.5%; SDS-PAGE reductibility electrophoresis molecular weight is 36KD, single district band; Than 3200U/mg alive, hemorrhage malicious 50U/ml mouse is subcutaneous no hemorrhage; 0.22um freeze-drying is preserved after the membrane filtration degerming, obtains hemocoagulase 10980U altogether.

(3) pharmacodynamics test of Agkistrodon acutus hemocoagulase atrox of the present invention and result

(1) observes of the effect of ahylysantinfarctase 36KD hemocoagulase with test tube method to the rabbit whole blood setting time

Whole blood is meant the time that picks up counting when coagulation of blood does not flow time of coagulation when blood enters syringe.

Tried healthy new zealand rabbit and be divided into agkistrodon acutus 36KD hemocoagulase group, import reptilase control group, physiological saline control group and agkistrodon acutus fiber eliminating enzyme control group at random.Before the administration, after getting blood 1.2ml, respectively injects rabbit ear central artery 2 dry clean tube respectively.Get agkistrodon acutus 36KD hemocoagulase, reptilase, physiological saline and fiber eliminating enzyme, be mixed with 1U/ml solution with water for injection respectively, by rabbit body weight 1U/Kg rabbit ear edge intravenous administration, at 30min, 60min gets blood 1.2ml from rabbit ear central artery respectively and respectively injects 2 dry clean tube, observes whole blood time of coagulation.

The result shows, ahylysantinfarctase 36KD hemocoagulase with an intravenous injection of 1U/Kg after 30min, 60min, obviously shorten the rabbit whole blood setting time.After the administration, 30-60min reaches maximum effect, keeps 6-12hr, and whole blood clotting time recovers normal gradually then; The reptilase control group clotting time also obviously shortens; The physiological saline group clotting time does not have change; The agkistrodon acutus fiber eliminating enzyme then obviously prolongs the clotting time.(seeing the following form 1,2,3,4)

(2) observe of the influence of ahylysantinfarctase hemocoagulase with the Pick Wire method to rabbit fibrinogen formation time

The Pick Wire time is meant to get and bleeds on slide glass, and every interval certain hour is provoked blood with syringe needle, provokes the time of fiber protein yarn to syringe needle.

In the experiment, administration and blood extracting method are identical with above-mentioned whole blood coagulation, get blood after, remove syringe needle, drip a 2-3 and bleed on the dried and clean slide glass, provoke blood once with dry syringe needle every 10-20sec, observed and recorded Pick Wire time of occurrence.

The result shows, the rabbit fibrin of ahylysantinfarctase hemocoagulase group and import reptilase control group occur with administration before compared obvious shortening, the physiological saline control group maintains original level, fiber eliminating enzyme then obviously prolongs the clotting time.(seeing the following form 1,2,3,4)

Table 1 agkistrodon acutus 3.6KD hemocoagulase group

Numbering	Body weight (kg)	Dosage (U)	Before the administration		Time behind the medicine (min)	After the administration		The result judges
									The Pick Wire time	Whole blood time of coagulation	The Pick Wire time	Whole blood time of coagulation
			No. 1	2.6		2.6	6 minutes 30 seconds						7 minutes 07 second	30	2 minutes 18 seconds	3 minutes 37 seconds	Obviously shorten
60	2 minutes 42 seconds	2 minutes 48 seconds			Obviously shorten
								No. 2	2.7	2.7	5 minutes 26 seconds	6 minutes 18 seconds		30	2 minutes 30 seconds	4 minutes 13 seconds	Obviously shorten
60	2 minutes 08 second	3 minutes 07 second	Obviously shorten
				No. 3	2.4	2.4	5 minutes						5 minutes 07 second	30	3 minutes 36 seconds	4 minutes	Obviously shorten
60	2 minutes 47 seconds	3 minutes 36 seconds	Obviously shorten

Table 2 import reptilase control group

Numbering	Body weight (kg)	Dosage (U)	Before the administration		Time behind the medicine (min)	After the administration		The result judges
									The Pick Wire time	Whole blood time of coagulation	The Pick Wire time	Whole blood time of coagulation
			No. 1	2.8		2.8	5 minutes						5 minutes 37 seconds	30	3 minutes	4 minutes 13 seconds	Obviously shorten
60	2 minutes 20 seconds	2 minutes 40 seconds			Obviously shorten
								No. 2	2.3	2.3	4 minutes 12 seconds	5 minutes 30 seconds		30	2 minutes 08 second	2 minutes 43 seconds	Obviously shorten
60	2 minutes 30 seconds	2 minutes 50 seconds	Obviously shorten
				No. 3	2.9	2.9	7 minutes 15 seconds						7 minutes 20 seconds	30	5 minutes 36 seconds	6 minutes 07 second	Obviously shorten
60	3 minutes 12 seconds	3 minutes 47 seconds	Obviously shorten

Table 3 physiological saline control group

Numbering	Body weight (kg)	Dosage (U)	Before the administration		Time behind the medicine (min)	After the administration		The result judges
									The Pick Wire time	Whole blood time of coagulation	The Pick Wire time	Whole blood time of coagulation
			No. 1	2.5		2.5	4 minutes 03 second						4 minutes 35 seconds	30	4 minutes 07 second	4 minutes 01 second	No significant difference
60	4 minutes 13 seconds	4 minutes 21 seconds			No significant difference
								No. 2	2.6	2.6	4 minutes 53 seconds	5 minutes 30 seconds		30	4 minutes 30 seconds	5 minutes 20 seconds	No significant difference
60	4 minutes 58 seconds	5 minutes 20 seconds	No significant difference
				No. 3	2.4	2.4	5 minutes 02 second						5 minutes 17 seconds	30	4 minutes 48 seconds	5 minutes 43 seconds	No significant difference
60	5 minutes 12 seconds	5 minutes 33 seconds	No significant difference

Below in the experiment, the little 2KD of ahylysantinfarctase fiber eliminating enzyme molecular weight ratio hemocoagulase can obviously prolong the clotting time, as table 4:

Table 4 agkistrodon acutus fiber eliminating enzyme control group

Numbering	Body weight (kg)	Dosage (U)	Before the administration		Time behind the medicine (min)	After the administration		The result judges
									The Pick Wire time	Whole blood time of coagulation	The Pick Wire time	Whole blood time of coagulation
			No. 1	2.9		2.9	6 minutes						6 minutes 27 seconds	30	7 minutes 14 seconds	9 minutes 15 seconds	Obviously prolong
60	9 minutes 46 seconds	11 minutes 34 seconds			Obviously prolong
								No. 2	2.7	2.7	5 minutes 6 seconds	5 minutes 42 seconds		30	7 minutes 50 seconds	10 minutes 10 seconds	Obviously prolong
60	7 minutes 58 seconds	9 minutes 57 seconds	Obviously prolong
				No. 3	2.7	2.7	5 minutes 15 seconds						5 minutes 17 seconds	30	8 minutes 12 seconds	9 minutes 09 second	Obviously prolong
60	9 minutes 12 seconds	9 minutes 30 seconds	Obviously prolong

(3) the ahylysantinfarctase hemocoagulase does not have obvious influence to prothrombin time (PT), activated partial thrombin time (APTT) and Fibrinogen; Do not activate the XIII factor.

(4) toxicology of ahylysantinfarctase hemocoagulase of the present invention and safety testing

Select 20 of 20 of healthy ICR mouse and SD rats, male and female half and half, be divided into 4 groups at random, every group 10, ahylysantinfarctase hemocoagulase of the present invention is mixed with 1U/ml solution once to behind mouse and rat intravenous injection 0.2ml and the abdominal injection 1ml with water for injection, does not observe toxic reaction and animal dead.

The subcutaneous injection of hemorrhage malicious 50U/ml mouse back, zootomy is not seen hemorrhage after 24 hours.

Carry out phospholipase A, L-amino-acid oxidase, phospholipase detection by state quality standard WS-350 (X-318) 2001, its content is 0.00.

Carry out undue toxicity, pyrogen, supersensitivity and do not have the mattress test by state-promulgated pharmacopoeia (version in 2005) related request, it is all qualified to detect.

(5) ahylysantinfarctase 36KD strand hemocoagulase aminoacid sequence table

SEQ ID NO 1：

Sequence signature:

Chain: strand

Structure: linearity

Molecule type: polypeptide

Terminal 15 aminoacid sequences of N-are described: SEQ ID NO:1

1 5 10 15

V I G G N E C D T N E E H F L

Claims (3)

1, ahylysantinfarctase 36KD strand hemocoagulase is characterized in that the electrophoresis through SDS-PAGE, and its molecular weight is the single colour band of 36KD ± 2KD, and has 90% homogeny at least with the amino acid whose N-terminal sequence of strand shown in the SEQ ID NO1.

2, ahylysantinfarctase 36KD strand hemocoagulase according to claim 1, it is characterized in that this hemocoagulase iso-electric point is 6.59, HPLC measures and is simple spike, and color atlas calculates by area normalization method, purity is greater than 98%, and enzyme is 2000U/mg-3000U/mg albumen than living.

3, the preparation method of ahylysantinfarctase 36KD strand hemocoagulase is characterized in that may further comprise the steps:

(1) take by weighing after the agkistrodon acutus snake venom adds Tris-HCl (PH8.0) damping fluid dissolving, the small-molecule substance and the inorganic salt of the following molecular weight of 1KD are removed in dialysis, are convenient to next step purifying;

(2) Metal Sepharose F.F affinity column on the supernatant liquor is collected active peak, removes most of non-Thrombin-like enzyme albumen, lipid, carbohydrate and other material, obtains the thick component of 36KD hemocoagulase and fiber eliminating enzyme;

(3) penetrate DEAE-Sepharose F.F ion exchange column on the peak, collect active peak, 36KD hemocoagulase and fiber eliminating enzyme are separated;

(4) will have Superdex 75 posts and Sephacryl S-100 molecular sieve column on active the 5th peak of 36KD hemocoagulase, collect the 2nd peak, obtain the 3.6KD strand hemocoagulase of purifying;

(5) Sephadex G-25 post desalination, degerming, freeze-drying obtain purity greater than 98% 36KD strand hemocoagulase.

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