JPS5932116B2 - Purification method of urokinase - Google Patents
Purification method of urokinaseInfo
- Publication number
- JPS5932116B2 JPS5932116B2 JP2819077A JP2819077A JPS5932116B2 JP S5932116 B2 JPS5932116 B2 JP S5932116B2 JP 2819077 A JP2819077 A JP 2819077A JP 2819077 A JP2819077 A JP 2819077A JP S5932116 B2 JPS5932116 B2 JP S5932116B2
- Authority
- JP
- Japan
- Prior art keywords
- urokinase
- column
- solution
- adsorbed
- crude
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
Landscapes
- Enzymes And Modification Thereof (AREA)
Description
【発明の詳細な説明】
本発明はウロキナーゼの精製法に関し、更に詳しくは人
尿またはそれに由来する粗製ウロキナーゼから高純度の
ウロキナーゼを工業的有利に取得する方法に関する。DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for purifying urokinase, and more particularly to a method for industrially advantageously obtaining highly pure urokinase from human urine or crude urokinase derived therefrom.
ウロキナーゼは血清中に含まれるプラスミノーゲンを活
性化してフィブリン溶解能を有するプラスミンを生成す
る機能があるため、線溶系の賦活剤として有用であり、
末梢動脈血栓症や心筋硬そく症などの治療に広く臨床使
用されている。Urokinase has the function of activating plasminogen contained in serum to produce plasmin, which has fibrinolytic ability, so it is useful as an activator of the fibrinolytic system.
It is widely used clinically to treat peripheral arterial thrombosis and myocardial stiffness.
またウロキナーゼは近年抗ガン剤の増強剤としての用途
も開発され、その需要は急速に増大している。Urokinase has also recently been developed for use as an enhancer for anticancer drugs, and its demand is rapidly increasing.
ウロキナーゼの精製法としては、燐酸セルロース、アン
バーライト■ＲＣ−５０、カルボキシメチルデキストラ
ン、カルボキシメチルセルロース等のイオン交換体を用
いる方法、シリカゲル、ガラスピーズ、多孔性ガラス、
カーデワックス処理多孔性ガラス、珪酸アルミニウムマ
グネシウム、硫酸バリウム、珪藻土、活性化珪藻土、ヒ
ドロキシアパタイト、ベントナイト等の物理化学的吸着
剤を用いる方法、アクリルニトリル系合成繊維、シアノ
アルキル化セルロース、ポリエチレンイミン含有ポリア
ミド繊維、寒天、アガロース等の特異的吸着剤を用いる
方法、管種々の方法が知られている。Methods for purifying urokinase include methods using ion exchangers such as cellulose phosphate, Amberlite RC-50, carboxymethyl dextran, and carboxymethyl cellulose, silica gel, glass beads, porous glass,
Cardewax-treated porous glass, method using physicochemical adsorbents such as magnesium aluminum silicate, barium sulfate, diatomaceous earth, activated diatomaceous earth, hydroxyapatite, bentonite, acrylonitrile synthetic fiber, cyanoalkylated cellulose, polyamide containing polyethyleneimine Various methods are known, including methods using specific adsorbents such as fibers, agar, and agarose.
本発明者らはウロキナーゼの精製法について種種研究を
重ねた結果、従来ウロキナーゼの精製に利用されたこと
のないスルホエチルセルロースがウロキナーゼに対して
特異的な吸着効果を有していることを見い出し、かかる
知見に基づいて、高純度のウロキナーゼを工業的有利に
取得する方法を確立するに至った。As a result of repeated research on purification methods for urokinase, the present inventors discovered that sulfoethylcellulose, which had not been previously used for purification of urokinase, has a specific adsorption effect on urokinase. Based on this knowledge, we have established an industrially advantageous method for obtaining highly purified urokinase.
すなわち、本発明は人尿またはそれに由来する粗ウロキ
ナーゼをスルホエチルセルロースと接触させてウロキナ
ーゼを吸着させた後、吸着せるウロキナーゼを溶出する
ことからなるウロキナーゼの精製法である。That is, the present invention is a method for purifying urokinase, which comprises bringing human urine or crude urokinase derived therein into contact with sulfoethylcellulose to adsorb urokinase, and then eluting the adsorbed urokinase.
以下、本発明方法を具体的に説明する。The method of the present invention will be specifically explained below.
まず人尿またはそれに由来する粗製ウロキナーゼ溶液を
、スルホエチルセルロースを充填１．たカラムに流下さ
せて、ウロキナーゼを吸着させる。First, human urine or a crude urokinase solution derived therefrom is filled with sulfoethylcellulose.1. Flow down the column to adsorb urokinase.
この吸着操作はｐＨ３〜７で行なうのが好ましく、また
カラムに流下する人尿または粗製ウロキナーゼ溶液の電
気伝導率は３０ ｍｒｒｈｏ ／ｃｍ 以下に調整し
てお（のが好ましい。This adsorption operation is preferably carried out at a pH of 3 to 7, and the electrical conductivity of the human urine or crude urokinase solution flowing down the column is preferably adjusted to 30 mrrho/cm or less.
この条件でウロキナーゼはスルホエチルセルロースに吸
着され、一部）不純物は吸着されずにそのまま溶出する
。Under these conditions, urokinase is adsorbed to sulfoethylcellulose, and some impurities are eluted as they are without being adsorbed.
次いで、カラムをｐＨ３〜８の稀薄塩類溶液（例えば電
気伝導率３０ｍｍｈｏ／ａｍ以下の燐酸緩衝液）で洗浄
する。The column is then washed with a dilute salt solution having a pH of 3 to 8 (for example, a phosphate buffer with an electrical conductivity of 30 mmho/am or less).
洗浄液の量はカラム容量の２〜３倍量が好ましい。The amount of washing solution is preferably 2 to 3 times the column volume.
この洗浄操作により不純物は完全に除かれるが、ウロキ
ナーゼを吸着されたまま残る。Although impurities are completely removed by this washing operation, urokinase remains adsorbed.
吸着しているウロキナーゼを溶出するには、カラムにｐ
Ｈ７，５〜１１の濃厚塩類溶液（例えば電気伝導率４０
ｍｍｈｏ／ｃＩｒＬ以上の燐酸緩衝液、食塩溶液）ま
たはアンモニア水を流下することによって容易に行なう
ことができる。To elute the adsorbed urokinase, add p to the column.
Concentrated salt solution of H7,5-11 (e.g. electrical conductivity 40
This can be easily carried out by flowing down a phosphate buffer solution, a saline solution of mmho/cIrL or higher, or aqueous ammonia.
尚、塩類溶液を用いて溶出するに際し、塩類溶液の濃度
を段階的に上昇させる濃度勾配法を用いることにより不
純物をより完全に除去できる場合がある。In addition, when performing elution using a salt solution, impurities may be more completely removed in some cases by using a concentration gradient method in which the concentration of the salt solution is increased stepwise.
かくして得られたウロキナーゼ溶出液は常法により硫安
塩析し、透析後、凍結乾燥することによりウロキナーゼ
を単離することができる。The urokinase eluate thus obtained is subjected to salting out with ammonium sulfate in a conventional manner, followed by dialysis and lyophilization to isolate urokinase.
尚、本発明の吸着操作及び溶出操作は上述の如きカラム
法以外にバッチ法によっても行なうことができる。Incidentally, the adsorption operation and elution operation of the present invention can be carried out not only by the above-mentioned column method but also by a batch method.
上記の如き本発明方法は、少量の樹脂で非常に高純度の
ウロキナーゼが得られ、かつ樹脂を繰り返し使用できる
等の利点がある。The method of the present invention as described above has the advantage that extremely high purity urokinase can be obtained with a small amount of resin, and the resin can be used repeatedly.
以下、実施例を挙げて本発明方法を説明するが、実施例
中ウロキナーゼの力価はプロラグらのフィブリン平板法
（Ｊ、Ｐｌｏｕｇら： Ｂｉｏｃｈｉｍ、 Ｂｉｏｐｈ
ｙｓ 、 Ａｃｔａ、２４．２７８（１，９５７））に
て測定し、力価表示はジョンソンらにより定められた国
際単位（Ａ、 Ｊ 、 Ｊ ｏｈｎｓｏｎら： Ｔｈｒ
ｏｍｂｏｓ 。The method of the present invention will be described below with reference to Examples. In the Examples, the titer of urokinase was determined using the fibrin plate method of Ploug et al.
ys, Acta, 24.278 (1,957)), and the titer is expressed in the international units defined by Johnson et al. (A, J, Johnson et al.: Thr
ombos.
Ｄ １ａｔｈｅｓ 、 ｈａｅ ｍｏｒｒｈ （Ｓ ｔ
ｕｔｔｇ 、 ）、２］、、２５９（１９６９））によ
った。D 1athes, hae morrh (S t
uttg, ), 2], 259 (1969)).
実施例 １
スルホエチルセルロース（商品名：ＳＥ−セルロース、
セルヴア社製）５グを水にて膨潤させた後、０．５Ｎ力
性ソーダ、次いで０．５ Ｎ塩酸にて活性化してＨ十型
とする。Example 1 Sulfoethyl cellulose (trade name: SE-cellulose,
After swelling 5g (manufactured by Selva) with water, it was activated with 0.5N hydrochloric acid and then with 0.5N hydrochloric acid to form an H-type.
これをカラム（］、、３２ｃｍｘ１４．６ＣＴＬ）に充
填し、０．０ ］、 Ｍ燐酸緩衝液（ｐＨ８，０）にて
緩衝化する。This was packed into a column (32 cm x 14.6 CTL) and buffered with 0.0 M phosphate buffer (pH 8.0).
このカラムに新鮮な人尿から抽出した粗製ウロキナーゼ
溶液５０ｍ１（５５０００単位、５５００単位／ｍｙ蛋
白質、電気伝導率２．５ ｍ ｍｈｏ／ｃｍ ）を流下
させる。50 ml of a crude urokinase solution extracted from fresh human urine (55000 units, 5500 units/my protein, electrical conductivity 2.5 m mho/cm ) is allowed to flow down this column.
カラムを０．０１Ｍ燐酸緩衝液（ｐＨ８，０） ６０ｍ
１．にて洗浄し、次いで０．４ Ｍ食塩を含んだ０．Ｏ
ＩＭ燐酸緩衝緩衝 ｐＨｓ、ｏ ）６ｏｍｌを流してカ
ラムに吸着した不純物を流出除去させる。Column with 0.01M phosphate buffer (pH 8,0) 60m
1. and then washed with 0.4M sodium chloride solution. O
6 oml of IM phosphate buffer (pHs, o) is passed through the column to remove impurities adsorbed on the column.
次いでカラムに０．７Ｍ食塩を含んだ０．０１Ｍ燐酸緩
衝液（ｐＨ８，０）４０ｍｌを流下してウロキナーゼを
溶出させる。Next, 40 ml of 0.01 M phosphate buffer (pH 8,0) containing 0.7 M sodium chloride is allowed to flow down the column to elute urokinase.
このようにして得られたウロキナーゼ溶液の比活性は３
４０００単位／ｍｅ蛋白質であり、また精製により、４
８４００単位のウロキナーゼを回収することができ、粗
製ウロキナーゼ溶液からの活性収率は８８％であった。The specific activity of the urokinase solution obtained in this way was 3
4000 units/me protein, and by purification, 4
8400 units of urokinase could be recovered, and the activity yield from the crude urokinase solution was 88%.
Claims (1)
ホエチルセルロースと接触させてウロキナーゼを吸着さ
せた後、吸着せるウロキナーゼを溶出することを特徴と
するウロキナーゼの精製法。1. A method for purifying urokinase, which comprises bringing human urine or crude urokinase derived therein into contact with sulfoethylcellulose to adsorb urokinase, and then eluting the adsorbed urokinase.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2819077A JPS5932116B2 (en) | 1977-03-14 | 1977-03-14 | Purification method of urokinase |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2819077A JPS5932116B2 (en) | 1977-03-14 | 1977-03-14 | Purification method of urokinase |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS53113081A JPS53113081A (en) | 1978-10-03 |
| JPS5932116B2 true JPS5932116B2 (en) | 1984-08-06 |
Family
ID=12241765
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2819077A Expired JPS5932116B2 (en) | 1977-03-14 | 1977-03-14 | Purification method of urokinase |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS5932116B2 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6449624U (en) * | 1987-09-24 | 1989-03-28 |
-
1977
- 1977-03-14 JP JP2819077A patent/JPS5932116B2/en not_active Expired
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6449624U (en) * | 1987-09-24 | 1989-03-28 |
Also Published As
| Publication number | Publication date |
|---|---|
| JPS53113081A (en) | 1978-10-03 |
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