JPS59109172A - Kallikrein purification method - Google Patents
Kallikrein purification methodInfo
- Publication number
- JPS59109172A JPS59109172A JP21811582A JP21811582A JPS59109172A JP S59109172 A JPS59109172 A JP S59109172A JP 21811582 A JP21811582 A JP 21811582A JP 21811582 A JP21811582 A JP 21811582A JP S59109172 A JPS59109172 A JP S59109172A
- Authority
- JP
- Japan
- Prior art keywords
- kallikrein
- spacer
- column
- acid
- sodium chloride
- 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.)
- Granted
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- Enzymes And Modification Thereof (AREA)
Abstract
Description
【発明の詳細な説明】
本発明はカリクレインの精製法に関するものであり、更
に詳しくは、哺乳動物の膵臓中に存在するカリクレイン
を簡単な操作での工業的規模において大量かつ高純度に
精製する方法に関するものである。DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for purifying kallikrein, and more specifically, a method for purifying kallikrein, which is present in the pancreas of mammals, in large quantities and with high purity on an industrial scale using simple operations. It is related to.
カリクレインは哺乳動物の各種組織で生産される蛋白分
wf酵素の一種であり、またキニン遊離酵素の一種とし
て知られている。遊離キニンには末梢血管拡張作用があ
り、従ってカリクレインは循環器系疾患の治療用に使用
されている。カリクレインは通常、哺乳動物臓器から抽
出されるので、そσ−〕粗抽出物[は多くの異種蛋白質
が含まれている。なかでもキニン産生あるいはキニン分
解に関連したトリフ0シン及びキモトリプシンの混在は
治療の際の副作用を惹起させるおそれがあるため、抽出
物を医薬品として使用するには、更に十分な精製を行っ
て高純度のものとすることが必要である。Kallikrein is a type of protein wf enzyme produced in various tissues of mammals, and is also known as a type of kinin-releasing enzyme. Free kinin has a peripheral vasodilating effect, and kallikrein is therefore used for the treatment of cardiovascular diseases. Since kallikrein is usually extracted from mammalian organs, the crude extract contains many foreign proteins. In particular, the presence of trypsin and chymotrypsin, which are involved in kinin production or kinin decomposition, may cause side effects during treatment, so in order to use the extract as a medicine, it must be further purified to a high purity. It is necessary that the
従来、粗カリクレインから異種蛋白質及び類似酵素を除
去−」るための方法としてば、塩析法、デルI4過法、
イオン父換りロマトクラフ法(特開昭53−62893
)、等電点分別法(%開昭49−41583)、溶媒沈
殿法(特開昭52−102495)などの手段を組み合
せた方法や、また阻書剤を利用したアフィニティークロ
マトクラフ法(特開昭54−62612)アルギニン誘
導体結合担体を用いたクロマトグラフ法(%開昭56−
117791、特開昭57−50924)などが知られ
ている。しかし、これらの方法は操作が繁卸−であった
り、ノ欠率が低い又は試薬が高仙1であるなどの点でい
ずれも工業的製法として満足し得るものではなかった。Conventionally, methods for removing foreign proteins and similar enzymes from crude kallikrein include salting out method, del I4 filtration method,
Ion father replacement romatograph method (Japanese Patent Application Laid-open No. 53-62893
), isoelectric point fractionation method (% 49-41583), solvent precipitation method (JP 52-102495), and affinity chromatography using an inhibitor (JP 52-102495). 1972-62612) Chromatographic method using arginine derivative-bound carrier
117791, JP-A-57-50924), etc. are known. However, none of these methods was satisfactory as an industrial production method because the operations were complicated, the defect rate was low, and the reagent used was Kosen 1.
本発明者らは、より簡Jやで効率的なカリクレインσJ
精製法を提供1べく鋭意研究を行った結果、カリクレイ
ンがグアニジノ基に刻して特異的に親和性を有すること
、およびグアニジノ基を有する物質をリガンドとしてア
フィニティークロマトグラフィーを行うことにより、簡
単に、且っ高収率で〜j糾度のカリクレインが得られる
ことを見い出し、本発明を完成した。The present inventors have proposed a simpler and more efficient kallikrein σJ
As a result of intensive research to provide a purification method, we found that kallikrein has a specific affinity for guanidino groups, and that by performing affinity chromatography using a substance having a guanidino group as a ligand, it was found that They have also discovered that kallikrein with a hardness of ~j can be obtained in high yield, and have completed the present invention.
ゴーなわら、本発明は、カリクレイン含有液を次の式(
r+
〔式中、Rは−(−OH2→−n (ここでnは1〜5
の数を示ず)又は−C6H4−を示す〕
で表わされるグアニジノカルボン酸をスペーサーを介し
て水不溶性担体に結合させて得た吸着体に接触させてカ
リクレインを吸着せしめ、次いで吸着したカリクレイン
を溶離せしめることを特徴とするカリクレインの精製法
を提供1−るものである。However, in the present invention, the kallikrein-containing liquid can be prepared using the following formula (
r+ [In the formula, R is -(-OH2→-n (where n is 1 to 5
) or -C6H4-] A guanidinocarboxylic acid represented by is bonded to a water-insoluble carrier via a spacer, and kallikrein is adsorbed by contacting with the adsorbent, and then the adsorbed kallikrein is eluted. The present invention provides a method for purifying kallikrein, which is characterized by:
本発明で使用される吸着体は、アガロース、デキストリ
ン、セルロース等の水不溶性担体に、−個以上のアミノ
基を有するスペーサーを結合させ、次いで、このスペー
サーのアミノ基と化合物(11のカルボキシル基とを常
法によりペプ□チド結合させることにより調製される。The adsorbent used in the present invention is produced by bonding a spacer having - or more amino groups to a water-insoluble carrier such as agarose, dextrin, or cellulose, and then combining the amino group of this spacer with a compound (11 carboxyl groups). It is prepared by peptide linkage using a conventional method.
スペーサーとして使用される化合物としては、水不溶性
担体または活性化した水不溶性担体と結合し得る官能基
を有し、かつこれと結合した後、遊離のアミノ基が残存
する化合物であればよく、この様な化合物としては、α
、ω−ジアミノアルキレン例えば1.6−ジアミツヘキ
サン、1.5−ジアミノペンクン、1゜8−ジアミノオ
クタン、1.10−ジアミノデカン等が挙げられる。ま
た、スペーサーとアガロースを結合させた゛アミノへキ
シルアガロースがファルマシア・ファインケミカル社よ
り[AH−セファロースJの商品名で市販されており、
本発明ではこれを使用し、吸着体を調製することもでき
る。The compound used as a spacer may be any compound that has a functional group that can bind to a water-insoluble carrier or an activated water-insoluble carrier, and that leaves a free amino group after binding to this. As a compound like α
, ω-diaminoalkylene such as 1,6-diamithexane, 1,5-diaminopenkune, 1°8-diaminooctane, 1,10-diaminodecane and the like. In addition, aminohexyl agarose, which is a combination of a spacer and agarose, is commercially available from Pharmacia Fine Chemicals under the trade name AH-Sepharose J.
In the present invention, this can also be used to prepare an adsorbent.
リガンドである弐(11のグアニジノカルボン酸として
は、グアニジノ酢酸、グアニジノ酢酸、グアニジノ安息
香酸等が挙げられる。Examples of the guanidinocarboxylic acid of the ligand 2(11) include guanidinoacetic acid, guanidinoacetic acid, guanidinobenzoic acid, and the like.
本発明を実#+するには、上述の如くして調製した吸着
体をカラムに充填し、これにカリクレイン含有液を通液
してカリクレインを吸着させ、次いて゛これに溶出液を
注加してカリクレインを溶出させ机
カリクレイン含有液の通液は、カリクレインのm度、吸
着体の種類によっても異なるが、一般には流速30〜5
Q ml / hr とするのが好ましい。To put the present invention into practice, the adsorbent prepared as described above is packed in a column, a kallikrein-containing solution is passed through the column to adsorb kallikrein, and then an eluent is poured into it. The flow rate of the kallikrein-containing solution varies depending on the m degree of kallikrein and the type of adsorbent, but generally the flow rate is 30 to 5.
It is preferable to set it as Qml/hr.
また溶出液としては塩化ナトリウムを含む−4,5〜9
.0(特に好ましくはP)17.4 )の緩衝液を用い
るのが好ましい。溶出にあたっては、最初に塩化ナトリ
ウム濃度の低い溶出液(塩化ナトリウム濃1隻:約0,
1〜O2ろM)を通して、不純物のトリプシン、キモト
リフ0シン等の酵素及び蛋白質を溶出させ、次いでこれ
に地化す) リウム濃度の高い溶IJJ 7i (4
化 シト リ ウ 4 8に度 : 0.4 〜1.
0 M ) を通し′Cカリクレインを浴出させ
るのが好ましい。この場合l島化すトす内広濃度が漸次
増大するグラジェント方式(例えは塩化ナトリウム0.
2〜1.0M)によりカリクレインを船出させCもよい
。尚本発明で使用される緩衝1俵は特に限定されないが
、精製物を凍結乾燥−4る前にア士トンを加えて脱地濃
釉白する場合(」、トリス(ヒドロキシメチル)アミツ
メタン・塩酸緩衝液が好ましい。また使用した吸着体は
緩衝液で洗浄するだけで再使用が可能であり、特別な再
生作操作を必要としない。In addition, the eluent contains -4,5 to 9 sodium chloride.
.. It is preferable to use a buffer with a concentration of 0 (particularly preferably P) 17.4). For elution, first use an eluent with a low sodium chloride concentration (1 bottle of sodium chloride concentration: approximately 0,
Impurities such as enzymes and proteins such as trypsin and chymotrifosin are eluted through 1 to O2 filtration (M), and then concentrated to the solution IJJ 7i (4
Converting to 4 8 degrees: 0.4 to 1.
Preferably, the 'C kallikrein is leached out through 0 M). In this case, a gradient method is used in which the concentration of sodium chloride gradually increases as it becomes an island (for example, sodium chloride 0.
2 to 1.0M) may also be used to ship Kallikrein. The bale of buffer used in the present invention is not particularly limited, but when the purified product is deglazed and deglazed by adding acetone before freeze-drying, tris(hydroxymethyl)amitsumethane/hydrochloric acid is used. A buffer solution is preferred.Furthermore, the adsorbent used can be reused simply by washing with a buffer solution, and no special regeneration operation is required.
斜上の本発明方法は、1回の吸脱着操作をおこなうだけ
で、トリプシン、キモトリプシン等の不純物をほとんど
含まない高紳度のカリクレインを高収率で得ることので
きる優れたものである。The above-mentioned method of the present invention is an excellent method that allows a high yield of kallikrein containing almost no impurities such as trypsin and chymotrypsin to be obtained by performing only one adsorption/desorption operation.
次に実施例を挙げ、本発明を更に詳しく説明する。Next, the present invention will be explained in more detail with reference to Examples.
実施例1
(11豚膵臓抽出原末25gに0.2係均化す) IJ
ウム水溶液250 mAを加え充分に攪拌して懸濁させ
、2N塩酸でpH4,5として遠心分離する。得られた
上清アンモニア水を用いてpf(7,4とした後、アセ
トン40〜55憾の分画沈降を行い、得られた沈殿を水
1oomiに溶かし、1N−水酸化ナトリウムでpH7
,4とし、カリクレイン含有液を得る。Example 1 (11 porcine pancreas extracted bulk powder 25g equalized by 0.2) IJ
Add 250 mA of an aqueous solution of 250 mA and stir thoroughly to suspend the mixture, adjust the pH to 4.5 with 2N hydrochloric acid, and centrifuge. After adjusting the pf to 7.4 using the obtained supernatant aqueous ammonia, fractional precipitation was carried out using acetone of 40 to 55%, the obtained precipitate was dissolved in 1 ml of water, and the pH was adjusted to 7.
, 4 to obtain a kallikrein-containing liquid.
(11)一方AHセファロース4B(ファルマ幻′・フ
ァインケミカル社&’)15.?を0.5 M 塩化ナ
トリウム水溶液に入れて膨潤させた後、吸引1過し、0
.5M4化ナトリウム水浴液、次いで脱イオン水で洗浄
する。得られた膨潤ゲル約6Qm/に脱イオン水1oo
mzを加えて懸濁する。これにグアニジノ凸′1酸70
m9 ([l+6 m m01e )を脱イオン水2
5鯰に溶かした溶液を加えlN−4酸又は1N−水酸化
ナトリウムで−44,5とする。これに1−エチル−ろ
−(3′−ジメチルアミノプロピル)カルボシイミド6
.4gを脱イオン水5 mlに溶かした溶液を加え、p
li 5.OK保ちながら室温で一夜攪拌する。(11) On the other hand, AH Sepharose 4B (Pharma Gen', Fine Chemical &') 15. ? was swollen in a 0.5 M sodium chloride aqueous solution, filtered with suction for 1 time, and 0.
.. Wash with 5M sodium tetrachloride water bath and then with deionized water. Approximately 6Qm of the resulting swollen gel was added to 100ml of deionized water.
Add mz and suspend. In this, guanidinoconvex'1 acid 70
m9 ([l+6 m m01e) in deionized water 2
5 Add the solution dissolved in catfish and adjust to -44.5 with 1N-4 acid or 1N-sodium hydroxide. To this, 1-ethyl-ro-(3'-dimethylaminopropyl)carbosiimide 6
.. Add a solution of 4 g dissolved in 5 ml deionized water, and
li 5. Stir overnight at room temperature while keeping OK.
反応混合物を吸引1過し、0.1M酢酸緩衝液(pJJ
4.0 : I M−塩化ナトリウム)および0.1
M )リス(ヒドロキシメチル)アミノメタン塩酸緩衝
液(pH8JJ : I M 堪化す) IJウム)で
交互に洗浄した後、吸引f過するとグアニジノ酢酸結合
AH・セファロース4Bが得られる。The reaction mixture was aspirated once and diluted with 0.1M acetate buffer (pJJ
4.0: IM-sodium chloride) and 0.1
M) After alternately washing with lith(hydroxymethyl)aminomethane hydrochloride buffer (pH 8JJ: IM), guanidinoacetic acid-bound AH/Sepharose 4B is obtained by passing through suction.
fllll (Il+で得られたグアニジノ酢酸結合
AH・セファロース4 Bをカラム(2φ×ろ0CrI
L)に充填り、、+11で得たカリクレイン含有液をこ
のカラムに通液し、50m!・A トリス(ヒドロキシ
メチル)アミノメタン・塩酸緩衝液(pf47.4 )
次いで0.2 M塩化ナトリウムを含む同緩衝液でカラ
ムを洗う。flllll (guanidinoacetic acid-bound AH Sepharose 4B obtained with
L) was filled, and the kallikrein-containing solution obtained in +11 was passed through this column, and 50 m!・A Tris(hydroxymethyl)aminomethane/hydrochloric acid buffer (pf47.4)
The column is then washed with the same buffer containing 0.2 M sodium chloride.
次に0.2〜1.0 M 塩化ナトリウム溶液(25(
IIA−250ml)でグラジェント溶出を行う。集め
たカリクレイン分画にアセトンを加えて脱環濃縮し、凍
結乾燥して精製カリクレイ724〜(総括性8160に
、U* )を得た。Then 0.2-1.0 M sodium chloride solution (25 (
Perform gradient elution with IIA-250 ml). Acetone was added to the collected kallikrein fractions to remove ring and concentrate, followed by freeze-drying to obtain purified kallikrein 724~ (overall 8160, U*).
*に、U:カリクレインユニット
実施例2
実施例1 fillにおけるグアニジノ酢酸70mgを
グアニジノ安息香酸129m&に変える以外は全(同様
にしC得たグアニジノ安、は香酸結合A)l・セファロ
ース4Bをカラム(2X30CnL)に充填し、実施例
1(1)と同様にして得たカリクレイン含有液をこのカ
ラムに通液し、50mM+−リス(ヒドロキシメチル)
アミノメタン・塩酸緩衝液(pH7,4)、次いで0.
2M塙化す) IJウムを含む同緩衝液でカラムを洗浄
した。次に(1,4Mh=化ナトナトリウムむ同緩衝液
でカリクレインを溶出し、集めたカリクレイン分画にア
セトンを加えて脱塩濃縮後、凍結乾燥し゛〔精製カリク
レイン164mp(総括性9676 K、U )を得た
。*, U: kallikrein unit Example 2 All except changing 70 mg of guanidinoacetic acid in Example 1 fill to 129 m& of guanidinobenzoic acid (guanidinoamic acid obtained in the same manner as C, is aromatic acid bonded A) l. Sepharose 4B was added to the column ( A kallikrein-containing solution obtained in the same manner as in Example 1 (1) was passed through this column, and 50 mM +-lith(hydroxymethyl) was added to the column.
aminomethane/hydrochloric acid buffer (pH 7.4), then 0.
The column was washed with the same buffer containing 2M IJum. Next, kallikrein was eluted with the same buffer containing sodium chloride (1,4Mh), and acetone was added to the collected kallikrein fractions to desalt and concentrate, followed by lyophilization. I got it.
実施例3
実施例1 fillにおけるグアニジノ酢酸70rng
をグアニジノ酪酸87mノに変える以外は全(同様にし
て得たグアニジノ酪酸結合AI(・セファロース4Bを
カラム(2×60cm)に充填する。次いで実施例1(
1)でアセトン40〜55係を63〜67係に変える以
外は全(同様にして得たカリクレイン含有液を上記のカ
ラムに通液し、10mM)リス(ヒドロキシメチル)ア
ミノメタ/−塩酸緩衝液(pH7,4)、次いで0.2
5 M塩化ナトリウムを含む同緩衝液でカラムを洗浄し
たO更にo、5vtta化ナトリウムを含む同緩衝液で
カリクレインを溶出後、集めたカリクレイン分画にアセ
トンを加えて脱塙濃縮し、都農カリクレイン72mノ(
総括性9 3 6 0 K、U ) を 得プこ
。Example 3 Example 1 70 rng of guanidinoacetic acid in fill
A column (2 x 60 cm) was filled with guanidinobutyric acid-conjugated AI (Sepharose 4B) obtained in the same manner, except that 87m of guanidinobutyric acid was used. Then, Example 1 (
1) except that the acetone 40-55 ratio was changed to 63-67 ratio (the kallikrein-containing solution obtained in the same manner was passed through the above column, and 10mM) was added to the lis(hydroxymethyl)aminometh/-hydrochloric acid buffer ( pH 7.4), then 0.2
The column was washed with the same buffer containing 5 M sodium chloride. After eluting kallikrein with the same buffer containing 5 M sodium chloride, acetone was added to the collected kallikrein fractions, which was then concentrated to remove Tsuno kallikrein 72m. of(
Overall performance: 9360 K, U).
実施例4
豚膵臓原末25gに0.2 ’1鳩化ノート・リウム水
溶#250威を加えて充分に撹拌して懸濁させ、2N地
酸でpf44.5として遠心分離すξ)1、得られた十
清をアンモニア水を用いpH7゜4とした後、硫酸アン
モニウムを加えて硫安分画を行う。得られた沈殿を水に
溶かし、DEAF・セルロースを加えてカリクレインを
I)lit AFi・セルロースに吸着させ、2%塩化
ナトリウム水溶液でDEAF セルロースからカリク
レインを脱着させる。次いでこのカリクレイン含有液を
アセト733〜67%の分画沈殿を行い、得られる沈殿
を水25mAに溶かし、p)17.4とする。Example 4 To 25 g of pig pancreas bulk powder, add 0.2'1 pigeon-hatched Noterium aqueous solution #250, stir thoroughly to suspend, and centrifuge at pf 44.5 with 2N base acid ξ) 1. After adjusting the pH of the resulting tensein to 7.4 using aqueous ammonia, ammonium sulfate is added to carry out ammonium sulfate fractionation. The obtained precipitate is dissolved in water, DEAF/cellulose is added to adsorb kallikrein to I)lit AFi/cellulose, and kallikrein is desorbed from DEAF cellulose with a 2% aqueous sodium chloride solution. Next, this kallikrein-containing solution is subjected to fractional precipitation of 733 to 67% acetate, and the resulting precipitate is dissolved in 25 mA of water to give p) 17.4.
この溶液を実施例1 fil+と同様にして得たグアニ
ジノ酢酸結合AH・セファロース4Bを充填したカラム
(2φx3QCTL)に通液し、このカラムを10mM
トリス(ヒドロキシメチル)アミノメタン−塩酸緩
衝爵(pH7,4)、次いで0.25 M塩化ナトIJ
、:ツムを含む同緩衝液で洗浄する。0.5M塙化ナ
トリウムを含む同緩衝液でカリクレインを溶出し、集め
たカリクレイン分画にアセトンを加え、脱塩濃縮し、凍
結乾燥して精與カリクレイン4η(総括性4120 K
、U )を得た。このものはトリプシン、キモトリプシ
ンを全く含まず純度の高いものであった。This solution was passed through a column (2φ
Tris(hydroxymethyl)aminomethane-hydrochloric acid buffer (pH 7,4), then 0.25 M sodium chloride IJ
,: Wash with the same buffer containing Tsum. Kallikrein was eluted with the same buffer containing 0.5 M sodium chloride, and acetone was added to the collected kallikrein fractions, desalted and concentrated, and lyophilized to obtain purified kallikrein 4η (total strength: 4120 K).
, U) were obtained. This product contained no trypsin or chymotrypsin and was highly pure.
実施例5
実施例1〜4で得た精製カリクレインについてそのカリ
クレイン活性、トリプシン活性及びキモトリフ0シン活
性を測定した。更に、カリクレイン活性の測定結果から
、その比活性、回収率、精製倍率を計算した。この結果
を第1表に示す。Example 5 The purified kallikrein obtained in Examples 1 to 4 was measured for its kallikrein activity, trypsin activity, and chymotrifosin activity. Furthermore, the specific activity, recovery rate, and purification ratio were calculated from the measurement results of kallikrein activity. The results are shown in Table 1.
(1) カリクレインの生理活性
9屋等の方法〔ず・ジャーナル・オプ・バイオケミスト
リー(J、Biochem、 〕 558巻201貞
、1956年〕に従って行1工った。(1) Physiological activity of kallikrein 9 A row was prepared according to the method of et al. [J. Biochem., Vol. 558, 201 Sada, 1956].
(2)トリプシン活性
高見の方法〔生化学、41巻、777員、1969年)
に従って行った(ベンゾイル−DL−アルギニン・P・
ニトロアニリド・HCJを基質とし、67°Cで15分
間反応させた)。(2) Trypsin activity Takami's method [Biochemistry, vol. 41, 777 members, 1969]
(benzoyl-DL-arginine P.
(Nitroanilide HCJ was used as a substrate and the reaction was carried out at 67°C for 15 minutes).
(3) キモトリプシン活性
佐々木等の方法〔アナリテイカルバイオケミス ト リ
イ (Anal、 Bj、ochem、 )
E3 6 巻 1 59ji 、1978年]に従っ
て行なった(N−ペンシイイル−b−シロシル−P−ニ
トロアニリドを基質とし37°Cで300分間反応せた
)。(3) Chymotrypsin activity Sasaki et al.'s method [Analytical Biochemistry (Anal, Bj, ochem, )]
E3, Volume 6, Volume 1, 59ji, 1978] (N-pencyyl-b-silosyl-P-nitroanilide was used as a substrate and the reaction was carried out at 37°C for 300 minutes).
以下余白 Margin below
Claims (1)
、Rは+(3H2−)−n(ここでnは1〜5の数を示
す)又は−06H,−を示1゛〕 で表わされるグアニジノカルボン酸をスペーサーを介し
て水不溶性担体に結合させて得た吸着体に接触させてカ
リクレインを吸着せしめ、次いで吸着したカリクレイン
を溶離せしめることを特徴とするカリクレインの精製法
。 (2) スペーサーがα、ω−ジアミノアルキレンよ
り導かれたものである特許請求の範囲第1項記載の方法
。 (3) スペーサーが1.6−ジアミノへ・キサン、
1゜5−ジアミノペンタン、1,8−ジアミノオクタン
、1,10−ジアミノデカンのいずれかから導かれたも
のである特許請求の範囲第1項又は第2項記載の方法。[Claims] (11) A kallikrein-containing liquid is prepared by the following general formula (IlL formula, where R is +(3H2-)-n (where n represents a number from 1 to 5) or -06H,- Purification of kallikrein, characterized by adsorbing kallikrein by bringing it into contact with an adsorbent obtained by bonding a guanidinocarboxylic acid represented by the formula 1 to a water-insoluble carrier via a spacer, and then eluting the adsorbed kallikrein. (2) The method according to claim 1, wherein the spacer is derived from α,ω-diaminoalkylene. (3) The spacer is 1,6-diaminohexane,
3. The method according to claim 1 or 2, which is derived from any one of 1°5-diaminopentane, 1,8-diaminooctane, and 1,10-diaminodecane.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP21811582A JPS59109172A (en) | 1982-12-13 | 1982-12-13 | Kallikrein purification method |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP21811582A JPS59109172A (en) | 1982-12-13 | 1982-12-13 | Kallikrein purification method |
Publications (2)
Publication Number | Publication Date |
---|---|
JPS59109172A true JPS59109172A (en) | 1984-06-23 |
JPH0260316B2 JPH0260316B2 (en) | 1990-12-14 |
Family
ID=16714854
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP21811582A Granted JPS59109172A (en) | 1982-12-13 | 1982-12-13 | Kallikrein purification method |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPS59109172A (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1987002709A1 (en) * | 1985-10-24 | 1987-05-07 | Biotechnology Research Partners, Ltd. | Blood tests diagnostic for hypertension |
JP2008091798A (en) * | 2006-10-04 | 2008-04-17 | Olympus Corp | Electric circuit device and its manufacturing method |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH05121958A (en) * | 1991-10-29 | 1993-05-18 | Saitama Nippon Denki Kk | Distortion compensation control system for linear amplifier |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS504227A (en) * | 1972-12-19 | 1975-01-17 | ||
JPS5276481A (en) * | 1975-12-18 | 1977-06-27 | Otsuka Pharmaceut Co Ltd | Production of high purity urokinase |
JPS5592355A (en) * | 1978-12-22 | 1980-07-12 | Nippon Soda Co Ltd | Enzyme-adsorbing substance and purification of enzyme using the same |
-
1982
- 1982-12-13 JP JP21811582A patent/JPS59109172A/en active Granted
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS504227A (en) * | 1972-12-19 | 1975-01-17 | ||
JPS5276481A (en) * | 1975-12-18 | 1977-06-27 | Otsuka Pharmaceut Co Ltd | Production of high purity urokinase |
JPS5592355A (en) * | 1978-12-22 | 1980-07-12 | Nippon Soda Co Ltd | Enzyme-adsorbing substance and purification of enzyme using the same |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1987002709A1 (en) * | 1985-10-24 | 1987-05-07 | Biotechnology Research Partners, Ltd. | Blood tests diagnostic for hypertension |
JP2008091798A (en) * | 2006-10-04 | 2008-04-17 | Olympus Corp | Electric circuit device and its manufacturing method |
Also Published As
Publication number | Publication date |
---|---|
JPH0260316B2 (en) | 1990-12-14 |
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