JPS58124798A - Purification method of alpha2-plasmin inhibitor - Google Patents

Abstract

PURPOSE:To prepare an alpha2-plasmin inhibitor which is an inhibitor of enzymic plasmin in high purity and yield by easy operations, by bringing a solution of the alpha2-plasmin inhibitor containing impurities into contact with an immobilized ligand, and purifying the alpha2-plasmin inhibitor. CONSTITUTION:A solution of an alpha2-plasmin inhibitor (hereinafter referred to as alpha2-PI) containing impurities is brought into contact with an immobilized ligand of formulaI(the water-insoluble polysaccharide is cellulose gel, crosslinked dextran gel, agarose gel or crosslinked agarose gel) or formula II (the water-soluble polysaccharide is dextran or starch) to purify the alpha2-PI. For example, the immobilized ligand is suspended in a buffer solution and packed in a column, and the resultant suspension of the alpha2PI containing impurities in the buffer solution is then passed through the column. The buffer solution is then passed through the column to elute and separate the impurities and then alpha2-PI.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to α2-plasmin inhibitor (hereinafter α, -P
Abbreviated as I. ). α2-PI is described by Aoki et al. (J. Biol. Ohem, vol. 251, 59
56 pages.

(1976) is a protein that exists in trace amounts in mammalian body fluids (mainly blood) and is an inhibitor that blocks the enzymatic activity of the fibrinolytic enzyme plasmin. Its most important physiological function is to deactivate the enzyme plasmin, which acts to restore blood flow fluidity after blood coagulation (hemostasis). It is known that patients who are genetically deficient in α,-PI or whose concentration is pathologically reduced are prone to severe bleeding, and the importance of measuring the blood concentration of α2-PI has recently been highlighted. It has been recognized and its therapeutic application is being considered.

However, even though α2-PI is present in plasma, which has a relatively high concentration, its normal value is extremely small at approximately 6 m9/10 o ml, making it difficult to prepare a standard product for concentration measurement, let alone as a therapeutic drug. It shows.

Currently relatively effective purification methods include affinity chromatography of immobilized plasminogen, which utilizes the weak binding between α2-PI and plasminogen, a precursor of plasmin, and human plasminogen. There is an affinity chromatography method in which plasminoken is hydrolyzed with elastase and the site where plasminoken binds to fibrin is immobilized, and an immunological purification method using an anti-α2-PI antibody. However, these methods are limited to α2-P, which can bind to immobilized plasminogen.
I is only 80% of the total concentration, and plasminogen has the property of being easily converted to plasmin, so it is not suitable for continuous treatment. Due to the complexity of immobilization through such methods, reproducibility and speed are poor, or sufficient anti-α
Since the antibody is obtained from a foreign animal after preparing a highly purified PI product, it is necessary to use the antibody in the antibody titer adjustment panel to prevent the contamination of proteins derived from a foreign animal when obtaining α, -PI. It is difficult to say that it is useful because it involves a lot of operations.

The present inventors used blue sepharose (Sweten, sold by PharmanA) to remove albumin present in large quantities in the α2-PI crude preparation, but due to the difference in pH during use, α2-PI We discovered that the behavior of PI changes. Therefore, by varying the pH, α,, -P I
After investigating the behavior of the protein, it was found that at pH 7.2 or above, not only albumin but also most proteins other than α2-PI and α,-PI were separated, and the present invention was completed.

In the present invention, α, -P I solution containing impurities can be prepared using the general formula (however, water-insoluble polysaccharide refers to cellulose gel, cross-linked dextrough gel, agarose gel, cross-linked agarose gel) or (however, water-soluble polysaccharide and (denotes dextran or starch)
This is an industrially advantageous α2-PI purification method that allows high-purity α2-PI to be obtained in high yield through a simple operation of contacting with I bath solution and developing with a buffer solution in the range of pH 7.2 to 95. .

Although the present invention can be carried out by either a column method or a batch method, the present invention will be explained by taking the column method as an example.

First, a column is filled with a suspension of the immobilized ligand described above in a buffer solution (for example, Tris buffer, pH 8). Next, the α2-PI bath solution containing impurities dissolved in the same buffer solution is allowed to flow down the column, and then about twice the column volume of the buffer solution is allowed to flow down, and the elution of the impurities is almost completed.

Then, by further flowing down the same buffer solution, α2-PI
is eluted.

For carrying out the present invention, the adhesive immobilized product is, for example, J.
Chromatogr, Volume 69, Page 209, 1
It can be obtained by the method described in 1972. That is.

Cibacron Blue F3 GA (released by Sigma, product name: Reactive Blue) 2 f to 6o
ml of water and heated to 60°C with stirring, a.
Expand to 5 oml + YSLf, =loy Sephatex G2
00■ (Published by Pharmacia, Sweden) (Crosslinked Textlauhe is added dropwise and stirred for an additional 30 minutes. Next, 457 sodium chloride is added and stirred for 60 minutes. This mixture is heated to 80°C and the carbonic acid I of 4) is added dropwise. -IJum was added and stirred at the same temperature for 2 hours to obtain a ligand immobilized product in which Cibacron Blue F8GA and Sephadex were bound.

If this operation is performed using water-insoluble polysaccharides such as cellulose gel, agarose gel, cross-linked agarose gel, etc., each ligand immobilized product can be obtained, but Cibacron Blue F
i3 OA was cross-linked with agarose gel-bound Blue Sepharose ■ (Pharmacia, Sweden) or agarose gel-bound Madrex Blue A (
It is convenient to use a commercially available product (trade name, Amicon, USA). On the other hand, in the case of inclusion in acrylamide gel, for example, Cibacron Blue F 3 GA and dextran are bonded in the same manner as described above for bonding Cibacron Blue F3GA and Sephadex. 9.5 ml of this binding solution.

Add 0.475% acrylamide gel to 0.0257% N,N'-methylenehis-acrylamide.

Next, 20 μl of potassium persulfate and 80 μl of β-dimethylaminopropionitrile were added, degassed at 0° C., and left at 0° C. overnight. After the polymerization reaction, the gel is crushed and washed with 0.05M acetic acid to obtain a Gantt-immobilized product in which Cibacron Blue 1.3GA-dextran is included in the acrylamide gel.

Further, the buffer used for development may be one commonly used, such as Tris buffer, phosphate buffer, or borate buffer.

The following table summarizes the purification degree and yield of α, -PI when the pH was varied using Blue Sepharose.

In addition, when pi-1 is 7.0 or less, only albumin is removed, and other proteins and α, -PI cannot be separated at all. Further, at pH 9.5 or higher, α2-PI is deactivated, which is not preferable.

The degree of purification was expressed by specific activity, which is the total plasmin activity inhibition activity divided by the total protein amount. In other words, the inhibitory activity that deactivates 1 unit of plasmin is considered to be 1 inhibitory activity, and the total inhibitory activity is 28
The absorbance at 0 nm was calculated based on the amount of protein. Furthermore, the degree of increase in purification was determined by dividing the specific activity after contact with the specific activity before contact with Blue Sepharose.

On the other hand, the identity of α2-PI was confirmed by Ophthalony method using anti-human rabbit antiserum.

As described above, the method of the present invention is an industrially advantageous method that is easy to operate and can provide high yield and high purity α2-PI.

The present invention will be explained below with reference to examples.

Example 1 One liter of human plasma is salted out with ammonium sulfate to obtain a pseudoglobulin fraction. The pseudoglobulin fraction was dissolved in 50mM To'J Tris buffer (8.0), dialyzed against the same solution, and diethylaminoethyl-Sephadex A equilibrated with the same buffer.
-50 (sold by Pharmacia) (diameter 5 crn x height 3
0 c7rL) for partial purification. After further concentrating by ultrafiltration, it was applied to a column packed with blue sepharose (diameter 2.6 cIn x height 60 Ci) equilibrated with 50 mM Tris buffer (pH 8,0), developed with the same buffer, and fractionated. A fraction of 8 ml was collected using a collector. Refined α2
-PI was recovered between fraction numbers 47 and 57 and impurities between 21 and 46. The increase in purity at this time was 80 times, and the yield was 67%.

Examples 2 to 4 Instead of the blue Sepharose in Example 1, Cibacron Blue F8 GA-Textran was included in an acrylamide gel, Kant immobilized product (Example 2), and f-Bacron Blue F +3 GA tocellulose gel were used. binding or Gantt immobilization (Example 3), and Cibacron Blue F8
[alpha]2-PI was purified from partially purified [alpha],-PI in the same manner as in Example 1 by combining GA with cross-linked Text Langel or using Gantt immobilized material (Example 4).

Claims (1)

[Claims] (1) An α2-plasmin inhibitor solution containing impurities is prepared with the general formula (however, water-insoluble polysaccharides are cellulose gel, cross-linked dextrin gel,
agarose gel or cross-linked agarose gel) or (water-soluble polysaccharide refers to dextran or starch) to separate proteins other than m-plasmin inhibitor and α2-plasmin inhibitor. A method for purifying α2-plasmin inhibitor, characterized by: