JPS62163690A - Production of human plasmin-alpha2-plasmin inhibitor complex and method for separating same - Google Patents

Abstract

PURPOSE:To form human plasmin-alpha2-plasmin inhibitor complex stably, by adding a specific drug to human blood or plasma and to separate human alpha2-plasmin inhibitor as human plasmin-alpha-2-plasmin inhibitor complex efficiently, by bringing the reaction mixture into contact with a column and subjecting to gel filtration. CONSTITUTION:Human blood or plasma is optionally blended with an inactivator of human-alpha2-macroglobulin and mixed with plasminogen activator to form human plasmin-alpha2-plasmin inhibitor complex. Then, the reaction mixture is brought into contact with a hydroxyapatite column and subjected to gel filtration so that human plasmin-alpha2-plasmin inhibitor complex is separated and collected.

Description

DETAILED DESCRIPTION OF THE INVENTION a. Field of Industrial Application The present invention relates to a method for producing and purifying human plasmin-α,-plasmin inhibitor complex. More specifically, the present invention relates to the production of a plasmin-human α-2 plasmin inhibitor complex by adding plasminogen 7 knapeter to human plasma or blood, and a method for selectively separating the complex.

b. Prior Art Human α,2-plasmin inhibitor was first purified in a single core by Aomizu and Terii, and! It is a powerful lasmin inhibitor that instantly inhibits the sterase activity of the fibrillary enzyme plasmid (plaamin), and is a molecule containing 11.7% sugar.
cjdo, which is known to be one of the glycoproteins of the present application.
roi &Aoki; The Journal
of Biolo-gical Chemistry
1251, 5956-5h6sH976)].

It is known that m1 human m-plasmin inhibitor has an active position of 56 in three plants.

The first is a site that prevents plasmin from dissolving N-cells (hereinafter this may be referred to as the "reactive side").
CB, Woman &Co11en;'rhe
Journal of Biological Chem
istryw 254, 9291-9297 (197
9)], and the second is the plasmin binding site CB on the terminal side of the carboxyl group, Wiman &D, Co11en.
;European Journal of Bi
ochemistry + 84 r573 578 (
1978)], and the third is L Y, which is the fifurin binding bb position at the end of the aeno group.

Salutm + et al, + Thrombo
! 1is Research v l 6 279-2
82 (1979) #Sho].

Human α22-plasmin inhibitor inhibits plasmin activity almost instantaneously, and -2-plasmin inhibitor binds to plasmin at a ratio of l=1 to form a complex.

(B, Woman & L), Co11an; T
he Journal of I$iological
Cbemiatry, 254.9291-9297 (1
979)+D, Co11en; Thrombogi
s and Haemoataaia * 43 m
77-89 (19803-Reference) 9. Intravascular donation 1. ! In conditions where plasminogen is activated, such as in thrombolytic therapy using J(DIC) or urokinase, active plasmin/α,-plasmin inhibitor reacts and forms a complex.

Recently, the quantification of plasmin-α1-plasmin inhibitor complex in plasma has been carried out using clot bath disassembly monitor and DIC.
It is considered to be effective in diagnosing (for example, N, A
, Booth & B, Bennett 2Brtt
iah Journal of Haematolog
y I 50 + 537-541 (1982))
.

Therefore, if the plasmin-α, -plasmin inhibitor complex h in the blood could be determined accurately and easily, it would be useful for preventing, diagnosing, and preventing diseases of woodcutter.

As conventionally known methods for measuring plasmin-α,-plasmin inhibitor post-association, the fA l g) method is a method using two-dimensional cross-immunization and chromophoresis, and the second method is a method using anti-blood Y#. This is based on the so-called sandwich method, in which the antibody is kept constant and the yeast-accumulative antibody method is applied.

It would be extremely advantageous for diagnostic testing and the preparation of kits therefor if a large number of standardized standards of 10,000 plasmin-α,2-plasmin inhibitor complexes could be easily obtained.

The previously known method for producing and separating human plasmin-α1-plasmin inhibitor complex involves adding plasmin to plasma from which plasminogen has been removed to generate a plasmin-α,-plasmin inhibitor complex. Lysine Sephas Ka, 50mM<
1) It is a method to elute with Z-7 Mi/Kabun Kai and finally obtain NWM product by Glu filtration method (B, Woman
&D,Co11en ;The Journal
of Biological Chemistry +
254 + 9291-9297 (1979) & Teru)
.

However, in this method, Glu-plasmin and Lys-plasmin also adsorbed to the lysine sepharose, and these
Eluted with plasmin-022-plasmin inhibitor complex by 1ljlK.

The amount of these proteins [the operation was one-shot, and the above]
:) Method 5 has the disadvantage that the plasmin added to plasma lacks aphasia stability during storage, and there is also a problem in administering an appropriate amount of plasmin to the feces. More importantly, the formation of human plasmid/-α,-macroguprin bale aggregates occurs simultaneously, making it difficult to separate and purify the complex.

Structure of the C0 Invention Therefore, the present inventors conducted research on the production of human plasmin-α, -plasmin inhibitor complex, its isolation, and f# production, and discovered that human plasminogen 7 cutipator was added to human plasma. As a result, a polyphagous plasmin-α,-plasmin inhibitor complex is formed, and the human plasmin-α,-plasmin inhibitor complex adsorbed on lysine sepharose (7 minocapron (increasing i once linearly) It was found that G/u-plasminogen and Lye-plasminogen were separated and released from the bath.

Furthermore, using the above-mentioned lysine sepharose, min.
human plasmin-α present in the human plasmin-AT2-plasmin inhibitor complex fraction,
- Macoog A7 phosphorus complex and plasminogen can be separated by high performance liquid chromatography using a hydroxyapatite column and gel filtration (by directly integrating the human plasmin-AT2-plasmin inhibitor complex) I also discovered Shiuroko.

Furthermore, in order to form a human plasmin-α,-plasmin inhibitor complex, an inactivating agent for human-α-omacroglobulin is added in advance (and a temporary human plasmin-α,-macroglobulin complex is formed). It has been found that the formation can be significantly inhibited and the yield of human plasmin-α,-plasmin inhibitor complex can be increased.

The present invention has been achieved based on the above findings, and the first invention is a human plasmin-α,-plasmin inhibitor complex characterized by adding plasminogen 7 activator to human blood or plasma. The invention of $2 is a method for producing human body, and the invention of $2 is to add human α, - to human blood or plasma.
The third invention is a method for producing a human plasmin-α,-plasmin/inhibitor complex, the characteristics of which include adding a macrop globulin inactivator and then detecting a plasminogen activator. The plasminogen 7 activator is added to human blood or plasma, and the reaction mixture obtained is then contacted with a hydroxy 7 patite column, and then applied to the gel twice. Human α.

2-Plasmin inhibitor human plasmin-α,-
This is a method for separating plasmin inhibitor as a complex, and the generation of stripe 4 is achieved by adding human macroglobulin inactivator 6 to human blood or plasma, then adding plasminogen 7 activator, and then adding the plasminogen 7 activator to human blood or plasma. The resulting reaction mixture was layered on a hydroxyapatite column and then passed through gel GJ to separate the layers as human pseudo-plasmin-alpha, 2-plasmin inhibitor added into it from human blood or plasma at %m. This is the way to do it.

The history of the method of the present invention will be explained in detail below.

Formation: K Sh 9tt in a human dish (pooled plasma) was heated with two sheets of paper and passed through a Sephas 4B column, pre-filled with excess buffer (0.15M Na
Plasminogen-depleted plasma is obtained by subsequent flow through a diteitalicin Sepharose column equilibrated with 50-Tris-kcl (pH 7,4) containing Cj. α in this plasma
, about 1/2 the number of moles of 2-plasmin inhibitor is newly added to plasminogen. While stirring at mold temperature, 912 U of urokinase per plasma was subcutaneously added to the plasma in several portions every 0 minutes.

#t#ale degree is 1mM DFP, IG after kk
Add plutease inhibitor to give V151777'Otinine + 5mM benzamidine and stop the reaction.

Through the above operation, plasminogen is converted to plasmin by urokinase, and this plasmin is temporarily
Forms post-association with plasmin inhibitor.

In the above procedure Vc, urokinase is used as the plasminogen 7 activator.

In addition, there are other 7 activators, such as tissue plasminogen 7 activator.
ganactivator) + vascular ring-derived plasminogen 7 activator (vascular p
Plasmin, which is a protein having a lysine binding site, is passed through a lysine Sephas to pass the plasma containing the plasmin-alpha-plasmin/inhibitor complex obtained by the above method to plasmin, which is a protein having a lysine-binding site. Plasminogen, human lasmin-2-plasmin inhibitor complex and human plasmin-α,-macroglobulin complex are transferred to lysine sepase. On the other hand, human plasmin inhibitor that does not form a complex is not adsorbed and can be separated from the above-mentioned protein. This lysine Sepharose is washed with a suitable washing solution (eg 1M Naα.

30 V/+77 pua 'f-near'k miyamu+5
Wash with 0 mM Tris-14CI pH 7,2). Next, from OmM of l-7 minocaproic acid to 2
Applying a direct gradient of 5 + ssM, Lysine Sepha 0-
The human plasmin/α,-plasmin inhibitor complex is eluted from the plasma membrane. Through this operation, plasmin and plasminogen present in plasma and plasmin/
It is possible to separate the α,-plasmin inhibitor complex. Then, the dispersed human plasmin-α and plasmin inhibitor complexes were concentrated,
Excess * buffer (e.g. 10mM phosphate buffer apH 6,8
) After dialysis against K, trace amounts of plasminogen are separated and removed using Tsumugi-soku liquid magnetography.

The column is a hydroxyapatite column with phosphoric acid 10
in a direct gradient from mM to 200 mM.

The complex fraction and plasminogen were separated. Subsequently, this complex fraction is filtered to separate the human plasmin-α,-plasmin inhibitor complex and the human plasmin-pseudo-macroguprin complex.

Instead of using the above-mentioned lysine sepase, any other insoluble carrier can be used as long as it has the lysine binding site Kfi of each protein. 100F from OmM? 1M range,
Preferably, 0+sM to 25mM is appropriate. Furthermore, the gradient of phosphoric acid from one column of hydroxyl 7-batite column under bath pressure is within the range of OmM to 500 FIIM (7), preferably 10 mM to 200 mM.

In the method of the present invention described above, human plasmin-α, -
8Q&C to form plasmin activator 41 combination
The purity and yield of the human plasmin-α,-plasmin inhibitor complex can be improved by adding an inactivating agent for human α-iomacroglobulin, such as plasma ft-edited methylamine.

Although the above α1 method was described using human plasma as a raw material, the method according to the present invention can be similarly applied to human blood.

A-According to the invention, a human plasmin-α,-plasmin inhibitor complex can be produced from human blood or human plasma, and the loaded complex can be selectively and easily separated. .

Examples of the method of the present invention/! -Explain in detail.

Add the purified plasminogen preparation of K 9.30119 to 250V of human plasma passed through lysine sepharose, gradually dissolve the urokinase while stirring at will, add human plasmin-α , - generated a plasmin inhibitor complex.

After adding 228.000 Units of p-kinase, the final quality was 1mM D F P + 1
0 Unit/j'j aprotinin* 5 mM benzamidine was added to stop one reaction. Next, 265μ of plasma containing the human plasmin-α,-plasmin inhibitor complex obtained by adding p-kinase was poured into a column of lysine Sepharose with a column volume of 50 μm.
M NnC1+ 10 Untt/"j 50 mM 'L'r+s -HCl containing 7pGl+Nia p)1
7.4 and baby wash pptz%.

The human plasmin-mid2-plasmin inhibitor complex was leached with a direct concentration gradient of t-7 minopron from mM to 25 mM concentrations. Through this operation, the complex with plasmi' + Giy-plasmi/-gun and Lye-plasminogen was separated and eluted, and the resulting complex fraction was subjected to A rapid body chromatography using a hydroxyapatite column. Ivy.

Proteins adsorbed on the hydroxyapatite column were washed out using a direct gradient from 10mM phosphoric acid solution pH 6.8 to 10mM phosphoric acid solution pH 6.8 containing ILIM NaCl. The extracted complex fraction was dialyzed, and after Isogumi, it was sent to Gerro Mausoleum (G 4000SW
Column: High performance liquid chromatography, made in late year W)
The human grasmin-pseudomacroglobulin complex was isolated and the human plasmin-α, -plasmin inhibitor complex was synthesized. The purity test was carried out using SDS polyacryl 7mide with a gel magnetism of 7.5 using air bed motion.

Human plasmin inhibitor and lysine sepharose were coated with human plasmin inhibitor by gel filtration and gel filtration. , hydroxyapatite, using gel filtration and finally human plasmin of ~1.82-
An α,-plasmin inhibitor conjugate was obtained.

In the elution of the human plasmin-α,-plasmin inhibitor complex from lysine Sepharose in Example 1 above, each fraction had an absorbance (280 nru) of
The relationship between the magnetic field of 8-7 minocabroic acid (t-ACA) and the magnetic field of The flow rate of 7 was 2 s, 7 M4/hr. In Figure 1, minute 11 &A is human plus ξ - α
, - Macp globulin plate assembly and human plasmin - ~-
This is a fraction containing a plasmin inhibitor.

Fraction B is a fraction containing human plasmin-α, -plasmin inhibitor complex, and Giu-plasminogen, and fraction C is a fraction containing Lys-plasminogen.

In addition, in Example 1 above, 5D
The results of S-polyacrylamide electrophoresis are shown in FIG.

In FIG. 2, sample number 1 is fraction A (lysine sepharose bath product), and sample number 2 is the fraction A obtained after contacting the fraction A with a hydroxyapatite column and then gelling.

Sample No. 3 is Min-B (Lysine Ceph 7ρ-S eluate), Sample No. 4 is after the above-mentioned Min 11 IIB was brought into contact with a hydroxyapatite column and then gelled, and Sample No. 45 is after Giu -Plasminogen/
and sample number 6 is a molecular ffi marker. Note that sample numbers 1 to 5 were not subjected to reduction treatment, and sample number 6 was subjected to redundancy treatment.

Implementation-” 2 Human normal face 111g1Lt and Bluff, Mino-mono removal R
Mix 20μ of serum and add 2M Tris-MCI p
H7,4 5N, monomethylamine hydrochloride 43.1 #
was added to give a final concentration of 1M to 3M. While stirring at 4°C, urokinase was added to the mixture to gradually activate plasminogen. When the plasminogen activity was 5X or less than the plasminogen activity in the starting reaction mixture, protease inhibitors were added to the reaction mixture so that the final concentration was 1 mM DFP + 10U/11j aprotinin and 10mM benzamidine. has been stopped. i! The amount of urokinase was 864 Units per 1 aL of plasma, which is equivalent to 20 hours.

20mM of plasma containing this human plasmin-2-plasmin inhibitor complex! J's loose liquid pi-
17.4 (0,15MNa(J, 10 U/31
After thorough dialysis against 401 (containing 47 brotinin and 1mM benzamidine), the lysine column was passed through a lysine column (volume 3-011t) to adsorb proteins that act as lysine binding sites. Squirrel mild solution pH 7,4 (0,15M NaCj?, 10
U/-7 flotinin, 1mM benzamidine)
15011t + 20mM) squirrel
Slow m g p)1 7.4 (I M NnC
l.

10U/Aprotinin Il? PIM benzamidium/l
O, 05316 (including Twaen 20) 50011
4 + 20mM)! J Sti*1eL
pH7.4 (0.15M NaC/, l OU/
Nail Ab1 (containing notinine) 25011t was used.

Next, lysine cephalysine G + -s or C) was added with a gradient of 25 mM to 25 mM
The protein was extracted. The eluted fraction was dialyzed, and 200mM from Rinka 10 Wuwl was diluted with 200mM using a simple column cartridge (7.6mφ x 100Ryu; manufactured by Mitsui Raiatsu). The complex was washed out with a gradient. Next, the human plasmin-α, -Mcμgl=furin complex was separated using high-performance liquid chromatography using a transparent gel filtration column (G-4000SW column; manufactured by Yoso Tei Co., Ltd.). , 240 μl of human plasmin-α, 2-plasmin inhibitor a combination manufactured by 'hi was obtained.

Figure 2 shows the results of a polyacrylamide cage test for the human plasmin-2-plasmin inhibitor complex σJ k d, which was isolated and destroyed by the inhibitor complex glue Gin Sepharose.

Claims (1)

[Claims] 1. Human plasmin-α_ characterized by adding a plasminogen activator to human blood or plasma
2- Method for producing plasmin inhibitor conjugate. 2. A method for producing a human plasmin-α_1-plasmin inhibitor complex, which comprises adding a human α_2-macroglobulin inactivator to human blood or plasma, and then adding a plasminogen activator. 3. Contained therein from human blood or plasma, characterized in that a plasminogen activator is added to human blood or plasma, and the resulting reaction mixture is then contacted with a hydroxyapatite column and then gel-filtered. A method for separating human α_2-plasmin inhibitor as a human plasmin-α_2-plasmin inhibitor complex. 4. Adding a human α_2-macroglobulin inactivator to human blood or plasma, then adding a plasminogen activator, and then contacting the resulting reaction mixture with a hydroxyapatite column, followed by gel filtration. A method for separating human α_2-plasmin inhibitor contained therein as a human plasmin-α_2-plasmin inhibitor complex from human blood or plasma, characterized in that: