JPS597694B2 - Purification method of antithrombin 3 using inactivated thrombin gel - Google Patents

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 will use an affinity gel based on inactivated thrombin in which the reaction center serine residue of thrombin has been converted to a dehydroalanine residue, and use the affinity of antithrombin with thrombin to separate and purify it. 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 associated with albumin, α, and β.
and γ-globulin and fibrinogen.

Antithrombin ■ is a glycoprotein belonging to α2-globulins.

It is normally only contained in plasma at a rate of 30 to 50 mg/dl.

However, by reacting with thrombin, activated factor X, etc. and forming a complex, it inhibits the plasma coagulation reaction and plays a major role in regulating the coagulation reaction within blood vessels.

Conventional methods for separating and purifying antithrombin■ include (1) fractional precipitation by adding neutral salts such as ammonium sulfate and water-soluble polymers such as polyethylene glycol, and (2) ion-exchange chromatography using the charge of molecules. (3) Molecular sieve chromatography, which utilizes the size of molecules; (4) Electrophoresis, which utilizes the difference in isoelectric points of molecules; and (5) Affinity stomatography, which utilizes affinity with heparin. One or a combination of these methods is used.

Among these methods, except for the affinity chromatography method (5), other combinations are difficult to separate and remove extraneous proteins and require repeated operations, resulting in a small recovery amount. The disadvantage is that it is complicated.

On the other hand, the affinity chromatography method, which separates antithrombin based on its specific affinity with the ligand, uses the specific properties of antithrombin■, so the purification process is simple, impurities can be easily separated, and the activity can be recovered. The rate also has good advantages.

By the way, the conventionally used heparin-based affinitake stomatography method takes advantage of the fact that antithrombin ■ binds to heparin.
It is simple and efficient compared to other methods.

However, when antithrombin (2) is purified from the sub-fraction by the cold ethanol method, which is currently widely used industrially, 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 a structure to be used in affinity stomatography, it is desirable to use a structure near the arginine reaction center residue, which is another structural site in antithrombin (2).

The present inventors have focused on the above-mentioned points, and as a result of intensive research to improve these shortcomings, the present inventors have determined that plasma and cold ethanol fractions can be collected using affinity chromatography 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 (2) to form a covalent complex, but has an affinity for it, instead of Gund, which is conventionally used in affinity stomatography. The present invention provides a method for quickly, simply, and efficiently separating and purifying antithrombin by contacting a mixed solution consisting of a buffer solution, a neutral salt solution, and plasma proteins to specifically adsorb antithrombin and eluting it. .

The present invention will be explained in detail below.

The inactivated thrombin used in the present invention is purified human thrombin with Tris-HCl MUU of pH 8.0 and ionic strength of 0.05. Then, it was reacted with phenylmethanesulfonyl fluoride to form an ester bond with the active center serine residue, resulting in the 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 stomatography was performed using agarose gel based on benzamidine, and a fraction showing affinity was collected and used as anhydrothrompin.

In addition, the purified thrombin in the present invention can be obtained by the low-temperature ethanol method (E, J. Cohh: Journal
of American Chemical Society
ty, 68:459 (1946)), the prothrombin complex was purified by polyethylene glycol precipitation and BaC2 precipitation, and activated with thromboplastin extracted from bovine brain. Thrombin purified by DEAE Sephadex column chromatography and benzamine Sepharose column chromatography 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 gels (trade names: Sepharose 4B and 6B), cross-linked dextran gels (trade names: Sephadex G100, G150, G200), cross-linked acrylamide gels (trade names: Sephacryl S200, S300), and the like.

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 IO and a neutral salt solution is used.

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

Substrate analogs may include penzamidine, nitobenzamidine, aminobenzamidine.

0. INNaOH is used at a pH of 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.

Blood clots when it leaves the body, and this is because fibrinogen turns into fibrin and forms blood clots.

Therefore, fibrin is precipitated and attached to the carrier during chromatography.

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.

Fibrinogen is removed from the plasma using ethanol at a low temperature, and the plasma is subjected to precipitation and concentration treatment, and then dissolved in an elution buffer and stored at ℃.

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, and after removing fipurinogen in advance, it is diluted with water so that the salt strength is 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, polyvinylpyrrolitone, polyacrylic acid, hydroxyethyl cellulose, and hydroxyethyl starch.

In addition, as neutral salts, NaCl, KCI, NH4Cl
, Na2SO4, K2SO4, (NH4)2S04.

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

As described above, a solution containing an eluate and plasma proteins is passed through or mixed, adsorbed onto an inactivated thrombin gel, and the eluate is collected to perform affinity chromatography.

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 in various ways, but since the present invention is based on affinity chromatography, the flow rate is set to 0. More preferably, it is 5 ml/cr;l or less.

Further increasing the flow rate may be achieved by adding 1 to 5 units/1 in the eluent.
This is possible by coexisting rll heparin.

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

In addition, when an aqueous solution containing a substrate analogue is used as the eluate, after separation and purification using this method, p
It is preferable to remove the substance by performing dialysis or molecular sieve chromatography in a buffer solution with a pH of 7.0 and an ionic strength of 1.0 or higher.

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 ■ in this operation is washed with an eluent on a glass filter, etc.
Antithrombin ■ is recovered by washing with an eluate in accordance with the column method described above.

The above operations are preferably performed at low temperatures in order to stably preserve the activity of antithrombin ①.

As explained above, the present invention uses an inactivated thrombin gel and brings it into contact with various plasma protein mixtures to specifically adsorb antithrombin and elute it, thereby quickly, easily, and efficiently. 《It can be separated and 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 (710,000 units, 30
0 drops) was dissolved in 50rrLM Tris buffer (pH 8.0), and 1000 times the amount of phenylmethanesulfonyl fluoride was added at 21°C and left for 30 minutes to eliminate thrombin activity.

Thrombin esterase activity was measured and found to be 99.

95% was lost. Then add 50mM}lith buffer (p
Desalting was carried out using a desalting column (trade name: Sephadex G-25) equilibrated with H9.0) and allowed to stand overnight at 4°C.
The solution was passed through a penzamidine agarose column (gel bed volume: 30 ml) equilibrated with pH 7.5), washed with the same buffer solution, aliquoted into 15 rIll portions, and analyzed at 280 nm.
As shown in FIG. 1, most of the inactivated thrombin was adsorbed to this column, indicating that it had substrate binding ability.

As shown in Figure 1, this was eluted with 0.2M penzamidi 7 (pH 7.5), dialyzed against 50mM Tris buffer (pH 7.5) containing IMNaCl, and
Penzamidine was removed to give anhydrothrompine.

Next, anhydrothrombin was added to antithrombin (III) in advance, incubated at 37°C, mixed with thrombin, added to fibrinogen solution, and clotting time was measured to determine the inhibition rate of the activity and antithrombin activity was measured.

Antithrombin activity was decreased by heating together 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 anhydrodrombin was dissolved in 0.1M NaHC03 (p
H 8.3 ) and activated cross-linked agarose gel (
Anhydrothrombin Sepharose gel was synthesized by mixing with activated CH Sepharose 4B (trade name).

Example 2 Plasma was heated to 60°C for 3 minutes, centrifuged at a low temperature, the supernatant was collected and filtered using a filter with a pore size of 0.2 μ, and 4771 liters of the plasma was inactivated using an inactivated thrombin Sepharose column (Kell bed). Volume: 4,771 liters)
After washing with 50TrLM Tris buffer (pH 7.5) and the same buffer containing 0.1M NaCl, MNa
50 mM acetate buffer (pH 5.5) containing C1
) to elute the adsorbed proteins.

The flow rate was 1rnl/min.

As a result, the eluate was measured by ultraviolet absorbance at a wavelength of 280 nrn, and as shown in FIG. 2, there was a fraction eluted at pH 5.5 in addition to a peak consisting of unrefined proteins.

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 showed that antithrombin ■ was highly pure, containing only a very small amount of impurity protein. It was confirmed that it was a fraction.

As shown in Table 1, the activity recovery rate of antithrombin ■ from the added heat-defibrinated plasma to the antithrombin ■ fraction was 90%, and the activity value per protein amount measured by the Folin method was 90%. It was increased 175 times compared to fipurin plasma.

A trace amount of extraneous protein observed in the electrophoretic image was removed by anion exchange resin column chromatography (trade name: DEAE Sephadex A-50), and even highly purified antithrombin (2) could be obtained.

(See Table 1) Example 3 The eluent used in Example 2 was changed to 0.2M penzamidine (pH 5.5), and the same operations and measurements as in Example 2 were performed except for fDL.

However, the antithrombin ■ fraction was dialyzed against 50rrLM Tris buffer (pH 7.5) containing IMNaCl.

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 systematic fractionation method for human plasma protein fractionation using the low-temperature ethanol method was developed by Cohn et al. (E, J. Cohn: Journal
of American Chemical Society
ty, 68:459 (1946)) and is still widely used industrially.

[4 m of IV-1 paste (wet weight 1 g) fractionated using this method]
The product was dissolved in 50771M Tris buffer (pH 7.5), filtered using a filter having a pore size of 0.2μ, and antithrombin ■ was separated and purified in the same manner as in Example 2.

As a result of absorbance measurement at a wavelength of 280 nm, it was confirmed that the sequentially eluted proteins were divided into peaks similar to those in Example 1.

As in Example 2, the peak eluted with the eluate of pH 5.5 and ionic strength 4 was collected, and as shown in Table 2,
The activity recovery rate of antithrombin (III) was 80%, confirming that it was efficiently recovered.

In addition, the ratio of antithrombin ■ activity per protein increased 177 times compared to before addition to the column.

(See Table 2) Example 5 Trisaminomethane was added to plasma to a final concentration of 50 mM. Adjust the pH to 7.5 with IN hydrochloric acid, add 50% polyethylene glycol 4000 solution while cooling,
The final concentration of polyethylene glycol is 15%.

The supernatant was collected by centrifugation at a low temperature, filtered using a filter, and 4TfLl was added to an inactivated thrombin gel column (gel bed volume: 4 ml). It was found that it can be recovered with an efficiency of

The recovery rate is 85% and the specific activity is 1 before and after chromatography.
It has increased 65 times.

Example 6 The same operations and measurements as in Example 2 were performed except that the eluent used in Example 2 was changed to 50TrLM Tris buffer (pH 8.0).

The resulting antithrombin (2) 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 IMNaCl-containing 5
The same operations and measurements as in Example 2 were performed except that the 0 mM IJ acid buffer (pH 6.0) was used.

As a result, the purity of antithrombin (■) was almost the same as in the example, but the recovery rate of activity was 50%, which was lower than that of cattle.

Example 8 (NH4)2SO4 powder was added to plasma at low temperature to a final concentration of 20%, left for 30 minutes, centrifuged, the supernatant was taken, and 50mM To'JS buffer (pH 7.5) was added. ) and diluted 100 times.

Then, use a filter to filter 400ml of Example 2.
It was possible to purify antithrombin ■ with a purity similar to that of the antithrombin ■ with a recovery rate of about 80%.

Example 9 Antithrombin ■ was purified by a batch method.

In the same manner as in Example 2, 100 Tnl of plasma from which fibrinogen had been removed by heating was obtained.

Add 50ml of anhydrothrombin gel to it and
The mixture was stirred at ℃ for 3 hours.

Collect the gel with suction on a glass filter, and add 250 ml of 0. 50mM containing 1M NaC 1
}After washing with Squirrel buffer (pH 7.5), 100r
The antithrombin ■ fraction was eluted with 50mM acetate buffer (pH 5.5) of /Ll.

When 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. .

Furthermore, when DEAE Sephadex column stomatography was performed, a highly pure specimen was obtained.

Example 10 Polyethylene glycol was added to plasma in the same manner as in Example 5, fibrinogen was removed, and 150 ml of the same was added.
Antithrombin Ⅰ was separated and purified by the batch method in the same manner as in Example 9.

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

Comparative Example 1 Cohn IV-1 Paste (wet weight: 5 ml) was placed in a column (gel bed volume: 20 ml) packed with agarose gel (trade name: 1 Heparin Sepharose (CL6B)) with heparin as a ligand.
) in 20ml of 50rrLM Tris buffer (pH 7.
5) and added after filtration.

After washing the column with 1001 rLl of the same buffer,
The adsorbed protein was eluted with the same buffer with a linear concentration gradient of NaCl.

The flow rate is 0. The flow rate was 3 ml/min.

The eluate was aliquoted into 5 ml portions, and the absorbance at 280 nm and antithrombin activity were measured.

As a result, as shown in FIG. 4, two eluted protein peaks each showed antithrombin activity.

Therefore, antithrombin ■ is divided into three peaks, but only the third peak is collected as the antithrombin ■ fraction due to its purity.

As shown in Table 3, the antithrombin ■ activity per protein in this fraction increased 150 times compared to before addition, indicating that it was a highly pure sample.

However, the recovery rate of antithrombin ■ activity is as low as 30%, and the efficiency is poor.

(See Table 3)

[Brief explanation of the drawing]

FIG. 1 is a penzamidine Sepharose affinity chromatogram of anhydrothrombin. ■． Anhydrothrombin Figures 2 and 4 are chromatograms of a plasma protein mixture separated by anhydrothrombin Sepharose affinity chromatography. 2. Antithrombin ■ Figure 3 is a polyacrylamide disk electrophoresis image of antithrombin ■ purified by anhydrothrombin Sepharose affinity chromatography.

Claims (1)

[Scope of Claims] 1. An eluent containing a buffer, a neutral salt solution, and a plasma protein mixture having a pH of 6 to 8 and an ionic strength of 0 to 1 is contacted with an adsorbent in which anhydrothrombin is bound to a hydrophilic carrier. 1. A method for separating and purifying antithrombin (2), which is characterized by specifically adsorbing antithrombin (2) by allowing antithrombin (2) to be absorbed, and eluting it with a neutral salt solution or a substrate analogue having a pH of 4.0 to 5.5. However, the term "substrate analogue" as used herein refers to a competitive inhibitor of thrombin. 2. The method according to claim 1, wherein the buffer solution is Tris buffer, phosphate buffer, or acetate buffer. 3 Neutral salt solutions include NaC 1 , KCl , NH
4C 1, (NH4) 2So, the method according to claim 1 or 2. 4 Claim 1 in which the substrate analog is penzamidine, aminobenzamidine, nitrobenzamidine
The method described in section. 5. The method according to any one of claims 1 to 4, wherein the solution containing plasma proteins is an aqueous solution. 6. The method according to any one of claims 1 to 4, wherein the solution containing plasma proteins is a solution containing a neutral salt. 7. The method according to any one of claims 1 to 4, wherein the solution containing plasma proteins is an aqueous solution containing a water-soluble polymer such as polyethylene glycol.