JPS6338200B2 - - Google Patents
Info
- Publication number
- JPS6338200B2 JPS6338200B2 JP57150701A JP15070182A JPS6338200B2 JP S6338200 B2 JPS6338200 B2 JP S6338200B2 JP 57150701 A JP57150701 A JP 57150701A JP 15070182 A JP15070182 A JP 15070182A JP S6338200 B2 JPS6338200 B2 JP S6338200B2
- Authority
- JP
- Japan
- Prior art keywords
- hydroxyanilide
- glutamyl
- compound
- leucyl
- activity
- 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
- 238000000034 method Methods 0.000 claims description 50
- 230000000694 effects Effects 0.000 claims description 46
- 239000000758 substrate Substances 0.000 claims description 40
- -1 amino compound Chemical class 0.000 claims description 38
- 238000004040 coloring Methods 0.000 claims description 27
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 claims description 22
- 150000001448 anilines Chemical class 0.000 claims description 22
- 102000035195 Peptidases Human genes 0.000 claims description 21
- 108091005804 Peptidases Proteins 0.000 claims description 21
- 235000019833 protease Nutrition 0.000 claims description 21
- 238000005259 measurement Methods 0.000 claims description 20
- 230000001590 oxidative effect Effects 0.000 claims description 17
- 150000001875 compounds Chemical class 0.000 claims description 14
- 229960003136 leucine Drugs 0.000 claims description 12
- 239000004395 L-leucine Substances 0.000 claims description 10
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- 239000012085 test solution Substances 0.000 claims description 10
- 238000009833 condensation Methods 0.000 claims description 8
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- 102000006640 gamma-Glutamyltransferase Human genes 0.000 claims description 8
- 238000000691 measurement method Methods 0.000 claims description 8
- 239000000126 substance Substances 0.000 claims description 8
- 102000004400 Aminopeptidases Human genes 0.000 claims description 7
- 108090000915 Aminopeptidases Proteins 0.000 claims description 7
- 101710107035 Gamma-glutamyltranspeptidase Proteins 0.000 claims description 7
- 101710173228 Glutathione hydrolase proenzyme Proteins 0.000 claims description 7
- 150000003839 salts Chemical class 0.000 claims description 7
- 125000003440 L-leucyl group Chemical group O=C([*])[C@](N([H])[H])([H])C([H])([H])C(C([H])([H])[H])([H])C([H])([H])[H] 0.000 claims description 5
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- PAYRUJLWNCNPSJ-UHFFFAOYSA-N N-phenyl amine Natural products NC1=CC=CC=C1 PAYRUJLWNCNPSJ-UHFFFAOYSA-N 0.000 claims description 3
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- 125000000291 glutamic acid group Chemical group N[C@@H](CCC(O)=O)C(=O)* 0.000 claims description 2
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- 108020004206 Gamma-glutamyltransferase Proteins 0.000 claims 1
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- 108010004098 Leucyl aminopeptidase Proteins 0.000 description 43
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- JQWHASGSAFIOCM-UHFFFAOYSA-M sodium periodate Chemical compound [Na+].[O-]I(=O)(=O)=O JQWHASGSAFIOCM-UHFFFAOYSA-M 0.000 description 36
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- IXCSERBJSXMMFS-UHFFFAOYSA-N hydrogen chloride Substances Cl.Cl IXCSERBJSXMMFS-UHFFFAOYSA-N 0.000 description 6
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- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 5
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- 235000017557 sodium bicarbonate Nutrition 0.000 description 5
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- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 4
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- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
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- FJKROLUGYXJWQN-UHFFFAOYSA-N papa-hydroxy-benzoic acid Natural products OC(=O)C1=CC=C(O)C=C1 FJKROLUGYXJWQN-UHFFFAOYSA-N 0.000 description 3
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- IUVKMZGDUIUOCP-BTNSXGMBSA-N quinbolone Chemical compound O([C@H]1CC[C@H]2[C@H]3[C@@H]([C@]4(C=CC(=O)C=C4CC3)C)CC[C@@]21C)C1=CCCC1 IUVKMZGDUIUOCP-BTNSXGMBSA-N 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 229960004889 salicylic acid Drugs 0.000 description 1
- 238000010898 silica gel chromatography Methods 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 235000010288 sodium nitrite Nutrition 0.000 description 1
- 229940083618 sodium nitroprusside Drugs 0.000 description 1
- IAAKNVCARVEIFS-UHFFFAOYSA-M sodium;4-hydroxynaphthalene-1-sulfonate Chemical compound [Na+].C1=CC=C2C(O)=CC=C(S([O-])(=O)=O)C2=C1 IAAKNVCARVEIFS-UHFFFAOYSA-M 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 239000011975 tartaric acid Substances 0.000 description 1
- 235000002906 tartaric acid Nutrition 0.000 description 1
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 201000005112 urinary bladder cancer Diseases 0.000 description 1
- 150000003739 xylenols Chemical class 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
- Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Description
【発明の詳細な説明】
本発明は、被験液に含有されているL−ロイシ
ンアミノペプチダーゼおよびγ−グルタミルトラ
ンスペプチダーゼからなる群より選ばれるペプチ
ダーゼの活性測定において、式
(式中、RはL−ロイシンアミノペプチダーゼ活
性測定の場合にL−ロイシル基、γ−グルタミル
トランスペプチダーゼ活性測定の場合にγ−L−
グルタミル基を示し、XおよびYは同一かまたは
異なりハロゲン原子を示す)で表わされるアミド
化合物を合成基質とする被験中のペプチダーゼの
新規な活性測定法に関する。Detailed Description of the Invention The present invention provides a method for measuring the activity of a peptidase selected from the group consisting of L-leucine aminopeptidase and γ-glutamyl transpeptidase contained in a test solution. (In the formula, R is an L-leucyl group in the case of L-leucine aminopeptidase activity measurement, and γ-L- in the case of γ-glutamyl transpeptidase activity measurement.
The present invention relates to a novel method for measuring the activity of a peptidase under test using as a synthetic substrate an amide compound represented by a glutamyl group, and X and Y are the same or different and represent a halogen atom.
ペプチダーゼは一般的にはペプチドのペプチド
結合に作用してアミノ末端から切断してアミノ酸
またはより低級のペプチドを遊離させる酵素の総
称として古くから知られている。例えばロイシン
アミノペプチダーゼ(臨床化学においてTrue
LAPともいう)やアリルアミダーゼ(臨床化学
においてClinical LAPともいう、以下時として
AAと称する)などのアミノペプチダーゼ(以下
True LAPとClinical LAPを併せて単にLAPと
称する)シスチンアミノペプチダーゼ、プロリン
アミノペプチダーゼ、アルギニンアミノペプチダ
ーゼ、アラニンアミノペプチダーゼ、γ−グルタ
ミルトランスペプチダーゼ(γ−GTP)などが
知られている。 Peptidases have long been known as a general term for enzymes that act on the peptide bonds of peptides and cleave them from the amino terminus to release amino acids or lower peptides. For example, leucine aminopeptidase (True in clinical chemistry)
(also referred to as LAP) and allylamidase (also referred to as Clinical LAP in clinical chemistry, hereinafter sometimes referred to as
Aminopeptidases (hereinafter referred to as AA) such as
Cystine aminopeptidase, proline aminopeptidase, arginine aminopeptidase, alanine aminopeptidase, and γ-glutamyl transpeptidase (γ-GTP) are known.
上記酵素のうち、特にロイシンアミノペプチダ
ーゼ(LAP)やγ−グルタミルトランスペプチ
ダーゼ(γ−GTP)は、生体内のあらゆる組織
に広く分布し、血清中にも存在しており、病的条
件によつて増加することが知られており、臨床検
査上重要な酵素活性測定項目の対称となつている
酵素である。 Among the enzymes mentioned above, leucine aminopeptidase (LAP) and γ-glutamyl transpeptidase (γ-GTP) in particular are widely distributed in all tissues in the body, and are also present in serum. This is an enzyme that is known to increase in enzyme activity and is the subject of important enzyme activity measurement items in clinical tests.
LAPはL−ペプチド、特にアミノ末端がL−
ロイシンまたはこれに関係あるアミノ酸であるL
−ペプチドの遊離アミノ基をもつアミノ末端残基
を加水分解してL−ロイシンを遊離させる酵素で
あつて、生体内の各組織に広く分布し、血清中に
も存在していて、血清中の酵素量は生体の状態に
より著しく変動することが知られており、急性肝
炎、肝癌、転移性肝癌、肝硬変症、胆道症などで
はLAP活性が増加する。そこでLAP活性がかか
る疾病の1つの指標となり、この酵素活性を測定
することは、これら疾病の診断に必要欠くべから
ざる検査法の1つになつている。 LAP is an L-peptide, especially the amino terminus is L-
L, which is leucine or a related amino acid
- An enzyme that hydrolyzes the amino-terminal residue of a peptide that has a free amino group to release L-leucine, and is widely distributed in various tissues in the body and is also present in serum. It is known that the amount of the enzyme varies significantly depending on the state of the living body, and LAP activity increases in acute hepatitis, liver cancer, metastatic liver cancer, liver cirrhosis, biliary tract disease, etc. Therefore, LAP activity is one of the indicators of such diseases, and measuring this enzyme activity has become one of the indispensable testing methods for diagnosing these diseases.
γ−GTPは生体内でγ−グルタミルペプチド
の代謝に関与する酵素であり、γ−グルタミルペ
プチドを加水分解すると共に、γ−グルタミル基
を他のペプチドあるいはアミノ酸に転移させる酵
素であつて、生体内の各組織に広く分布し、血清
中にも存在する。γ−GTPは病的条件により著
しく変動することが知られており、血清γ−
GTPは肝汁うつ滞型肝炎、閉塞性黄疸、原発性
肝癌、転移性肝癌などでは、著しい高値を示し、
特に慢性肝炎の非活動型では低値を示すが、活動
型では高値を示すなど一般に慢性の肝疾患に特異
的であり、従来臨床的に用いられて来た種々の逸
脱酵素とは全く異なる診断学的意義を有し、かか
る血清γ−GTP活性の測定はこのような疾病の
診断および病態把握に必要欠くべからざる検査法
の1つになつている。 γ-GTP is an enzyme involved in the metabolism of γ-glutamyl peptide in vivo, and is an enzyme that not only hydrolyzes γ-glutamyl peptide but also transfers the γ-glutamyl group to other peptides or amino acids. It is widely distributed in various tissues and is also present in serum. γ-GTP is known to vary significantly depending on pathological conditions, and serum γ-GTP
GTP shows markedly high levels in hepatostatic hepatitis, obstructive jaundice, primary liver cancer, metastatic liver cancer, etc.
In particular, inactive forms of chronic hepatitis show low levels, but active forms show high levels, and are generally specific to chronic liver diseases, making this diagnosis completely different from the various deviant enzymes that have been used clinically in the past. It has scientific significance, and measurement of serum γ-GTP activity has become one of the indispensable testing methods for diagnosing and understanding the pathology of such diseases.
従来、LAP活性の測定法は多数報告されてい
るが、それらの大部分は合成基質よりLAPによ
つて遊離されるアミン化合物を比色定量すること
によりLAP活性値を求める方法であつて、それ
に用いる合成基質としてL−ロイシル−p−ニト
ロアニリドを用い、LAPの酵素作用により生成
するp−ニトロアニリンの黄色を比色定量する方
法が挙げられるが、この比色定量の際、その合成
基質の呈色波長がオーバー・ラツプする欠点があ
り、また血清成分、特にビリルビン系色素による
測定の影響も免れることが出来ない欠点があつ
た。さらに、合成基質としてL−ロイシル−β−
ナフチルアミドを用いる方法が挙げられるが、こ
の方法では生成するβ−ナフチルアミンに5−ニ
トロ−2−アミノメトキシベンゼンジアゾテート
などをカツプリングさせて色素を形成させるかあ
るいは生成するβ−ナフチルアミンを亜硝酸ナト
リウムでジアゾ化し、N−(1−ナフチル)−エチ
レンジアミンにカツプリングさせるか、もしくは
p−ジメチルアミノベンズアルデヒドまたはp−
ジメチルアミノシンナムアルデヒドを縮合させて
色素を形成せしめ、次いでこれを比色定量する方
法であるが、反応過程が複雑で、かつ厳密な操作
を必要とするため、検査法として尚不便であるば
かりでなく、標準物質たるβ−ナフチルアミンの
毒性が著しく、近年膀胱の腫瘍や癌を発生するこ
とが明らかとなつたため、発癌物質として、その
使用に特に厳密な注意を用するなどの欠点があつ
た。 Conventionally, many methods for measuring LAP activity have been reported, but most of them are methods for determining LAP activity values by colorimetrically quantifying amine compounds liberated by LAP from synthetic substrates; One method is to colorimetrically quantify the yellow color of p-nitroaniline produced by the enzymatic action of LAP using L-leucyl-p-nitroanilide as the synthetic substrate. It has the disadvantage that the coloring wavelengths overlap, and it also has the disadvantage that measurement cannot be avoided by the influence of serum components, especially bilirubin dyes. Furthermore, L-leucyl-β-
A method using naphthylamide is mentioned, but in this method, the generated β-naphthylamine is coupled with 5-nitro-2-aminomethoxybenzenediazotate to form a dye, or the generated β-naphthylamine is combined with sodium nitrite. or p-dimethylaminobenzaldehyde or p-
This method involves condensing dimethylaminocinnamaldehyde to form a dye, which is then colorimetrically quantified, but the reaction process is complicated and requires strict operations, making it inconvenient as a testing method. However, the toxicity of β-naphthylamine, which is a standard substance, is extremely toxic, and it has recently become clear that it causes bladder tumors and cancer, so it has the drawback of requiring particularly strict precautions when using it as a carcinogen.
一方、γ−GTP活性の測定法も多数報告され
ているが、それらの大部分は合成基質よりγ−
GTPによつて遊離されるアミン化合物を比色定
量することによりγ−GTP活性値を求める方法
であつて、それを用いる合成基質としてγ−L−
グルタミル−p−ニトロアニリドを用い、γ−
GTPの酵素作用により生成するp−ニトロアニ
リンの黄色を410nmで比色定量する方法が挙げ
られるが、血清成分、特にビリルビン系色素など
の影響を避けるため、各検体に対して検体ブラン
クを厳密に測定しなければならず、正確な測定値
を得ることが困難であるという欠点があつた。ま
た生成するp−ニトロアニリンをp−ジメチルア
ミノシンナムアルデヒド、p−ジメチルアミノベ
ンズアルデヒドなどのアルデヒド化合物と縮合さ
せて長波長測の赤色で測定する方法も挙げられる
が、発色感度に対する温度の影響が大きく、測定
値の再現性に問題があつた。また生成するp−ニ
トロアニリンをジアゾ化し、3,5−キシレノー
ルと縮合させて生ずる赤色を測定する方法が提案
されたが反応操作段階が多く簡便性に欠けるとい
う欠点があつた。さらに合成基質としてγ−L−
グルタミル−β−ナフチルアミドを用いる方法が
挙げられるが、これらの方法としてγ−GTPに
より遊離したβ−ナフチルアミンをジアゾニウム
塩として比色定量する方法、3−メチル−2−ベ
ンゾチアゾリノンヒドラゾンと酸化剤で発色させ
て比色定量する方法、または前記アルデヒド化合
物を発色剤として縮合させて比色定量する方法が
あるが、前記と同様β−ナフチルアミンの発癌性
が一般に指摘されているところであり、操作が煩
雑で測定に長時間を要するなどの点で実用性に欠
けている。 On the other hand, many methods for measuring γ-GTP activity have been reported, but most of them use γ-GTP rather than synthetic substrates.
A method for determining the γ-GTP activity value by colorimetrically quantifying amine compounds liberated by GTP.
Using glutamyl-p-nitroanilide, γ-
One method is to colorimetrically quantify the yellow color of p-nitroaniline produced by the enzymatic action of GTP at 410 nm, but in order to avoid the influence of serum components, especially bilirubin pigments, it is necessary to strictly prepare a sample blank for each sample. The drawback was that it was difficult to obtain accurate measurements. Another method is to condense the produced p-nitroaniline with an aldehyde compound such as p-dimethylaminocinnamaldehyde or p-dimethylaminobenzaldehyde and measure the long wavelength red color, but the temperature has a large effect on the color sensitivity. There were problems with the reproducibility of measured values. A method has also been proposed in which the resulting p-nitroaniline is diazotized and condensed with 3,5-xylenol to measure the resulting red color, but this method has the drawback of requiring many reaction steps and lacking in simplicity. Furthermore, γ-L-
Examples include methods using glutamyl-β-naphthylamide, including colorimetric determination of β-naphthylamine liberated by γ-GTP as a diazonium salt, and oxidation with 3-methyl-2-benzothiazolinone hydrazone. There is a method of colorimetric determination by developing a color with a coloring agent, or a method of colorimetric determination by condensing the aldehyde compound as a coloring agent, but as mentioned above, the carcinogenicity of β-naphthylamine is generally pointed out, and the procedure is difficult. It lacks practicality in that it is complicated and takes a long time to measure.
本発明者らは、かかる欠点を有する従来の
LAPやγ−GTP活性測定法を改良すべく鋭意研
究を続けた結果、LAPやγ−GTP活性測定に優
れた性質を有する新規な合成基質を見い出し、本
発明を完成したものである。 The present inventors have discovered that the conventional
As a result of intensive research aimed at improving methods for measuring LAP and γ-GTP activities, we discovered a new synthetic substrate with excellent properties for measuring LAP and γ-GTP activities, and completed the present invention.
本発明は、被験液に含有されているLAPおよ
びγ−GTPからなる群より選ばれるペプチダー
ゼの活性測定において、式
(式中、R、XおよびYは前記と同じ意味を有す
る)で表わされるアミド化合物またはその塩に、
被験液中に含有されているペプチダーゼを作用さ
せて、式
(式中、XおよびYは前記と同じ意味を有する)
で表わされるアニリン誘導体を遊離せしめ、次い
で該アニリン誘導体を化学的手段により発色させ
て生成する発色化合物を比色定量することを特徴
とする新規な合成基質を用いるLAPおよびγ−
GTPからなる群より選ばれるペプチダーゼの活
性測定法である。 The present invention provides a method for measuring the activity of a peptidase selected from the group consisting of LAP and γ-GTP contained in a test solution. (wherein R, X and Y have the same meanings as above) or a salt thereof,
By allowing the peptidase contained in the test solution to act, the formula (In the formula, X and Y have the same meanings as above)
LAP and gamma-
This is a method for measuring the activity of peptidase selected from the group consisting of GTP.
本発明においてペプチダーゼの合成基質として
用いられるアミド化合物〔〕は、ペプチド合成
の常法によつて製造することができる。即ち、L
−ロイシンのカルボキシル基やL−グルタミル酸
のγ−カルボキシル基とアニリン誘導体〔〕と
の縮合反応により得られる。 The amide compound [ ] used as a synthetic substrate for peptidase in the present invention can be produced by a conventional method for peptide synthesis. That is, L
- Obtained by a condensation reaction between the carboxyl group of leucine or the γ-carboxyl group of L-glutamic acid and an aniline derivative [].
上記の縮合反応に際しては、予め反応に関与し
てはならない官能基、即ちL−ロイシンのα−ア
ミノ基やL−グルタミン酸のα−アミノ基および
α−カルボキシル基を保護するのがよい。α−ア
ミノ基の保護基としては通常ペプチド合成に用い
られるα−アミノ保護基が用いられる。例えばt
−ブチルオキシカルボニル、t−アミノオキシカ
ルボニル、ベンジルオキシカルボニル、p−ニト
ロベンジルオキシカルボニル、アダマンタチルオ
キシカルボニル、p−メトキシベンジルオキシカ
ルボニル、フタロイル、o−ニトロフエニルチオ
基などが挙げられる。L−グルタミン酸のα−カ
ルボキシル基はメチルエステル、エチルエステ
ル、t−ブチルエステル、ベンジルエステル、p
−ニトロベンジルエステル、p−メトキシベンジ
ルエステルなどで保護するのが好ましいが、脱離
の際α−アミノ基の保護基と共に一段階で脱離さ
れる条件で脱離されるような保護基で保護するの
が特に好ましい。例えばα−アミノ基をベンジル
オキシカルボニル基、α−カルボキシル基をベン
ジルエステルで保護するのがその一例である。 In the above condensation reaction, it is preferable to protect in advance functional groups that should not participate in the reaction, ie, the α-amino group of L-leucine and the α-amino group and α-carboxyl group of L-glutamic acid. As the protecting group for the α-amino group, an α-amino protecting group commonly used in peptide synthesis is used. For example, t
Examples include -butyloxycarbonyl, t-aminooxycarbonyl, benzyloxycarbonyl, p-nitrobenzyloxycarbonyl, adamantatyloxycarbonyl, p-methoxybenzyloxycarbonyl, phthaloyl, and o-nitrophenylthio groups. The α-carboxyl group of L-glutamic acid is methyl ester, ethyl ester, t-butyl ester, benzyl ester, p
- It is preferable to protect with nitrobenzyl ester, p-methoxybenzyl ester, etc., but it is preferable to protect with a protecting group that can be removed in one step together with the protecting group of the α-amino group. is particularly preferred. For example, an α-amino group is protected with a benzyloxycarbonyl group, and an α-carboxyl group is protected with a benzyl ester.
上記縮合反応に用いるアニリン誘導体〔〕の
例としては、3,5−ジブロモ−4−ヒドロキシ
アニリン、3,6−ジクロロ−4−ヒドロキシア
ニリン、3−クロロ−5−ブロモ−4−ヒドロキ
シアニリンなどが挙げられる。 Examples of the aniline derivative [] used in the above condensation reaction include 3,5-dibromo-4-hydroxyaniline, 3,6-dichloro-4-hydroxyaniline, and 3-chloro-5-bromo-4-hydroxyaniline. Can be mentioned.
上記の縮合反応は、α−アミノ基が保護された
L−ロイシンのカルボキシル基またはα−アミノ
基およびα−カルボキシル基が保護されたL−グ
ルタミン酸のγ−カルボキシル基を酸ハライド、
酸無水物、酸アジド、酸イミダゾリド、活性エス
テル、例えばシアノメチルエステル、p−ニトロ
フエニルエステル、2,4−ジニトロフエニルエ
ステル、N−ヒドロキシスクシンイミドエステ
ル、N−ヒドロキシフタルイミドエステルなどの
活性化アシル誘導体に変換してアニリン誘導体
〔〕と反応させるか、あるいはカルボジイミド、
例えばウオーターソルブ・カルボジイミド塩酸塩
〔1−エチル−3−(3−ジメチルアミノプロピ
ル)カルボジイミド塩酸塩、N,N′−ジシクロ
ヘキシルカルボジイミド、N,N′−カルボニル
イミダゾール、イソオキサゾリウム塩、例えばウ
ツドワード試薬などの縮合剤の存在下上記の保護
されたL−ロイシンまたはL−グルタミン酸とア
ニリン誘導体〔〕を反応させることにより行わ
れる。 In the above condensation reaction, the carboxyl group of L-leucine with an α-amino group protected or the γ-carboxyl group of L-glutamic acid with an α-amino group and an α-carboxyl group protected with an acid halide,
Acid anhydrides, acid azides, acid imidazolides, activated esters, such as activated acyls such as cyanomethyl ester, p-nitrophenyl ester, 2,4-dinitrophenyl ester, N-hydroxysuccinimide ester, N-hydroxyphthalimide ester, etc. Either convert it into a derivative and react it with an aniline derivative [ ] or carbodiimide,
For example, watersolve carbodiimide hydrochloride [1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, N,N'-dicyclohexylcarbodiimide, N,N'-carbonylimidazole, isoxazolium salts, e.g. Woodward's reagent This is carried out by reacting the above-mentioned protected L-leucine or L-glutamic acid with the aniline derivative [ ] in the presence of a condensing agent such as .
上記の縮合反応においては、通常不活性有機溶
媒、例えばジメチルホルムアミド、ジメチルアセ
トアミド、ジメチルスルホキシド、テトラヒドロ
フラン、ジオキサン、ベンゼン、クロロホルム、
ジクロロメタン、ジクロロエタンなどの溶媒中、
両者ほぼ等量を加え、室温またはそれ以下の温度
で反応させることにより行われる。上記の反応経
過はシリカゲルなどの薄層クロマトグラフイー
(TLC)、高速液体クロマトグラフイー(HPLC)
などにより追跡できるので、出発物質のいずれか
の消失を持つて適宜反応を終了すればよい。 In the above condensation reaction, inert organic solvents such as dimethylformamide, dimethylacetamide, dimethylsulfoxide, tetrahydrofuran, dioxane, benzene, chloroform,
In solvents such as dichloromethane and dichloroethane,
This is carried out by adding approximately equal amounts of both and reacting at room temperature or lower temperature. The above reaction process can be performed using thin layer chromatography (TLC) such as silica gel or high performance liquid chromatography (HPLC).
The reaction can be tracked by such methods, so the reaction can be appropriately terminated when any of the starting materials disappear.
このようにして得られた反応生成物は、反応溶
媒を留去するかまたは留去することなく、非親水
性有機溶媒、例えばクロロホルム、ジクロロメタ
ン、酢酸エチル、酢酸ブチル、メチルイソブチル
ケトン、ベンゼン、ジエチルエーテルなどに溶か
し、酸性水およびアルカリ性水で洗浄した後、溶
媒を留去することにより採取できる。さらに精製
する必要がある場合には、適当な再結溶媒で再結
晶化するか、あるいはシリカゲル、活性アルミ
ナ、吸着樹脂などの吸着剤を用いるカラムクロマ
トグラフイーにより精製することができる。 The reaction product thus obtained can be treated with non-hydrophilic organic solvents, such as chloroform, dichloromethane, ethyl acetate, butyl acetate, methyl isobutyl ketone, benzene, diethyl, with or without distilling off the reaction solvent. It can be collected by dissolving it in ether etc., washing it with acidic water and alkaline water, and then distilling off the solvent. If further purification is required, it can be purified by recrystallization with a suitable recrystallization solvent or by column chromatography using an adsorbent such as silica gel, activated alumina, or adsorption resin.
次いで得られた反応生成物の保護基を脱離する
のであるが、この脱離化はペプチド化学における
保護基の脱離化法を用いて行われる。例えばα−
アミノ保護基がt−ブチルオキシカルボニル基で
ある場合には、2N塩化水素の酢酸溶液、トリフ
ルオロ酢酸、ギ酸などを用いる方法、ベンジルオ
キシカルボニル基である場合には、パラジウム−
炭素触媒を用いる接触還元による方法、臭化水素
酸の酢酸溶液を用いる方法により行えばよい。L
−グルタミン酸のα−カルボキシル基の保護基が
ベンジルエステルである場合には、パラジウム−
炭素触媒を用いる触媒還元による方法で行えばよ
い。 Next, the protective group of the resulting reaction product is removed, and this removal is carried out using a protective group removal method in peptide chemistry. For example α−
When the amino protecting group is a t-butyloxycarbonyl group, a method using a 2N hydrogen chloride acetic acid solution, trifluoroacetic acid, formic acid, etc. is used, and when the amino protecting group is a benzyloxycarbonyl group, a method using palladium-
This may be carried out by a method using a catalytic reduction using a carbon catalyst or a method using an acetic acid solution of hydrobromic acid. L
- When the protecting group for the α-carboxyl group of glutamic acid is benzyl ester, palladium-
It may be carried out by a method of catalytic reduction using a carbon catalyst.
このようにして得られたアミド化合物〔〕を
反応液から採取するには、先ず保護基の脱離化が
酸分解による場合には、中和し、接触還元による
場合には触媒を除去した後、非親水性有機溶媒、
例えばクロロホルム、ジクロロメタン、ジクロロ
エタン、酢酸エチル、酢酸ブチル、メチルイソブ
チルケトン、ベンゼン、ジエチルエーテルなどの
溶媒中、酸性水およびアルカリ性水で洗浄した
後、溶媒を留去することにより採取できる。さら
に精製する必要がある場合には、適当な再結溶媒
で再結晶化するか、あるいはシリカゲル、活性ア
ルミナ、吸着樹脂などの吸着剤を用いるカラムク
ロマトグラフイーにより精製することができる。 In order to collect the amide compound [] obtained in this way from the reaction solution, first, if the protecting group is removed by acid decomposition, it is neutralized, and if it is by catalytic reduction, the catalyst is removed. , non-hydrophilic organic solvent,
For example, it can be collected by washing with acidic water and alkaline water in a solvent such as chloroform, dichloromethane, dichloroethane, ethyl acetate, butyl acetate, methyl isobutyl ketone, benzene, or diethyl ether, and then distilling off the solvent. If further purification is required, it can be purified by recrystallization with a suitable recrystallization solvent or by column chromatography using an adsorbent such as silica gel, activated alumina, or adsorption resin.
本アミド化合物〔〕は必要に応じ、塩酸、硫
酸、硝酸、リン酸などの無機酸との塩、ギ酸、酢
酸、プロピオン酸、リンゴ酸、クエン酸、酒石
酸、シユウ酸などの有機酸との塩を形成すること
ができる。 This amide compound [ ] may be used as a salt with an inorganic acid such as hydrochloric acid, sulfuric acid, nitric acid, or phosphoric acid, or a salt with an organic acid such as formic acid, acetic acid, propionic acid, malic acid, citric acid, tartaric acid, or oxalic acid. can be formed.
次に、本アミド化合物〔〕を用いるペプチダ
ーゼ活性測定法について述べる。本測定法におい
ては、上記アミド化合物〔〕またはその塩に被
験液に含有されているLAP−およびγ−GTPか
らなる群より選ばれるペプチダーゼの作用により
遊離されるアニリン誘導体〔〕を定量するに当
り、化学的発色法を用いて発色化合物に導き、比
色定量するものである。 Next, a method for measuring peptidase activity using the present amide compound [] will be described. In this measurement method, the aniline derivative [] released by the action of a peptidase selected from the group consisting of LAP- and γ-GTP contained in the above-mentioned amide compound [] or its salt in the test solution is determined. , a chemical coloring method is used to derive a coloring compound and perform colorimetric determination.
上記の化学的発色法としては、カプラー共存下
でアニリン誘導体〔〕と酸化縮合させて発色化
合物に導き比色定量するのが好ましい。使用する
カプラーとしては、アニリン誘導体〔〕と酸化
縮合して発色化合物を形成する芳香族化合物であ
れば、どのような化合物でもよいが、これらのう
ち代表的な芳香族化合物としてはフエノール系化
合物、アミノフエノール系化合物、アニリン系化
合物またはナフトール系化合物が挙げられる。フ
エノール系化合物の例としては、フエノール、サ
リチル酸、m−ヒドロキシ安息香酸、p−ヒドロ
キシ安息香酸、2,6−ジヒドロキシ安息香酸、
サリチル酸メチル、o−クレゾール、m−クレゾ
ール、p−クレゾール、o−エチルフエノール、
m−エチルフエノール、2,3−キシレノール、
2,4−キシレノール、2,5−キシレノール
3,5−キシレノール、2,6−キシレノール、
o−メトキシフエノール、m−メトキシフエノー
ル、p−メトキシフエノール、2,6−ジメトキ
シフエノール、o−クロロフエノール、m−クロ
ロフエノール、p−クロロフエノール、2,4−
ジクロロフエノール、2,6−ジクロロフエノー
ル、o−ブロモフエノール、m−ブロモフエノー
ル、p−ブロモフエノール、2,4−ジブロモフ
エノール、2,6−ジブロモフエノール、2−メ
チル−6−クロロフエノール、2−クロロ−5−
メチルフエノール、o−カルボキシメチルフエノ
ール、2−ヒドロキシ−4−アミノエチルフエノ
ールなどが挙げられ、アミノフエノール系化合物
の例としては、4−クロロ−2−アミノフエノー
ル、N,N−ジエチル−m−アミノフエノール、
4−メチル−2−アミノフエノール、5−アミノ
−2−ヒドロキシ安息香酸、2−アミノ−3−ヒ
ドロキシ安息香酸、o−アミノフエノール、m−
アミノフエノール、p−アミノフエノールなどが
挙げられ、アニリン系化合物の例としては、アニ
リン、o−トルイジン、m−トルイジン、p−ト
ルイジン、N−メチルアニリン、N−エチルアニ
リン、N,N−ジメチルアニリン、N,N−ジエ
チルアニリン、N,N−ジメチル−o−トルイジ
ン、N,N−ジメチル−p−トルイジン、N,N
−ジエチル−o−トルイジン、N,N−ジメチル
−m−トルイジン、N,N−ジエチル−p−トル
イジン、o−クロロアニリン、m−クロロアニリ
ン、m−ブロモアニリン、アントラニル酸、3−
アミノ安息香酸、p−ジメチルアミノ安息香酸、
p−クロロ−o−トルイジン、3−アミノ−4−
メチル安息香酸、m−フエニレンジアミン、N,
N−ジメチル−m−フエニレンジアミン、2−メ
チル−m−フエニレンジアミン、4−メチル−o
−フエニレンジアミン、4−メチル−m−フエニ
レンジアミン、2−クロロ−m−フエニレンジア
ミン、3−クロロ−o−トルイジン、2−メトキ
シ−5−クロロアニリン、o−エチルアニリン、
2,5−ジエトキシアニリン、N−エチル−N−
ヒドロキシエチルアニリン、N−エチル−N−ヒ
ドロキシエチル−m−トルイジンなどが挙げら
れ、ナフトール系化合物の例としてはα−ナフト
ール、β−ナフトール、2−カルボキシ−1−ナ
フトール、4−クロロ−1−ナフトール、1−ヒ
ドロキシ−2−ナフトエ酸、1−ナフトール−2
−スルホン酸、1−ナフトール−3−スルホン
酸、1−ナフトール−4−スルホン酸、2−ナフ
トール−6−スルホン酸、2−ナフトール−3,
6−ジスルホン酸などが挙げられる。 As for the above-mentioned chemical coloring method, it is preferable to carry out oxidative condensation with the aniline derivative [ ] in the presence of a coupler to form a coloring compound, which is then subjected to colorimetric determination. The coupler to be used may be any aromatic compound as long as it forms a color-forming compound through oxidative condensation with the aniline derivative [ ], but typical aromatic compounds include phenolic compounds, Examples include aminophenol compounds, aniline compounds, and naphthol compounds. Examples of phenolic compounds include phenol, salicylic acid, m-hydroxybenzoic acid, p-hydroxybenzoic acid, 2,6-dihydroxybenzoic acid,
Methyl salicylate, o-cresol, m-cresol, p-cresol, o-ethylphenol,
m-ethylphenol, 2,3-xylenol,
2,4-xylenol, 2,5-xylenol, 3,5-xylenol, 2,6-xylenol,
o-methoxyphenol, m-methoxyphenol, p-methoxyphenol, 2,6-dimethoxyphenol, o-chlorophenol, m-chlorophenol, p-chlorophenol, 2,4-
Dichlorophenol, 2,6-dichlorophenol, o-bromophenol, m-bromophenol, p-bromophenol, 2,4-dibromophenol, 2,6-dibromophenol, 2-methyl-6-chlorophenol, 2- Chloro-5-
Examples of aminophenol compounds include methylphenol, o-carboxymethylphenol, 2-hydroxy-4-aminoethylphenol, and examples of aminophenol compounds include 4-chloro-2-aminophenol, N,N-diethyl-m-amino phenol,
4-methyl-2-aminophenol, 5-amino-2-hydroxybenzoic acid, 2-amino-3-hydroxybenzoic acid, o-aminophenol, m-
Examples of aniline compounds include aniline, o-toluidine, m-toluidine, p-toluidine, N-methylaniline, N-ethylaniline, N,N-dimethylaniline. , N,N-diethylaniline, N,N-dimethyl-o-toluidine, N,N-dimethyl-p-toluidine, N,N
-diethyl-o-toluidine, N,N-dimethyl-m-toluidine, N,N-diethyl-p-toluidine, o-chloroaniline, m-chloroaniline, m-bromoaniline, anthranilic acid, 3-
Aminobenzoic acid, p-dimethylaminobenzoic acid,
p-chloro-o-toluidine, 3-amino-4-
Methylbenzoic acid, m-phenylenediamine, N,
N-dimethyl-m-phenylenediamine, 2-methyl-m-phenylenediamine, 4-methyl-o
-phenylenediamine, 4-methyl-m-phenylenediamine, 2-chloro-m-phenylenediamine, 3-chloro-o-toluidine, 2-methoxy-5-chloroaniline, o-ethylaniline,
2,5-diethoxyaniline, N-ethyl-N-
Examples include hydroxyethylaniline, N-ethyl-N-hydroxyethyl-m-toluidine, and examples of naphthol compounds include α-naphthol, β-naphthol, 2-carboxy-1-naphthol, 4-chloro-1- naphthol, 1-hydroxy-2-naphthoic acid, 1-naphthol-2
-sulfonic acid, 1-naphthol-3-sulfonic acid, 1-naphthol-4-sulfonic acid, 2-naphthol-6-sulfonic acid, 2-naphthol-3,
Examples include 6-disulfonic acid.
上記の酸化縮合はPHを約4〜12の範囲内で反応
を行わせることにより発色反応は定量的に完結す
る。上記PHの維持のために緩衝液またはアルカリ
水溶液としては炭酸塩緩衝液、リン酸塩緩衝液、
ホウ酸塩緩衝液、水酸化アルカリ水溶液などが有
効に使用できる。 In the above oxidative condensation, the coloring reaction is quantitatively completed by carrying out the reaction at a pH within the range of about 4 to 12. In order to maintain the above pH, carbonate buffer, phosphate buffer,
Borate buffers, aqueous alkaline hydroxide solutions, etc. can be effectively used.
上記の酸化縮合させるためにはアニリン誘導体
〔〕とカプラーを酸化縮合することのできる酸
化剤の存在下で行われる。このような酸化剤とし
ては、通常過ヨウ素酸塩、クロラミンT、次亜塩
素酸塩などのハロゲン系酸化剤もしくは過硫酸塩
などの過酸化物系酸化剤、過酸化水素などが挙げ
られるが、メタ過ヨウ素酸ナトリウムは好適な一
例である。 The above oxidative condensation is carried out in the presence of an oxidizing agent capable of oxidizing and condensing the aniline derivative [] and the coupler. Such oxidizing agents usually include halogen-based oxidizing agents such as periodate, chloramine T, hypochlorite, peroxide-based oxidizing agents such as persulfate, hydrogen peroxide, etc. Sodium metaperiodate is a suitable example.
アニリン誘導体〔〕と上記カプラーとの酸化
縮合により生成する発色化合物は、カプラーの種
類により極大吸収波長が約550〜770nmに巾広く
分布するが、通常は殆んど570〜680nmに極大吸
収を有する有色系色素であり、呈色も極めて高感
度で安定性も良く、しかも温度による変動も殆ん
どなく、生体試験料中のビリルビンなどの挾雑物
による影響も受けにくいため、正の誤差も殆んど
受けることがないので、LAPやγ−GTPのペプ
チダーゼ活性測定に極めて好結果を与える。 The color-forming compound produced by the oxidative condensation of the aniline derivative [] and the above coupler has a maximum absorption wavelength widely distributed in the range of about 550 to 770 nm depending on the type of coupler, but usually has maximum absorption in the range of 570 to 680 nm. It is a colored pigment, and its color development is extremely sensitive and stable, and there is almost no fluctuation due to temperature, and it is not easily affected by contaminants such as bilirubin in biological test materials, so there is no positive error. Since it is rarely affected, it gives very good results in measuring the peptidase activity of LAP and γ-GTP.
上記のアニリン誘導体〔〕を呈色定量する他
の方法としては、ペンタシアノ鉄錯塩を過酸化物
で処理して呈色液を調製して発色させる方法が用
いられる。上記の過酸化物としては過ヨウ素酸ナ
トリウム塩、過ヨウ素酸カリウム塩、過マンガン
酸カリウム、過酸化水素などが挙げられるが、過
酸化水素は最も好適な一例である。上記鉄錯塩は
重炭酸塩、例えば重炭酸ナトリウム、重炭酸リチ
ウム、重炭酸カリウムなどと低分子デキストラン
を配合するとさらに安定な鉄錯塩試薬が得られ
る。また、これらの配合試薬は凍結乾燥した場合
は、鉄錯塩が非常に安定化され、呈色用反応試薬
としてキツト化する場合は凍結乾燥試薬とするこ
とが望ましい。 Another method for coloring and quantifying the above-mentioned aniline derivatives is a method in which a pentacyanoiron complex salt is treated with a peroxide to prepare a coloring solution to develop color. Examples of the above-mentioned peroxides include sodium periodate, potassium periodate, potassium permanganate, and hydrogen peroxide, with hydrogen peroxide being the most preferred example. When the above-mentioned iron complex salt is mixed with a bicarbonate such as sodium bicarbonate, lithium bicarbonate, potassium bicarbonate, etc., and low molecular weight dextran, a more stable iron complex reagent can be obtained. Furthermore, when these compounded reagents are freeze-dried, the iron complex salt is extremely stabilized, and when they are made into a kit as a coloring reaction reagent, it is desirable to use freeze-dried reagents.
上記の鉄錯塩を用いる呈色反応を行う場合に
は、酸性側で反応を進行する方が好ましい。使用
し得るPHとしては3〜7であるのが好ましく、4
〜5.5が最も好ましい。上記のPHを維持するため
に通常弱酸性の緩衝液が用いられるが、特に乳酸
塩、クエン酸塩、シユウ酸塩などの緩衝液が好ま
しい。緩衝液の濃度は0.01〜1Mの範囲内で使用
し得るが、0.05〜0.4Mの範囲の濃度が特に好ま
しい。 When carrying out a color reaction using the above-mentioned iron complex salt, it is preferable to proceed with the reaction on the acidic side. The pH that can be used is preferably 3 to 7, and 4
~5.5 is most preferred. A weakly acidic buffer is usually used to maintain the above pH, and lactate, citrate, and oxalate buffers are particularly preferred. Buffer concentrations may be used in the range 0.01-1M, but concentrations in the range 0.05-0.4M are particularly preferred.
上記の鉄錯塩とアニリン誘導体〔〕との反応
によつて形成される発色化合物は極大吸収波長が
700nm付近に分布し、色調が安定なためLAPや
γ−GTPのペプチダ−ゼ活性側定に適している。 The color-forming compound formed by the reaction between the above iron complex salt and the aniline derivative [] has a maximum absorption wavelength.
It is distributed around 700 nm and has a stable color tone, making it suitable for measuring the peptidase activity of LAP and γ-GTP.
さらにナトリウムペンタシアノアコフエリアー
トNa2〔Fe(CN)5・H2O〕を遊離したアニリン誘
導体〔〕と反応させて発色化合物を形成せし
め、その発色化合物の量を比色定量してLAPや
γ−GTPのペプチダ−ゼ活性側を行なつてもよ
い。 Furthermore, sodium pentacyanoacopheliato Na 2 [Fe(CN) 5 H 2 O] is reacted with the liberated aniline derivative [ ] to form a color-forming compound, and the amount of the color-forming compound is determined colorimetrically. The peptidase activity side of γ-GTP may also be performed.
さらにまた、芳香族アミンを発色定量する方
法、例えばジアゾカツプリング法、アルデヒド系
化合物を反応させて生成するシツク塩基を定量す
る方法など公知の種々の化学的発色法を適用して
アニリン誘導体〔〕を発色定量することにより
LAPやγ−GTPのペプチダ−ゼ活性測定を行つ
てもよい。 Furthermore, aniline derivatives can be obtained by applying various known chemical coloring methods, such as methods for colorimetrically quantifying aromatic amines, such as diazo coupling methods and methods for quantifying thick bases produced by reacting aldehyde compounds. By colorimetrically quantifying
LAP or γ-GTP peptidase activity may also be measured.
次に、LAPの活性測定について更に詳しく説
明する。LAPの活性測定を行うに当つては、先
ずLAPの合成基質であるL−ロイシル−3,5
−ジハロゲノ−4−ヒドロキシアニリドに被験液
中のLAPを酵素反応させてアニリン誘導体〔〕
を遊離させる。被験液としては血清を0.01〜5ml
の範囲内で用いられる。上記酵素反応は通常37℃
付近で5分以上反応させればよい。この反応の至
適PHは6.5〜8.0の範囲にあるから、この範囲内で
適宜PHを設定すればよい。PHを保持するための緩
衝液としては、リン酸塩、バルビタール、ホウ酸
塩、トリスヒドロキシメチルアミノメタンなどの
緩衝液が用いられる。 Next, the measurement of LAP activity will be explained in more detail. To measure the activity of LAP, first, L-leucyl-3,5, which is a synthetic substrate of LAP,
-Aniline derivatives are produced by enzymatically reacting LAP in the test solution with dihalogeno-4-hydroxyanilide []
release. 0.01 to 5 ml of serum as the test solution
Used within the scope of. The above enzyme reaction is usually carried out at 37°C.
It is sufficient to allow the reaction to occur nearby for 5 minutes or more. Since the optimum pH for this reaction is in the range of 6.5 to 8.0, the pH may be appropriately set within this range. As a buffer for maintaining pH, a buffer such as phosphate, barbital, borate, trishydroxymethylaminomethane, etc. is used.
生成したアニリン誘導体〔〕を定量するに当
つては、p−キシレノールなどのカプラー共存下
でアルカリの条件下メタ過ヨウ素酸ナトリウムな
どの酸化剤で酸化縮合させて発色化合物を形成し
て比色定量してもよく、また過酸化水素などの酸
化剤でペンタシアノアミノフエロエートを酸化し
て得た呈色試薬を用いて発色させ、比色定量して
もよい。 To quantify the generated aniline derivative [], perform oxidative condensation with an oxidizing agent such as sodium metaperiodate under alkaline conditions in the presence of a coupler such as p-xylenol to form a color-forming compound, and perform colorimetric determination. Alternatively, a color reagent obtained by oxidizing pentacyanoaminoferroate with an oxidizing agent such as hydrogen peroxide may be used to develop a color, and colorimetric determination may be performed.
次にγ−GTPの活性測定について更に詳しく
説明する。γ−GTPの活性測定を行うに当つて
は、先ずγ−GTPの合成基質であるγ−L−グ
ルタミル−3,5−ジハロゲノ−4−ヒドロキシ
アニリドに被検液中のγ−GTPを酵素反応させ
てアニリン誘導体〔〕を遊離させる。被験液と
しては血清を0.01〜5mlの範囲内で用いられる。
上記酵素反応は通常37℃付近で5分以上反応させ
ればよい。この反応の至適PHは7.3〜9.0の範囲に
あるから、この範囲内で適宜PHを設定すればよ
い。反応に際しては受容体としてアミノ酸やペプ
チド、例えばグリシルグリシンの適当量を含むPH
7.5〜9.0の緩衝液中で反応させ、試料中のγ−
GTP活性に比例して生成するアニリン誘導体
〔〕を定量すればよい。PHを保持するための緩
衝液としては、リン酸塩、バルビタール、ホウ酸
塩、炭酸塩、トリエタノールアミン、グリシン、
トリスヒドロキシメチルアミノメタンなどの緩衝
液が用いられる。 Next, the measurement of γ-GTP activity will be explained in more detail. To measure the activity of γ-GTP, first, γ-GTP in the test solution is subjected to an enzymatic reaction with γ-L-glutamyl-3,5-dihalogeno-4-hydroxyanilide, which is a synthetic substrate for γ-GTP. to liberate the aniline derivative []. Serum is used as the test solution in a range of 0.01 to 5 ml.
The above enzymatic reaction may normally be carried out at around 37°C for 5 minutes or more. Since the optimum pH for this reaction is in the range of 7.3 to 9.0, the pH may be appropriately set within this range. During the reaction, a PH containing an appropriate amount of amino acids or peptides, such as glycylglycine, is used as a receptor.
The reaction is carried out in a buffer solution of 7.5 to 9.0, and the γ-
The aniline derivative produced in proportion to GTP activity may be quantified. Buffers to maintain pH include phosphate, barbital, borate, carbonate, triethanolamine, glycine,
A buffer such as trishydroxymethylaminomethane is used.
生成したアニリン誘導体〔〕を定量するに当
つては、LAPの活性測定におけると同様な化学
的発色法を用いて発色化合物に導き、比色定量す
ればよい。 In order to quantify the produced aniline derivative [], it is sufficient to use a chemical coloring method similar to that used in measuring LAP activity to derive a colored compound and perform colorimetric determination.
前記したように本発明の合成基質を用いる測定
法は、各反応工程が簡便なため、速やかに且つ正
確なLAP、γ−GTPのペプチダ−ゼ活性値を測
定することができるばかりでなく、生成する発色
化合物が通常500〜750nm附近に極大吸収を有す
るため、生成試料中の夾雑物による影響を受けに
くく、ペプチダ−ゼ活性測定において精度の高い
方法である。 As mentioned above, the measurement method using the synthetic substrate of the present invention is not only capable of quickly and accurately measuring LAP and γ-GTP peptidase activity values because each reaction step is simple, but also allows for the rapid and accurate measurement of LAP and γ-GTP peptidase activity values. Since the color-forming compound usually has a maximum absorption in the vicinity of 500 to 750 nm, it is not easily affected by contaminants in the product sample and is a highly accurate method for measuring peptidase activity.
次に実施例および参考例を挙げて本発明を具体
的に説明するが、これにより何ら本発明を限定す
るものではない。 Next, the present invention will be specifically explained with reference to Examples and Reference Examples, but the present invention is not limited thereto in any way.
尚、実施例および参考例中に記載の略記号は次
の意味を有する。 In addition, the abbreviations described in Examples and Reference Examples have the following meanings.
Leu;L−ロイシル
r−Glu;γ−グルタミル
BOC;t−ブチルオキシカルボニル
OSu;N−ビドロキシスクシンイミドエステル
AcOH;酢酸
参考例 1
L−ロイシル−3,5−ジクロロ−4−ヒドロ
キシアニリド・塩酸塩
2,6−ジクロロ−4−アミノフエノール1.78
g(10ミリモル)および炭酸水素ナトリウム1.26
g(15ミリモル)を水25mlに溶かし、これに0〜
5℃に冷却しつつBOC−Leu−OSu3.28g(10ミ
リモル)のジオキサン(25ml)溶液を撹拌下滴し
た。室温で一夜撹拌した後、30℃以下でジオキサ
ンを減圧下留去した。残渣を酢酸エチル200mlに
溶かし、飽和炭酸水素ナトリウム水溶液、水、
1N塩酸、飽和食塩水の順で各50mlで3回づつ洗
浄した。酢酸エチル層を無水硫酸マグネシウムで
乾燥した後、減圧濃縮してBOC−L−ロイシル
−3,5−ジクロロ−4−ヒドロキシアニリド
2.96gを得た。次いで、これを2N−HCl/
AcOH15mlに溶かし、室温で2時間撹拌した後、
乾燥ジエチルエーテル100mlを加えて結晶化し、
乾燥エーテルで2回デカンテーシヨンした。得ら
れた結晶を減圧乾燥してL−ロイシル−3,5−
ジクロロ−4−ヒドロキシアニリド・塩酸塩を得
た。Leu; L-leucyl r-Glu; γ-glutamyl BOC; t-butyloxycarbonyl OSu; N-hydroxysuccinimide ester AcOH; Acetic acid reference example 1 L-leucyl-3,5-dichloro-4-hydroxyanilide hydrochloride 2,6-dichloro-4-aminophenol 1.78
g (10 mmol) and sodium bicarbonate 1.26
Dissolve g (15 mmol) in 25 ml of water and add 0 to
While cooling to 5° C., a solution of 3.28 g (10 mmol) of BOC-Leu-OSu in dioxane (25 ml) was added dropwise with stirring. After stirring overnight at room temperature, dioxane was distilled off under reduced pressure at a temperature below 30°C. Dissolve the residue in 200 ml of ethyl acetate, add saturated aqueous sodium bicarbonate solution, water,
It was washed three times with 50 ml each of 1N hydrochloric acid and saturated saline, in that order. After drying the ethyl acetate layer over anhydrous magnesium sulfate, it was concentrated under reduced pressure to obtain BOC-L-leucyl-3,5-dichloro-4-hydroxyanilide.
2.96g was obtained. Next, this was mixed with 2N-HCl/
After dissolving in 15 ml of AcOH and stirring at room temperature for 2 hours,
Add 100ml of dry diethyl ether to crystallize,
Decanted twice with dry ether. The obtained crystals were dried under reduced pressure to obtain L-leucyl-3,5-
Dichloro-4-hydroxyanilide hydrochloride was obtained.
収量 1.89g(収率57.7%)
分子式 C12H16N2O2Cl2・HCl
融点;127〜133℃(分解)
シリカゲル薄層クロマトグラフイ(TLC);Rf=
0.63〔n−ブタノール−酢酸−水(4:1:
1)〕
参考例 2
L−ロイシル−3,5−ジブロモ−4−ヒドロ
キシアニリド・塩酸塩
2,6−ジブロモ−4−アミノフエノール4.90
g(18.3ミリモル)および炭酸水素ナトリウム
1.68g(20ミリモル)を水70mlに溶かし、これに
0〜5℃に冷却しつつBOC−Leu−OSu6.01g
(18.3ミリモル)のジオキサン(70ml)溶液を撹
拌下滴下した。室温で一夜撹拌した後、30℃以下
でジオキサンを源圧下留去した。残渣を酢酸エチ
ル400mlに溶かし、飽和炭酸水素ナトリウム水溶
液、水、1N塩酸、飽和食塩水の順で各100mlで3
回づつ洗浄した。酢酸エチル層を無水硫酸マグネ
シウムで乾燥した後、減圧濃縮してBOC−L−
ロイシル−3,5−ジブロモ−4−ヒドロキシア
ニリド5.8gを得た。次いでこれを2N−HCl/
AcOH30mlに溶かし、室温で2時間撹拌した後、
乾燥エーテルを加えて結晶化させてL−ロイシル
3,5−ジブロモ−4−ヒドロキシアニリド・塩
酸塩を得た。Yield 1.89g (yield 57.7%) Molecular formula C 12 H 16 N 2 O 2 Cl 2・HCl Melting point: 127-133℃ (decomposition) Silica gel thin layer chromatography (TLC); Rf=
0.63 [n-butanol-acetic acid-water (4:1:
1)] Reference example 2 L-leucyl-3,5-dibromo-4-hydroxyanilide hydrochloride 2,6-dibromo-4-aminophenol 4.90
g (18.3 mmol) and sodium bicarbonate
Dissolve 1.68 g (20 mmol) in 70 ml of water, and add 6.01 g of BOC-Leu-OSu to this while cooling to 0-5℃.
A solution of (18.3 mmol) in dioxane (70 ml) was added dropwise with stirring. After stirring overnight at room temperature, dioxane was distilled off under source pressure below 30°C. Dissolve the residue in 400 ml of ethyl acetate, and add 100 ml each of saturated aqueous sodium bicarbonate solution, water, 1N hydrochloric acid, and saturated saline in that order.
Washed several times. After drying the ethyl acetate layer over anhydrous magnesium sulfate, it was concentrated under reduced pressure to obtain BOC-L-
5.8 g of leucyl-3,5-dibromo-4-hydroxyanilide was obtained. Next, this was mixed with 2N-HCl/
After dissolving in 30 ml of AcOH and stirring at room temperature for 2 hours,
Dry ether was added for crystallization to obtain L-leucyl 3,5-dibromo-4-hydroxyanilide hydrochloride.
収量 2.50g(収率32.8%)
分子式 C12H16N2O2Br2・HCl(416.54)
シリカゲル薄層クロマトグラフイ(TLC);Rf=
0.65〔n−ブタノール:酢酸:水=4:1:1〕
融点;131〜134℃(分解)
参考例 3
γ−L−グルタミル−3,5−ジクロロ−4−
ヒドロキシアニリド
N,N−フタロイル−L−グルタミン酸無水物
5.16g(20ミリモル)および4−アミノ−2,6
−ジクロロフエノール3.56g(20ミリモル)をジ
オキサン50mlに溶かし、60℃で2時間撹拌した。
ジオキサンを減圧下留去し、残渣にヒドラジン・
ヒドラート1.5mlのメタノール(50ml)溶液を加
え、室温で2日間放置した。メタノールを減圧下
留去し、残渣に水を加えた後、0.5N−HClでPH
3に調節して析出したγ−L−グルタミル−3,
5−ジクロロ−4−ヒドロキシアニリド3.96gを
得た。Yield 2.50g (yield 32.8%) Molecular formula C 12 H 16 N 2 O 2 Br 2・HCl (416.54) Silica gel thin layer chromatography (TLC); Rf=
0.65 [n-butanol:acetic acid:water=4:1:1] Melting point: 131-134°C (decomposition) Reference example 3 γ-L-glutamyl-3,5-dichloro-4-
Hydroxyanilide N,N-phthaloyl-L-glutamic anhydride
5.16 g (20 mmol) and 4-amino-2,6
- 3.56 g (20 mmol) of dichlorophenol was dissolved in 50 ml of dioxane and stirred at 60°C for 2 hours.
Dioxane was distilled off under reduced pressure, and the residue contained hydrazine and
A solution of 1.5 ml of hydrate in methanol (50 ml) was added, and the mixture was left at room temperature for 2 days. Methanol was distilled off under reduced pressure, water was added to the residue, and the pH was adjusted with 0.5N HCl.
γ-L-glutamyl-3, which was precipitated by adjusting the
3.96 g of 5-dichloro-4-hydroxyanilide was obtained.
収量 3.96g(収率64.5%)
分子式 C11H12N2O4Cl2
融点;214〜217℃(分解)
シリカゲルTLC;Rf=0.49(n−ブタノール:酢
酸:水=4:1:1)
参考例 4
γ−L−グルタミル3,5−ジブロモ−4−ヒ
ドロキシアニリド
N,N−フタロイル−L−グルタミン酸無水物
2.18g(8.4ミリモル)および4−アミノ−2,
6−ジブロモフエノール2.26g(8.4ミリモル)
をジオキサン20mlに溶かし、60℃で1.5時間撹拌
した。ジオキサンを減圧下留去し、残渣にヒドラ
ジン・ヒドラート0.7mlのメタノール(20ml)溶
液を加え、室温で2日間放置した。メタノールを
減圧下留去し、残渣に水を加えた後、0.5N−
HClでPH3に調節して析出したγ−L−グルタミ
ル−3,5−ジブロモ−4−ヒドロキシアニリド
2.13gを得た。Yield 3.96g (yield 64.5%) Molecular formula C 11 H 12 N 2 O 4 Cl 2 Melting point: 214-217℃ (decomposition) Silica gel TLC; Rf = 0.49 (n-butanol:acetic acid:water = 4:1:1) Reference example 4 γ-L-glutamyl 3,5-dibromo-4-hydroxyanilide N,N-phthaloyl-L-glutamic anhydride
2.18 g (8.4 mmol) and 4-amino-2,
6-dibromophenol 2.26g (8.4mmol)
was dissolved in 20 ml of dioxane and stirred at 60°C for 1.5 hours. Dioxane was distilled off under reduced pressure, and a solution of 0.7 ml of hydrazine hydrate in methanol (20 ml) was added to the residue, and the mixture was allowed to stand at room temperature for 2 days. After distilling off methanol under reduced pressure and adding water to the residue, 0.5N−
γ-L-glutamyl-3,5-dibromo-4-hydroxyanilide precipitated after adjusting the pH to 3 with HCl
2.13g was obtained.
収量 2.13g(収率64.0%)
分子式 C11H12N2O4Br2
融点;191〜194℃(分解)〔日本薬局方一般試験
法融点測定の項に準じて測定した結果は199〜
200℃(分解)であつた。〕
シリカゲルTLC;Rf=0.53(n−ブタノール:酢
酸:水=4:1:1)
実施例 1
L−ロイシル−3,5−ジクロロ−4−ヒドロ
キシアニリド、メタ過ヨウ素酸ナトリウム、p
−キシレノールを用いるLAP活性測定
L−ロイシル−3,5−ジクロロ−4−ヒドロ
キシアニリド・塩酸塩を0.1Mリン酸緩衝液(PH
7.0)に溶解して基質溶液とした(5mM)。この
基質溶液1mlに、患者血清(236G−R単位の
LAP)20μを加え良く混合し、37℃にて20分間
反応させた。反応後、2mMメタ過ヨウ素酸ナト
リウム、10mMp−キシレノールを含有する0.2M
−KOH溶液からなる酸化試薬液3mlを加え反応
せしめて発色させた。Yield 2.13g (yield 64.0%) Molecular formula C 11 H 12 N 2 O 4 Br 2Melting point: 191-194℃ (decomposed) [Results measured according to the Japanese Pharmacopoeia General Tests Melting point measurement section are 199-194℃
It was at 200℃ (decomposition). ] Silica gel TLC; Rf=0.53 (n-butanol:acetic acid:water=4:1:1) Example 1 L-leucyl-3,5-dichloro-4-hydroxyanilide, sodium metaperiodate, p
- LAP activity measurement using xylenol L-leucyl-3,5-dichloro-4-hydroxyanilide hydrochloride was added to 0.1M phosphate buffer (PH
7.0) to prepare a substrate solution (5mM). Add patient serum (236 G-R units) to 1 ml of this substrate solution.
20μ of LAP) was added, mixed well, and reacted at 37°C for 20 minutes. After the reaction, 0.2M containing 2mM sodium metaperiodate, 10mM p-xylenol
3 ml of an oxidizing reagent solution consisting of -KOH solution was added to react and color was developed.
次いでこれを波長585nmにおける発色液の吸
光度を測定したところO.D585=0.18を得た。 Next, the absorbance of the coloring liquid at a wavelength of 585 nm was measured, and OD 585 =0.18 was obtained.
すなわち、本発明の合成基質は、臨床上、重要
とされているLAPに対して良く作用することが
示された。 In other words, the synthetic substrate of the present invention was shown to act well against LAP, which is clinically important.
実施例 2
L−ロイシル−3,5−ジブロモ−4−ヒドロ
キシアニリド、メタ過ヨウ素酸ナトリウム、p
−キシリレノールを用いるLAP活性測定
L−ロイシル−3,5−ジブロモ−4−ヒドロ
キシアニリド・塩酸塩を0.1Mリン酸緩衝液(PH
7.0)に溶解して基質溶液とした(5mM)。この
基質溶液1mlに、患者血清(250G−R単位の
LAP)20μを加え良く混合し、37℃にて20分間
反応させた。反応後、2mMメタ過ヨウ素酸ナト
リウム、10mM p−キシレノールを含有する
0.2N−KOH溶液からなる酸化試薬液3mlを加
え、発色させた。Example 2 L-leucyl-3,5-dibromo-4-hydroxyanilide, sodium metaperiodate, p
- LAP activity measurement using xylylenol L-leucyl-3,5-dibromo-4-hydroxyanilide hydrochloride was added to 0.1M phosphate buffer (PH
7.0) to prepare a substrate solution (5mM). Add patient serum (250 G-R units) to 1 ml of this substrate solution.
20μ of LAP) was added, mixed well, and reacted at 37°C for 20 minutes. After the reaction, containing 2mM sodium metaperiodate and 10mM p-xylenol.
3 ml of oxidizing reagent solution consisting of 0.2N-KOH solution was added to develop color.
次いで、これを波長585nmにおける発色液の
吸光度を測定したところO.D.=0.22を得た。 Next, when the absorbance of the coloring liquid was measured at a wavelength of 585 nm, OD=0.22 was obtained.
すなわち、本発明の合成基質は、臨床上、重要
とされているLAPに対して良く作用することが
示された。 In other words, the synthetic substrate of the present invention was shown to act well against LAP, which is clinically important.
実施例 3
γ−L−グルタミル−3,5−ジクロロ−4−
ヒドロキシアニリド、メタ過ヨウ素酸ナトリウ
ム、p−キシレノールを用いるγ−GTP活性
測定
γ−L−グルタミル−3,5−ジクロロ−4−
ヒドロキシアニリド(5mM)及びグリシルグシ
ン(100mM)を50mMトリス塩酸緩衝液(PH
8.0)に溶解して基質溶液とした。この基質溶液
0.5mlに、患者血清(130mU/mlのγ−GTP)
10μを加え良く混合し、37℃にて20分間反応さ
せた。反応後、2mMメタ過ヨウ素酸ナトリウ
ム、10mM p−キシレノールを含有する0.2N
−KOH溶液からなる酸化試薬液2mlを加えて発
色させた。Example 3 γ-L-glutamyl-3,5-dichloro-4-
γ-GTP activity measurement using hydroxyanilide, sodium metaperiodate, and p-xylenol γ-L-glutamyl-3,5-dichloro-4-
Hydroxyanilide (5mM) and glycylgucine (100mM) were dissolved in 50mM Tris-HCl buffer (PH
8.0) to prepare a substrate solution. This substrate solution
Patient serum (130 mU/ml γ-GTP) in 0.5 ml
10μ was added, mixed well, and reacted at 37°C for 20 minutes. After the reaction, 0.2N containing 2mM sodium metaperiodate, 10mM p-xylenol
2 ml of an oxidizing reagent solution consisting of -KOH solution was added to develop color.
次いで、これを波長585nmにおける発色液の
吸光度を測定したところO.D585=0.112を得た。 Next, when the absorbance of the coloring liquid was measured at a wavelength of 585 nm, OD 585 =0.112 was obtained.
すなわち、本発明の合成基質は、臨床上、重要
とされているγ−GTPに対して良く作用するこ
とが示された。 In other words, the synthetic substrate of the present invention was shown to act well on γ-GTP, which is clinically important.
実施例 4
γ−L−グルタミル−3,5−ジブロモ−4−
ヒドロキシアニリド、メタ過ヨウ素酸ナトリウ
ム、p−キシリレノールを用いるγ−GTP活性
測定γ−L−グルタミル−3,5−ジブロモ−4
−ヒドロキシアニリド(5mM)およびグリシル
グリシン(100mM)を50mMトリス塩酸緩衝液
(PH8.0)に溶解して基質溶液とした。この基質溶
液0.5mlに患者血清(130mU/mlのγ−GTP)
10μを加え良く混合し、37℃にて20分間反応さ
せた。反応後、2mMメタ過ヨウ素酸ナトリウ
ム、10mM p−キシレノールを含有する0.2N
−KOH溶液からなる酸化試薬液2mlを加えて発
色させた。Example 4 γ-L-glutamyl-3,5-dibromo-4-
γ-GTP activity measurement using hydroxyanilide, sodium metaperiodate, and p-xylylenol γ-L-glutamyl-3,5-dibromo-4
-Hydroxyanilide (5mM) and glycylglycine (100mM) were dissolved in 50mM Tris-HCl buffer (PH8.0) to prepare a substrate solution. Add patient serum (130 mU/ml γ-GTP) to 0.5 ml of this substrate solution.
10μ was added, mixed well, and reacted at 37°C for 20 minutes. After the reaction, 0.2N containing 2mM sodium metaperiodate, 10mM p-xylenol
2 ml of an oxidizing reagent solution consisting of -KOH solution was added to develop color.
次いで、これを波長585nmにおける発色液の
吸光度を測定したところO.D585=0.140を得た。 Next, when the absorbance of the coloring liquid was measured at a wavelength of 585 nm, OD 585 =0.140 was obtained.
すなわち、本発明の合成基質は、臨床上、重要
とされているγ−GTPに対して良く作用するこ
とが示された。 In other words, the synthetic substrate of the present invention was shown to act well on γ-GTP, which is clinically important.
実施例 5
L−ロイシル−3,5−ジブロモ−4−ヒドロ
キシアニリド、メタ過ヨウ素酸ナトリウム、2
−クロロ−5−メチルフエノールを用いる
LAP活性測定
L−ロイシル−3,5ジブロモ−4−ヒドロキ
シアニリド・塩酸塩を0.1Mリン酸緩衝液(PH
7.0)に溶解して基質溶液とした(5mM)。この
基質溶液1mlに、患者血清(250G−R単位の
LAP)20μを加え良く混合し、37℃にて20分間
反応させた。反応後、2mMメタ過ヨウ素酸ナト
リウム、5mM2−クロロ−5−メチルフエノー
ルを含有する0.2N−KOH溶液からなる酸化試薬
液3mlを加え発色させた。Example 5 L-leucyl-3,5-dibromo-4-hydroxyanilide, sodium metaperiodate, 2
-using chloro-5-methylphenol
LAP activity measurement L-leucyl-3,5 dibromo-4-hydroxyanilide hydrochloride was added to 0.1M phosphate buffer (PH
7.0) to prepare a substrate solution (5mM). Add patient serum (250 G-R units) to 1 ml of this substrate solution.
20μ of LAP) was added, mixed well, and reacted at 37°C for 20 minutes. After the reaction, 3 ml of an oxidizing reagent solution consisting of a 0.2N KOH solution containing 2mM sodium metaperiodate and 5mM 2-chloro-5-methylphenol was added to develop color.
次いで、これを波長635nmにおける発色液の
吸光度を測定したところ、O.D585=0.33を得た。 Next, when the absorbance of the coloring liquid was measured at a wavelength of 635 nm, OD 585 =0.33 was obtained.
すなわち、本発明の合成基質は、LAPに対し
て良く作用することが示された。 In other words, the synthetic substrate of the present invention was shown to act well on LAP.
実施例 6
γ−L−グルタミル−3,5−ジブロモ−4−
ヒドロキシアニリド、メタ過ヨウ素酸ナトリウ
ム、2−クロロ−5−メチルフエノールを用い
るγ−GTP活性測定
γ−L−グルタミル−3,5−ジブロモ−4−
5mMヒドロキシアニリド(5mM)およびグリ
シルグリシン(100mM)を50mMトリス塩酸緩
衝液(PH8.0)に溶解して基質溶液とした。この
基質溶液0.5mlに患者血清(130mU/mlのγ−
GTP)10μを加え良く混合し、37℃にて20分間
反応させた。反応後、2mMメタ過ヨウ素酸ナト
リウム、5mM2−クロロ−5−メチルフエノー
ルを含有する0.2N−KOH溶液からなる酸化試薬
液2mlを加えて発色させる。Example 6 γ-L-glutamyl-3,5-dibromo-4-
γ-GTP activity measurement using hydroxyanilide, sodium metaperiodate, and 2-chloro-5-methylphenol γ-L-glutamyl-3,5-dibromo-4-
5mM hydroxyanilide (5mM) and glycylglycine (100mM) were dissolved in 50mM Tris-HCl buffer (PH8.0) to prepare a substrate solution. Patient serum (130 mU/ml γ-
GTP) was added, mixed well, and reacted at 37°C for 20 minutes. After the reaction, 2 ml of an oxidizing reagent solution consisting of a 0.2N KOH solution containing 2mM sodium metaperiodate and 5mM 2-chloro-5-methylphenol is added to develop color.
次いで、これを波長635nmにおける発色液の
吸光度を測定したところO.D635=0.214を得た。 Next, when the absorbance of the coloring liquid was measured at a wavelength of 635 nm, OD 635 =0.214 was obtained.
すなわち、本発明の合成基質は、γ−GTPに
対して良く作用することが示された。 That is, the synthetic substrate of the present invention was shown to act well on γ-GTP.
実施例 7
L−ロイシル−3,5−ジブロモ−4−ヒドロ
キシアニリド、ナトリウムペンタシアノアミノ
フエロエートを用いるLAP活性測定
ナトリウムペンタシアノアミノフエロエート2
gを水20mlに溶解し、0.3%過酸化水素60mlを加
え、更に10%重炭酸ナトリウム液20mlを加えた。
次にデキストランT10(フアルマシア社製)4g
を加え、呈色原液を調製した。反応停止・呈色原
液1mlに1%食塩および0.5%Tween80を含有す
る0.2Mのクエン酸緩衝液(PH4.5)50mlを加えて
調製した。Example 7 LAP activity measurement using L-leucyl-3,5-dibromo-4-hydroxyanilide, sodium pentacyanoamin ferroate Sodium pentacyano amino ferroate 2
g was dissolved in 20 ml of water, 60 ml of 0.3% hydrogen peroxide was added, and an additional 20 ml of 10% sodium bicarbonate solution was added.
Next, 4g of Dextran T10 (manufactured by Pharmacia)
was added to prepare a coloring stock solution. A solution was prepared by adding 50 ml of 0.2M citrate buffer (PH4.5) containing 1% sodium chloride and 0.5% Tween 80 to 1 ml of the reaction termination/coloring stock solution.
0.1Mリン酸緩衝液(PH7.0)に溶解した5mM
L−ロイシル−3,5−ジブロモ−4−ヒドロ
キシアニリド・塩酸塩1mlに患者血清(250G−
R単位のLAP)20μを加え良く混合し、37℃に
て20分間反応させた。反応後、反応停止・呈色液
5mlを加え、室温に20分間放置し、呈色させた
後、700nmにて吸光度を測定した。その結果、
O.D700=0.21であつた。 5mM dissolved in 0.1M phosphate buffer (PH7.0)
Add patient serum (250G-
20μ of LAP (R unit) was added, mixed well, and reacted at 37°C for 20 minutes. After the reaction, 5 ml of reaction stop/coloring solution was added, and the mixture was left at room temperature for 20 minutes to develop color, and then the absorbance was measured at 700 nm. the result,
OD 700 = 0.21.
実施例 8
γ−L−グルタミル−3,5−ジブロモ−4−
ヒドロキシアニリド、ナトリウムペンタシアノ
アミノフエロエートを用いるγ−GTP活性測
定
50mMトリス塩酸緩衝液(PH8.0)に溶解した
5mM γ−L−グルタミル−3,5−ジブロモ
−4−ヒドロキシアニリド、100mMグリシルグ
リシン溶液1mlに患者血清(130mU/mlのγ−
GTP)20μを加えて良く混合し、37℃にて20分
間反応させた。反応後、実施例7に記載した反応
停止・呈色液5mlを加え、20分間放置し、呈色さ
せた後、700nmに吸光度を測定した。その結果、
O.D700=0.132であつた。Example 8 γ-L-glutamyl-3,5-dibromo-4-
γ-GTP activity measurement using hydroxyanilide, sodium pentacyanoaminoferroate. Patient serum (130 mU/ml γ-
GTP) was added, mixed well, and reacted at 37°C for 20 minutes. After the reaction, 5 ml of the reaction stopping/coloring solution described in Example 7 was added, and the mixture was allowed to stand for 20 minutes to develop color, and then the absorbance was measured at 700 nm. the result,
OD 700 = 0.132.
実施例 9
L−ロイシル−3,5−ジブロモ−4−ヒドロ
キシアニリド、ナトリウムペンタシアノアコフ
エリアートを用いるLAP活性測定法
L−ロイシル−3,5−ジブロモ−4−ヒドロ
キシアニリド・塩酸塩を0.1Mリン酸緩衝液(PH
7.0)に溶解して基質溶液(5mM)とした。こ
の基質溶液1mlに患者血清(250G−R単位の
LAP)20μを加え良く混合し、37℃にて15分間
反応させた。反応後、1%−ナトリウムペンタシ
アノアコフエリアート30mlに0.2M EDTA・2Na
(PH11)溶液90mlを加えて調製した溶液4mlを加
え37℃にて15分間反応させた。反応呈色後、
730nmにて吸光度を測定した。その結果、O.D730
=0.232であつた。Example 9 LAP activity measurement method using L-leucyl-3,5-dibromo-4-hydroxyanilide, sodium pentacyanoacopheriate L-leucyl-3,5-dibromo-4-hydroxyanilide hydrochloride at 0.1M Phosphate buffer (PH
7.0) to prepare a substrate solution (5mM). Add patient serum (250 G-R units) to 1 ml of this substrate solution.
20μ of LAP) was added, mixed well, and reacted at 37°C for 15 minutes. After the reaction, add 0.2M EDTA・2Na to 30ml of 1%-sodium pentacyanoacopheriate.
4 ml of the solution prepared by adding 90 ml of the (PH11) solution was added and allowed to react at 37°C for 15 minutes. After reaction coloration,
Absorbance was measured at 730 nm. As a result, OD 730
= 0.232.
1%−ナトリウムペンタシアノアコフエリアー
トは、1%−ニトロプルシツドナトリウムNa2
〔Fe(CN)5NO〕−1%炭酸ナトリウム液を15分
間、紫外線照射して作製した。 1%-sodium pentacyanoacopheriate is 1%-sodium nitroprusside Na2
[Fe(CN) 5 NO]-1% sodium carbonate solution was prepared by irradiating ultraviolet rays for 15 minutes.
実施例 10
γ−L−グルタミル−3,5−ジブロモ−4−
ヒドロキシアニリドを用いたγ−GTPの検量
線
γ−L−グルタミル−3,5−ジブロモ−4−
ヒドロキシアニリド(5mM)およびグリシルグ
リシン(100mM)を50mMトリス塩酸緩衝液
(PH8.0)に溶解して基質溶液とした。この基質溶
液0.5mlに血清γ−GTP(74〜370mU/ml)10μ
を加えて良く混合し、37℃にて20分間反応させ
た。反応後、2mMメタ過ヨウ素酸ナトリウム、
10mM p−キシレノールを含有する0.2N−
KOH溶液からなる酸化試薬液2mlを加えて発色
させた。Example 10 γ-L-glutamyl-3,5-dibromo-4-
Calibration curve of γ-GTP using hydroxyanilide γ-L-glutamyl-3,5-dibromo-4-
Hydroxyanilide (5mM) and glycylglycine (100mM) were dissolved in 50mM Tris-HCl buffer (PH8.0) to prepare a substrate solution. 10μ of serum γ-GTP (74-370mU/ml) was added to 0.5ml of this substrate solution.
was added, mixed well, and reacted at 37°C for 20 minutes. After the reaction, 2mM sodium metaperiodate,
0.2N containing 10mM p-xylenol
Color was developed by adding 2 ml of an oxidizing reagent solution consisting of a KOH solution.
次いで、これを波長585nmにおける発色液の
吸光度を測定した。その結果を第1図に示した。
この結果より血清γ−GTPが良好に測定される
ことが示された。 Next, the absorbance of the coloring liquid was measured at a wavelength of 585 nm. The results are shown in Figure 1.
This result showed that serum γ-GTP could be measured satisfactorily.
実施例 11
L−ロイジル−4−N,N−ジエチルアニリド
とL−ロイジル−3,5−ジブロモ−4−ヒド
ロキシアニリドを用いたLAP測定法の比較。Example 11 Comparison of LAP measurement methods using L-leusyl-4-N,N-diethylanilide and L-leusyl-3,5-dibromo-4-hydroxyanilide.
従来からLAP測定法に使用されている公知化
合物L−ロイシル−4−N,N−ジエチルアニリ
ドと本発明の参考例2の化合物を用いLAP測定
法の比較を行つた。 The LAP measurement method was compared using the known compound L-leucyl-4-N,N-diethylanilide, which has been conventionally used in the LAP measurement method, and the compound of Reference Example 2 of the present invention.
L−ロイシル−4−N,N−ジエチルアミノア
ニリド1.0mM、フエノール0.1%を含有するPH
7.0、0.1Mリン酸緩衝液を調製して基質溶液とし
た。この基質溶液2.0mlに種々の血清試料50μを
加えよく混合したのち、37℃、20分間反応させ
た。その後発色試薬〔1−ナフトール−2−スル
ホン酸(カプラ)、メタ過ヨウ素酸ナトリウム〕
1.0mlを加え、呈色(青色)した反応液を675nm
で吸光度を測定した。 PH containing 1.0mM L-leucyl-4-N,N-diethylaminoanilide, 0.1% phenol
A 7.0 and 0.1M phosphate buffer was prepared and used as a substrate solution. 50μ of various serum samples were added to 2.0ml of this substrate solution, mixed well, and then reacted at 37°C for 20 minutes. Then color reagent [1-naphthol-2-sulfonic acid (Capra), sodium metaperiodate]
Add 1.0 ml of the reaction solution and test the colored (blue) reaction solution at 675 nm.
The absorbance was measured.
別にL−ロイシル−3,5−ジブロモ−4−ヒ
ドロキシアニリド塩酸塩を0.1Mリン酸緩衝液
(PH7.0)に溶解して基質溶液とした(5mM)。
この基質溶液2.0mlを用いて上記と同じ血清試料
20μとよく混合し、37℃、15分間反応後、発色
試薬〔p−キシレノール(カプラー)、メタ過ヨ
ウ素酸ナトリウム〕2.0mlを加えて、呈色(青色)
した反応液を610nmで吸光度を測定した。 Separately, L-leucyl-3,5-dibromo-4-hydroxyanilide hydrochloride was dissolved in 0.1M phosphate buffer (PH7.0) to prepare a substrate solution (5mM).
The same serum sample as above using 2.0 ml of this substrate solution.
Mix well with 20μ and react for 15 minutes at 37℃, then add 2.0ml of coloring reagent [p-xylenol (coupler), sodium metaperiodate] to develop color (blue).
The absorbance of the reaction solution was measured at 610 nm.
上記両方法により測定した同一血清試料につい
ての吸光度をプロツトして第2図に示すごとき結
果を得た。 The absorbance of the same serum sample measured by both of the above methods was plotted and the results shown in FIG. 2 were obtained.
なお、血清試料として通常血清の他高システイ
ンアミノペプチダーゼ(CAP)血清を用いて測
定した。 In addition to normal serum, high cysteine aminopeptidase (CAP) serum was used as the serum sample for measurement.
その結果、本発明の合成基質はCAPにより作
用されることはない。本願明の合成基質を用いる
ことにより、公知基質の約10倍の感度でLAP活
性測定が可能であることが判つた。 As a result, the synthetic substrates of the invention are not acted upon by CAP. It has been found that by using the synthetic substrate of the present invention, it is possible to measure LAP activity with a sensitivity approximately 10 times greater than that of known substrates.
第1図はγ−GTPの検量線、第2図は本発明
の合成基質と公知の合成基質との測定法相関図で
ある。
FIG. 1 is a calibration curve for γ-GTP, and FIG. 2 is a correlation chart of measurement methods between the synthetic substrate of the present invention and a known synthetic substrate.
Claims (1)
ペプチダーゼおよびγ−グルタミルトランスペプ
チダーゼからなる群より選ばれるペプチダーゼの
活性測定において、 式 (式中、RはL−ロイシンアミノペプチダーゼ活
性測定の場合にL−ロイシル基、γ−グルタミル
トランスペプチダーゼ活性測定の場合にγ−L−
グルタミル基を示し、XおよびYは同一か異なり
ハロゲン原子を示す)で表わされるアミド化合物
またはその塩に、被験液に含有されているペプチ
ダーゼを作用させて、式 (式中、XおよびYは前記と同じ意味を有する)
で表わされるアニリン誘導体を遊離せしめ、次い
で該アニリン誘導体を化学的発色手段により発色
させて生成する発色化合物を比色定量することを
特徴とする新規な合成基質を用いるL−ロイシン
アミノペプチダーゼおよびγ−グルタミルトラン
スペプチダーゼからなる群より選ばれるペプチダ
ーゼの活性測定法。 2 アミノ化合物がL−ロイシル−3,5−ジハ
ロゲノ−4−ヒドロキシアニリドであり、ペプチ
ダーゼがL−ロイシンアミノペプチダーゼである
特許請求の範囲第1項記載の測定法。 3 L−ロイシル−3,5−ジハロゲノ−4−ヒ
ドロキシアニリドがL−ロイシル−3,5−ジブ
ロモ−4−ヒドロキシアニリドまたはL−ロイシ
ル−3,5−ジクロロ−4−ヒドロキシアニリド
である特許請求の範囲第2項記載の測定法。 4 アミド化合物がγ−L−グルタミル−3,5
−ジハロゲノ−4−ヒドロキシアニリドであり、
ペプチダーゼがγ−グルタミルトランスペプチダ
ーゼである特許請求の範囲第1項記載の測定法。 5 γ−L−グルタミル−3,5−ジハロゲノ−
4−ヒドロキシアニリドがγ−L−グルタミル−
3,5−ジブロモ−4−ヒドロキシアニリドまた
はγ−L−グルタミル−3,5−ジクロロ−4−
ヒドロキシアニリドである特許請求の範囲第2項
記載の測定法。 6 化学的発色手段がカプラーを共存させて酸化
縮合を行い、発色させる方法である特許請求の範
囲第1項記載の測定法。 7 カプラーが該アニリン誘導体と酸化縮合して
発色化合物を形成する芳香族化合物である特許請
求の範囲第6項記載の測定法。 8 芳香族化合物がフエノール系化合物、アミノ
フエノール系化合物、アニリン系化合物またはナ
フトール系化合物である特許請求の範囲第7項記
載の測定法。 9 化学的発色手段がペンタシアノ鉄錯塩を用い
て発色させる方法である特許請求の範囲第1項記
載の測定法。[Scope of Claims] 1. In measuring the activity of a peptidase selected from the group consisting of L-leucine aminopeptidase and γ-glutamyl transpeptidase contained in a test solution, the formula (In the formula, R is an L-leucyl group in the case of L-leucine aminopeptidase activity measurement, and γ-L- in the case of γ-glutamyl transpeptidase activity measurement.
glutamyl group, and X and Y are the same or different and represent a halogen atom), or a salt thereof, is reacted with peptidase contained in the test solution to obtain the formula (In the formula, X and Y have the same meanings as above)
L-leucine aminopeptidase and γ-using a novel synthetic substrate, characterized in that the aniline derivative represented by is liberated, the aniline derivative is then colored by a chemical coloring means, and the produced colored compound is colorimetrically quantified. A method for measuring the activity of a peptidase selected from the group consisting of glutamyl transpeptidases. 2. The measuring method according to claim 1, wherein the amino compound is L-leucyl-3,5-dihalogeno-4-hydroxyanilide and the peptidase is L-leucine aminopeptidase. 3 L-leucyl-3,5-dihalogeno-4-hydroxyanilide is L-leucyl-3,5-dibromo-4-hydroxyanilide or L-leucyl-3,5-dichloro-4-hydroxyanilide. The measurement method described in Scope 2. 4 The amide compound is γ-L-glutamyl-3,5
-dihalogeno-4-hydroxyanilide,
The measuring method according to claim 1, wherein the peptidase is γ-glutamyl transpeptidase. 5 γ-L-glutamyl-3,5-dihalogeno-
4-Hydroxyanilide is γ-L-glutamyl-
3,5-dibromo-4-hydroxyanilide or γ-L-glutamyl-3,5-dichloro-4-
The measuring method according to claim 2, which is a hydroxyanilide. 6. The measuring method according to claim 1, wherein the chemical coloring means is a method of performing oxidative condensation in the presence of a coupler to develop color. 7. The measuring method according to claim 6, wherein the coupler is an aromatic compound that undergoes oxidative condensation with the aniline derivative to form a color-forming compound. 8. The measuring method according to claim 7, wherein the aromatic compound is a phenol compound, an aminophenol compound, an aniline compound, or a naphthol compound. 9. The measuring method according to claim 1, wherein the chemical coloring means is a method of coloring using a pentacyanoiron complex salt.
Priority Applications (8)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP57150701A JPS5942350A (en) | 1982-09-01 | 1982-09-01 | Novel synthetic substrate and method for measuring activity |
US06/526,031 US4588836A (en) | 1982-09-01 | 1983-08-24 | Novel synthetic substrate and assay method using the same |
DE3348172A DE3348172C2 (en) | 1982-09-01 | 1983-09-01 | |
DE19833331588 DE3331588A1 (en) | 1982-09-01 | 1983-09-01 | AMID COMPOUNDS |
FR8314039A FR2532307B1 (en) | 1982-09-01 | 1983-09-01 | NOVEL AMIDE FOR USE AS SYNTHETIC SUBSTRATE AND ASSAY METHOD USING SAME |
IT22726/83A IT1171084B (en) | 1982-09-01 | 1983-09-01 | SYNTHETIC SUBSTRATE FOR THE PEPTIDASIC ACTIVITY TEST AND PEPTIDASE TEST PROCEDURE |
US06/713,242 US4675290A (en) | 1982-09-01 | 1985-03-18 | Assaying peptidase enzyme activity |
FR858508107A FR2567907B1 (en) | 1982-09-01 | 1985-05-30 | NOVEL AMIDE FOR USE AS SYNTHETIC SUBSTRATE AND ASSAY METHOD USING SAME |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP57150701A JPS5942350A (en) | 1982-09-01 | 1982-09-01 | Novel synthetic substrate and method for measuring activity |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP13749585A Division JPS6117550A (en) | 1985-06-24 | 1985-06-24 | Novel synthetic substrate |
Publications (2)
Publication Number | Publication Date |
---|---|
JPS5942350A JPS5942350A (en) | 1984-03-08 |
JPS6338200B2 true JPS6338200B2 (en) | 1988-07-28 |
Family
ID=15502523
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP57150701A Granted JPS5942350A (en) | 1982-09-01 | 1982-09-01 | Novel synthetic substrate and method for measuring activity |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPS5942350A (en) |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP4735038B2 (en) * | 2005-05-16 | 2011-07-27 | 日本精工株式会社 | Continuously variable transmission |
GB2454672A (en) * | 2007-11-13 | 2009-05-20 | Mologic Ltd | Chromogenic protease substrates |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS52148031A (en) * | 1976-06-01 | 1977-12-08 | Sankyo Co Ltd | Alpha-l-amino acid anilides |
JPS5452042A (en) * | 1977-09-28 | 1979-04-24 | Kanto Kagaku | Llleucyllpphydroxyanilide and measuring method by using it |
-
1982
- 1982-09-01 JP JP57150701A patent/JPS5942350A/en active Granted
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS52148031A (en) * | 1976-06-01 | 1977-12-08 | Sankyo Co Ltd | Alpha-l-amino acid anilides |
JPS5452042A (en) * | 1977-09-28 | 1979-04-24 | Kanto Kagaku | Llleucyllpphydroxyanilide and measuring method by using it |
Also Published As
Publication number | Publication date |
---|---|
JPS5942350A (en) | 1984-03-08 |
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