JPS5810522A - Purification of antithrombin 3 by deactivated thrombin gel - Google Patents

Abstract

PURPOSE:To separate and to purify antithrombin III efficiently, by adsorbing specifically antithrombin III on deactivated thrombin gel from a mixed solution of plasma and protein in affinity chromatography method, followed by eluting it. CONSTITUTION:In affinity chromatography, and elute having a pH of 6-8 and an ionic strength of 0-1, comprising a buffer solution, solution of neutral salt and a mixed solution of plasma and protein is brought into contact with the gel, and antithrombin III is specifically adsorbed on it. A gel obtained by linking chemically modified deactivated thrombin (e.g., antihydrothrombin) as an affinity ligand to a hydrophilic carrier is used as the gel. Antithrombin III is eluted with a solution of neutral salt in a pH of 4.0-5.5 or a substrate analog such as benzamidine, etc., so that it is separated and purified. Antithrombin III is one kind of glycoproteins paticipating in blood clotting.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to affinity chromatography,
Using a hydrophilic gel bound with inactivated thrombin as a ligand, an eluent consisting of a buffer solution, a neutral salt solution, and a plasma protein mixture can be passed through a column, or both can be mixed in a container using a batch method. The present invention relates to a method for separating and purifying antithrombin (2) by mixing the two and bringing them into contact with a gel to specifically adsorb antithrombin (2).

In more detail, we describe a method for separating and purifying antithrombin■ by utilizing its affinity with thrombin, using Afistiegel, which is based on inactivated thrombin in which the reaction center serine residue of thrombin has been converted to a dehydroalanine residue. This is related.

Blood consists of a formed component consisting of red blood cells, white blood cells, and platelets, and a non-formed component called plasma, and plasma contains more than 100 types of proteins. These proteins are electrophoretically classified into albumin, alpha, beta and r-globulins and fibrinogen.

Antitropin ■ is a glycoprotein belonging to α2-globulin. It is normally contained in plasma at a rate of only 30 to 50 mg/ml. However, by reacting with thrombin, activated factor X, etc. and forming a complex, it inhibits the blood coagulation reaction and plays a major role in regulating the coagulation reaction within blood vessels.

Conventional methods for separating and purifying antithrombin (IJ) include fractional precipitation by adding neutral salts such as ammonium sulfate and water-soluble polymers such as polyethylene glycol (2), ion-exchange chromatography using the charge of molecules (3) ) Molecular sieve chromatography method that utilizes the size of molecules (4) Electrophoresis method that utilizes the difference in isoelectric points of molecules (5)
Affinity chromatography methods that utilize affinity with heparin are known, and one or a combination of these methods is used.

Among these methods, with the exception of the affinity chromatography method (5), it is difficult to separate and remove contaminant proteins, and the operation must be repeated many times, resulting in a small recovery amount and complicated operations. There are drawbacks such as:

On the other hand, the affinity chromatography method, which separates by specific affinity with the ligand, utilizes the specific properties of antithrombin■, so the purification procedure is simple, contaminants can be easily separated, and the activity recovery rate is also good. have advantages.

By the way, the conventionally used heparin-based affinity chromatography method utilizes the binding of antithrombin to heparin.
It is simple and efficient compared to other methods. however,
Using this method, antithrombin
When purified, the yield is extremely low. Furthermore, even when purified from heat-treated plasma, the yield is low. In other words, this method relies on the structure of antithrombin ■ near the lysine residue, which is the heparin-binding site, and is therefore inefficient because the structure of this region is unstable against low-temperature ethanol treatment and heat treatment. Presumed. therefore,
As the structure used for affinity chromatography, it is desirable to use a structure near the arginine reaction center residue, which is another structural site in antithrombin (2).

The present inventors focused on the above-mentioned points, and as a result of intensive research to improve these shortcomings, we developed an affinity chromatography method using the arginine reaction center of antithrombin. The present invention was completed by discovering a method that can efficiently fractionate antithrombin (2) contained in a plasma protein mixture.

That is, the present invention uses thrombin, which does not react with inactivated thrombin, that is, antithrombin (■) to form a covalent complex, but has an affinity for it, instead of the ligand conventionally used in affinity chromatography, and uses a buffered thrombin. The purpose of the present invention is to provide a method for quickly, simply and efficiently separating and purifying antithrombin (2) by contacting a mixed solution consisting of a neutral salt solution, a neutral salt solution, and a plasma protein to specifically adsorb antithrombin (2) and eluting it.

The present invention will be explained in detail below.

Inactivated thrombin used in the present invention is obtained by reacting purified human thrombin with phenylmethanesulfonyl fluoride in a Tris-HCl buffer with a pH of 8.0 and an ionic strength of 0.0-5.
It formed an ester bond with the active center serine residue, causing a loss of thrombin activity. Thereafter, the pH was adjusted to 9.0 and kept at a low temperature to dissociate ester bonds and convert serine residues to dehydroalanine residues. Next, affinity chromatography was performed using agarose gel based on benzamidine, and a fraction showing affinity was collected and used as anhydrothrombin.

The purified thrombin used in the present invention can be obtained using a low-temperature ethanol method (E, J. Cohn: Journal of Am.
erican Chemical 5ociety, 6
8:459 (1946)), polyethylene glycol precipitate and Ba
The prothrombin complex was purified by Cl2 precipitation, activated with thrombin and lastin extracted from bovine brain, and then purified by DEAE Seph7dex column chromatography and benzamidine Sepharose column chromatography, and thrombin was used.

Antithrombin (2) is then specifically adsorbed by contacting a plasma protein mixture with a gel in which the ligand is immobilized on agarose, and is separated and purified by elution.

The carrier used to bind the ligand in the present invention can be arbitrarily selected from commonly used affinity carriers. Cross-linked agarose gel (trade name: Sepharose 4B and 6B) Cross-linked dextran gel (trade name:
Sephadex G100, G150, G200) Cross-linked acrylamide gel (Product name: Sephacryl 5200°5
300), etc.

In the present invention, for adsorption of plasma protein mixture, p
An eluent consisting of a buffer solution with H6 to 8 and an ionic strength of 0 to 1.0 and a neutral salt solution is used.

The buffer in the eluent can be any buffer having a pH in the range of 6 to 8, but Tris buffer and phosphate buffer are particularly preferred, and the neutral salt solution can be arbitrarily selected from commonly used neutral salt solutions. Although it can be selected, an aqueous solution of NaCl and KCI is particularly preferred.

In the present invention, the eluate for the adsorbed antithrombin (1) fraction uses a buffer solution with a pH of 5.0 to 5.5 and an ionic strength of 1 to 4, or a substrate analog with a pH of 4.0 to 5.5.

The buffer solution can be arbitrarily selected from a buffer solution exhibiting a pH range of pH 4.0 to 5.5, but acetic acid buffer is particularly good, and the neutral salt solution can be arbitrarily selected from commonly used neutral salt solutions. However, an aqueous solution of NaCl is preferred.

As a substrate analog, be/zuamidine! Mention may be made of nitrobenzamidine and laaminobenzamidine.

0. I N NaOH is used to adjust the pH to 4.0 to 5.5.

Next, the state of plasma proteins used in the present invention will be explained.

Fibrinogen is one of the plasma proteins. When blood leaves the body, it clots, and this is caused by fibrinogen forming a blood clot with fibrin. Therefore, fibrin is also deposited in chromatography and adheres to the carrier. In order to prevent this phenomenon, it is preferable to perform pretreatment to remove fibrin.

Plasma can be heated at 60° C. for several minutes to remove fibrinogen before use. Plasma can be used by removing fibrinogen with ethanol at low temperature, performing precipitation and concentration treatment, and then dissolving it in an elution buffer.

It can be used as a pretreatment to remove fibrinogen by coexisting a water-soluble polymer with plasma. Alternatively, it can be used by allowing a neutral salt to coexist, removing fibrinogen in advance, and diluting it with water so that the salt strength becomes 1.0 or less.

Therefore, the solution containing plasma proteins in the present invention is an aqueous solution, a water-soluble polymer, a neutral salt, or an aqueous solution containing alcohols.

Examples of water-soluble polymers include polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, polyacrylic acid, hydroxyethyl cellulose, and hydroxyethyl starch.

In addition, as neutral salts, NaCl, KCI, NH, C
1, Na25O<, K2S0. , (NH4)2S
Oi can be mentioned.

Examples of alcohols include alcohols having 2 to 5 carbon atoms.

As described above, affinity chromatography is performed by passing or mixing an eluent and a solution containing plasma proteins, adsorbing the solution onto an inactivated thrombin gel, and collecting the eluate as an eluate.

When a column method is used in the present invention, the inactivated thrombin gel is used by being packed into various columns of any size suitable for the throughput, including those packed into glass columns.

It is preferable to equilibrate the column by passing an eluent through it in advance.

The flow rate of the eluent can be varied, but since the present invention is based on affinity chromatography, the flow rate is preferably 0.5 ml/am2 or less. Further increasing the flow rate may be achieved by adding 1 to
This is possible by coexisting 5 units/ml of heparin.

The flow rate of the eluate can be varied within the range of the mechanical strength of the carrier used.

In addition, if an aqueous solution containing a substrate analogue is used as the eluate, after separation and purification by Kempo, p
It is preferable to remove the substance by dialysis or molecular sieve chromatography in a buffer with H7.0 and ionic strength of 1.0 or more.

Even when a batch method is used in the present invention, the amounts of eluent and gel containing plasma proteins can be varied depending on the amount to be processed. The time for mixing the gel and solution varies depending on the degree of stirring and the shape of the container, but it is generally desirable to mix the mixture for 2 hours or more. The gel that has adsorbed antithrombin (2) in this operation is washed with an eluent on a glass filter, and then washed with the eluent in accordance with the column method described above to recover antithrombin (4).

The above operations are desirably performed at low temperatures in order to stably preserve the activity of antithrombin (■).

As explained above, the present invention uses an inactivated thrombin gel and brings it into contact with a mixture of various plasma proteins to specifically adsorb antithrombin and elute it, thereby allowing rapid, simple, and efficient separation. It can be purified.

Hereinafter, the present invention will be explained in detail with reference to practical and comparative examples.

Example 1 Anhydrothrombin was prepared from human thrombin by the following method. Purified human α-thrombin (716
000 units, 300 mg) was dissolved in 50 mM Tris buffer (pH 8,0), and 100 times the amount of nophenylmethanesulfonyl fluorite was added at 21° C. and left for 30 minutes to eliminate thrombin activity. - When the esterase activity of thrombin was measured, 99.95% was lost. Then add 50mM Tris buffer (pH 9,0
Desalting column (trade name = Sephadex G) equilibrated with
-25) and was left as it was at 4°C for 1 day and night. This fraction was added to 50 mM Tris buffer (pH 7,5).
The solution was passed through a benzamidine agarone column (gel bed volume: 30 ml) equilibrated with the same buffer and washed with the same buffer. 5. m
When the absorbance at 280 rtm and the protein amount were measured by the Folin method, it was found that most of the inactivated thrombin was adsorbed to the column and had substrate binding ability, as shown in FIG. This is shown in Figure 1 and was eluted with 0.2M benzamidine (pH 7.5).

50mM Tris buffer (pH 7) containing 1M NaCl
, 5) to remove benzamidine and obtain anhydrothrombin.

Next, anhydrothrombin was added to antithrombin m in advance, incubated at 37°C, mixed with thrombin, added to fibrinogen solution, and the clotting time was measured to determine the inhibition rate of its activity. did. Antithrombin activity was decreased by heating with anhydrothrombin, indicating that antithrombin (■) and anhydrothrombin interact. Furthermore, since the activity did not increase even when anhydrothrombin was added to thrombin, it was confirmed that anhydrothrombin does not exhibit any activity.

The anhydrothrombin was dissolved in 0.1M NaHCO-(p
Anhydrothrombin sepharose scale was synthesized by dialyzing against H1s3) and mixing with activated cross-linked agarose gel (trade name - activated CH Sepharose 4B).

Example 2 Plasma was heated to 60°C for 3 minutes, centrifuged at low temperature, the supernatant was collected and filtered using a filter with a pore size of 0.2μ, and 4 ml of it was added to an inactivated thrombin Sepharose column (gel bed volume). 4 ml) K solution, 5
0mM Tris buffer (pH 7,5) followed by OIMN
After washing with the same buffer containing aCl,
Adsorbed proteins were eluted with 0 mM acetate buffer (pH 5,5). The flow rate was 1 ml/min. As a result, the eluate was measured by ultraviolet absorbance at a wavelength of 2F and 30 nm.As shown in Figure 2, in addition to the peaks of contaminant proteins,
There was a fraction eluted at 5.5. The antithrombin ■ activity of this fraction was measured, and it was determined that antithrombin ■ was contained, and as shown in Figure 3, the electrophoresis image revealed that the antithrombin ■ fraction was highly pure, containing only a very small amount of contaminant protein. It was confirmed that As shown in Table ■, the activity recovery rate of antithrombin ■ from the added heated membrane fibrin plasma to the antithrombin ■ fraction was 90%.
%, the activity value based on the amount of protein measured by the Folin method was 175 times higher than that of heated membrane fibrin disintegration. Trace amounts of contaminant proteins observed in electrophoretic images can be removed using anion exchange resin column chromatography (trade name: DEAE Sephadex A-50).
It was possible to obtain even more highly purified antithrombin (■) by removing the residue. (See Table 1) Table 1 Purification Example 3 of antithrombin ■ in Example 2 Same procedure as Example 2 except that the eluent used in Example 2 was changed to 0.2 M benzamidine (pH 5,5).・Measurements were made. However, the antithrombin ■ fraction is prepared using 50 mM Tris buffer (pH
7,5). As a result, it was confirmed that the purity was almost the same as in Example 2, and the activity of antithrombin (III) was recovered to the same extent.

Example 4 A method for fractionating human plasma proteins using a low-temperature ethanol method was developed by Cohn et al. (E, J. Cohn: Journal of Am
eri-can Chemical 5ociety
, 68:459 (1946)) is still widely used industrially.

The IV-1 paste (1 g wet weight) fractionated in this manner was dissolved in 4 ml of 50 mM Tris buffer (pH 7,5) and filtered using a filter with a pore size of 0.2 μ. Antithrombin ■ was separated and purified in the same manner as above. As a result of absorbance measurement at a wavelength of 230 nm, it was confirmed that the sequentially eluted proteins were divided into peaks similar to those in Example 1. pH 5.5 as in Example 2
When the peak eluted with the eluate with ionic strength 4 was collected, as shown in Table 2, the activity recovery rate of antithrombin ① was 80%, confirming that it was efficiently recovered. In addition, antithrombin per protein■
The activity ratio also increased 177 times compared to before addition to the column. (See Table 2) Table 2 Purification Example 5 of Antithrombin ■ in Example 4 Trisaminomethane was added to plasma to a final concentration of 5 omM, and 0. Adjust the pH to 7.5 with IN hydrochloric acid and cool to 5.
Process a 0% polyethylene glycol 4000 solution, and the final concentration of polyethylene glycol is 15. The supernatant was collected by centrifugation at low temperature, filtered using a filter, and 4 ml was added to an inactivated thrombin gel column (gel bed volume: 4 ml). : It was found that antithrombin m of the same purity can be recovered with the same efficiency. The recovery rate was 85g6 specific activity increased 165 times before and after chromatography.

Example 6 The same operations and measurements as in Example 2 were carried out except that the eluent used in Example 2 was changed to 50 mM IJ buffer (pH 8,0). The resulting antithrombin (1) had approximately the same purity as in the example, but the recovery rate of activity was 60.

Example 7 The eluent used in Example 2 was changed to 5 containing IMNaCI.
The same operations and measurements as in Example 2 were performed except that 0 mM U acid buffer (pH 6,0) was used. As a result, antithrombin ① had approximately the same purity as in the example, but the recovery rate of activity was slightly lower at 50%.

Example 8 Add (NH,)2S to plasma at low temperature to a final concentration of 20
Add O4 powder, leave for 30 minutes, centrifuge, remove the supernatant, and add 50 mM Tris buffer (pH 7.5).
Diluted 1:00. Then, filter 4 using a filter.
00ml with antithrombin of the same purity as in Example 2.
could be purified with a recovery rate of about 80q6.

Example 9 Cyanantithrombin m was purified using a batch method.

In the same manner as in Example 2, 100 ml of plasma from which fibrinogen had been removed by heating was obtained. 50 ml of anhydrothrombin gel was added thereto and stirred at 4°C for 3 hours.

The gel was collected with suction on a glass filter, washed with 250 ml of 50 mM Tris buffer (pH 7,5) containing 0.1 M NaCl, and then filtered into 100 ml of 50 m
The antithrombin ■ fraction was eluted with M acetate buffer (pH 5.5).

When the protein amount and antithrombin activity were measured, it was confirmed that the recovery rate was approximately 80%, that the specific activity increased 150 times before and after chromatography, and that the purity was close to that of Example 2. Ta.

Furthermore, when DEAE Sephadex column chromatography was performed, a highly pure standard sample was obtained.

Example 10 Polyethylene glycol was added to plasma in the same manner as in Example 5, fibrinogen was removed, and then 150 m
Antithrombin (2) was separated and purified using the same procedure as in Example 9 using the batch method.

Both the recovery rate and purity were comparable to those of Example 7.

Comparative Example 1 Agarose gel with heparin as a ligand (trade name: Heparin Sepharose (CL6 B)) A fully packed column (gel bed volume: 20 ml) was filled with 20 ml of Cohn IV-1 Paste (wet weight 5 g) at 50 mM). Hygiene liquid (p
'H7,5) and added after filtration. Same buffer 100
After washing the column with m1 0-2. Adsorbed proteins were eluted with the same buffer with a linear gradient of OMNaCl. The flow rate was 0.3 ml/min. The eluate was 5
Each ml of the sample was taken and the absorbance was measured at 230 nm and the antithrombin activity was measured.As shown in FIG. 4, the two eluted contaminant protein peaks each showed antithrombin activity. Therefore, antithrombin (2) is divided into three peaks, but due to its purity, only the third peak is collected as the antithrombin (m) fraction. As shown in Table 3, the antithrombin activity per protein in this fraction was 150% compared to that before addition.
It was found that the purity of the sample was high. However, the recovery rate of antithrombin ■ activity is as low as 30%, and the efficiency is poor.

(See Table 3) Table 3 Purification of antithrombin ■ in Comparative Example 1
The figure shows the benzamidine sepharose affinity chloratogen for anhydrothrombin.

(2) Anhydrothrombin Figures 2 and 4 are chromatograms of a plasma protein mixture separated by anhydrothrombin Sepharose affinity chromatography.

2. Antithrombin (■) Figure 3 (c) is a polyacrylamide disc electrophoresis image of antithrombin (■) purified by anhydrodrompin Sepharose affinity chromatography.

Claims (9)

[Claims] (1) Using the affinity chromatography method, antithrombin A method for separating and purifying antithrombin (2), which is characterized by specifically adsorbing (1) and eluting it with a neutral salt solution or a substrate analogue of pH 4.0 to 5.5. However, the term "substrate analogue" as used herein refers to a competitive inhibitor of thrombin. (2) The gel used for affinity chromatography is a gel that binds chemically modified inactivated thrombin as an affinity ligand to a hydrophilic carrier, and does not itself exhibit thrombin activity, but only has substrate binding ability. The method described in scope item (1). (3) The method according to claim (1) or (27), wherein the modified inactivated thrombin as the affinity ligand is anhydrothrombin. (4) Claims 11 to 3, wherein the buffer is Tris buffer, phosphate buffer, or acetate buffer.
The method described in Section 1. (5) The neutral salt solution contains NaCl, KCI, NH
4Cl, (NH4)2S04, the method according to claims (1) to (3). ~ (6) Claim No. 1 in which the substrate analog is benzamidine, aminobenzamidine, or nitrobenzamidine (
1j, the method described in Section 31. (7) The method according to any one of claims (1+) to (6), wherein the solution containing plasma proteins is an aqueous solution. (8) The method according to any one of claims (1) to (6), wherein the solution containing plasma proteins is a solution containing a neutral salt. (9) The method according to any one of claims (1) to (6), wherein the solution containing plasma proteins is an aqueous solution containing a water-soluble polymer such as polyethylene glycol.