JPH0243479B2 - - Google Patents
Info
- Publication number
- JPH0243479B2 JPH0243479B2 JP58103317A JP10331783A JPH0243479B2 JP H0243479 B2 JPH0243479 B2 JP H0243479B2 JP 58103317 A JP58103317 A JP 58103317A JP 10331783 A JP10331783 A JP 10331783A JP H0243479 B2 JPH0243479 B2 JP H0243479B2
- Authority
- JP
- Japan
- Prior art keywords
- chromogen
- bilirubin oxidase
- enzyme
- activity
- reaction
- 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 - Lifetime
Links
- 238000000034 method Methods 0.000 claims description 22
- 108010015428 Bilirubin oxidase Proteins 0.000 claims description 18
- 239000000758 substrate Substances 0.000 claims description 12
- 238000006482 condensation reaction Methods 0.000 claims description 11
- 239000003086 colorant Substances 0.000 claims description 7
- 230000005494 condensation Effects 0.000 claims description 2
- 238000009833 condensation Methods 0.000 claims description 2
- 238000006911 enzymatic reaction Methods 0.000 claims description 2
- 230000001590 oxidative effect Effects 0.000 claims description 2
- 125000003277 amino group Chemical group 0.000 claims 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims 1
- 125000005843 halogen group Chemical group 0.000 claims 1
- 125000004435 hydrogen atom Chemical group [H]* 0.000 claims 1
- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 claims 1
- 239000000049 pigment Substances 0.000 claims 1
- 230000000694 effects Effects 0.000 description 21
- 102000004190 Enzymes Human genes 0.000 description 19
- 108090000790 Enzymes Proteins 0.000 description 19
- 229940088598 enzyme Drugs 0.000 description 19
- 238000006243 chemical reaction Methods 0.000 description 12
- RLFWWDJHLFCNIJ-UHFFFAOYSA-N 4-aminoantipyrine Chemical compound CN1C(C)=C(N)C(=O)N1C1=CC=CC=C1 RLFWWDJHLFCNIJ-UHFFFAOYSA-N 0.000 description 11
- 238000002835 absorbance Methods 0.000 description 11
- 210000002966 serum Anatomy 0.000 description 9
- 102000002704 Leucyl aminopeptidase Human genes 0.000 description 7
- 108010004098 Leucyl aminopeptidase Proteins 0.000 description 7
- 238000011088 calibration curve Methods 0.000 description 6
- 239000003795 chemical substances by application Substances 0.000 description 5
- 239000000203 mixture Substances 0.000 description 4
- RUKJCCIJLIMGEP-ONEGZZNKSA-N 4-dimethylaminocinnamaldehyde Chemical compound CN(C)C1=CC=C(\C=C\C=O)C=C1 RUKJCCIJLIMGEP-ONEGZZNKSA-N 0.000 description 3
- FJKROLUGYXJWQN-UHFFFAOYSA-N 4-hydroxybenzoic acid Chemical compound OC(=O)C1=CC=C(O)C=C1 FJKROLUGYXJWQN-UHFFFAOYSA-N 0.000 description 3
- QNGVNLMMEQUVQK-UHFFFAOYSA-N 4-n,4-n-diethylbenzene-1,4-diamine Chemical compound CCN(CC)C1=CC=C(N)C=C1 QNGVNLMMEQUVQK-UHFFFAOYSA-N 0.000 description 3
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 3
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 3
- 101710107035 Gamma-glutamyltranspeptidase Proteins 0.000 description 3
- 101710173228 Glutathione hydrolase proenzyme Proteins 0.000 description 3
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 3
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 3
- 239000000975 dye Substances 0.000 description 3
- 102000006640 gamma-Glutamyltransferase Human genes 0.000 description 3
- 229910052700 potassium Inorganic materials 0.000 description 3
- 239000011591 potassium Substances 0.000 description 3
- 150000003739 xylenols Chemical class 0.000 description 3
- GZCWLCBFPRFLKL-UHFFFAOYSA-N 1-prop-2-ynoxypropan-2-ol Chemical compound CC(O)COCC#C GZCWLCBFPRFLKL-UHFFFAOYSA-N 0.000 description 2
- JBIJLHTVPXGSAM-UHFFFAOYSA-N 2-naphthylamine Chemical compound C1=CC=CC2=CC(N)=CC=C21 JBIJLHTVPXGSAM-UHFFFAOYSA-N 0.000 description 2
- TYMLOMAKGOJONV-UHFFFAOYSA-N 4-nitroaniline Chemical compound NC1=CC=C([N+]([O-])=O)C=C1 TYMLOMAKGOJONV-UHFFFAOYSA-N 0.000 description 2
- KZZWQCKYLNIOBT-UHFFFAOYSA-N 5-amino-2-nitrobenzoic acid Chemical compound NC1=CC=C([N+]([O-])=O)C(C(O)=O)=C1 KZZWQCKYLNIOBT-UHFFFAOYSA-N 0.000 description 2
- 102000013563 Acid Phosphatase Human genes 0.000 description 2
- 108010051457 Acid Phosphatase Proteins 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- BPYKTIZUTYGOLE-IFADSCNNSA-N Bilirubin Chemical compound N1C(=O)C(C)=C(C=C)\C1=C\C1=C(C)C(CCC(O)=O)=C(CC2=C(C(C)=C(\C=C/3C(=C(C=C)C(=O)N\3)C)N2)CCC(O)=O)N1 BPYKTIZUTYGOLE-IFADSCNNSA-N 0.000 description 2
- 102000003914 Cholinesterases Human genes 0.000 description 2
- 108090000322 Cholinesterases Proteins 0.000 description 2
- 102000004139 alpha-Amylases Human genes 0.000 description 2
- 108090000637 alpha-Amylases Proteins 0.000 description 2
- 229940024171 alpha-amylase Drugs 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 229940048961 cholinesterase Drugs 0.000 description 2
- YMAWOPBAYDPSLA-UHFFFAOYSA-N glycylglycine Chemical compound [NH3+]CC(=O)NCC([O-])=O YMAWOPBAYDPSLA-UHFFFAOYSA-N 0.000 description 2
- VBEGHXKAFSLLGE-UHFFFAOYSA-N n-phenylnitramide Chemical compound [O-][N+](=O)NC1=CC=CC=C1 VBEGHXKAFSLLGE-UHFFFAOYSA-N 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- JQWHASGSAFIOCM-UHFFFAOYSA-M sodium periodate Chemical compound [Na+].[O-]I(=O)(=O)=O JQWHASGSAFIOCM-UHFFFAOYSA-M 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- BAPAICNRGIBFJT-UHFFFAOYSA-O (4-Hydroxybenzoyl)choline Chemical compound C[N+](C)(C)CCOC(=O)C1=CC=C(O)C=C1 BAPAICNRGIBFJT-UHFFFAOYSA-O 0.000 description 1
- PWJNDVAKQLOWRZ-UHFFFAOYSA-N 1-hydroxynaphthalene-2-sulfonic acid Chemical compound C1=CC=C2C(O)=C(S(O)(=O)=O)C=CC2=C1 PWJNDVAKQLOWRZ-UHFFFAOYSA-N 0.000 description 1
- QWBBPBRQALCEIZ-UHFFFAOYSA-N 2,3-dimethylphenol Chemical compound CC1=CC=CC(O)=C1C QWBBPBRQALCEIZ-UHFFFAOYSA-N 0.000 description 1
- 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 1
- WXNZTHHGJRFXKQ-UHFFFAOYSA-N 4-chlorophenol Chemical compound OC1=CC=C(Cl)C=C1 WXNZTHHGJRFXKQ-UHFFFAOYSA-N 0.000 description 1
- 229940090248 4-hydroxybenzoic acid Drugs 0.000 description 1
- BTJIUGUIPKRLHP-UHFFFAOYSA-N 4-nitrophenol Chemical compound OC1=CC=C([N+]([O-])=O)C=C1 BTJIUGUIPKRLHP-UHFFFAOYSA-N 0.000 description 1
- KBZFDRWPMZESDI-UHFFFAOYSA-N 5-aminobenzene-1,3-dicarboxylic acid Chemical compound NC1=CC(C(O)=O)=CC(C(O)=O)=C1 KBZFDRWPMZESDI-UHFFFAOYSA-N 0.000 description 1
- GWZYPXHJIZCRAJ-UHFFFAOYSA-N Biliverdin Natural products CC1=C(C=C)C(=C/C2=NC(=Cc3[nH]c(C=C/4NC(=O)C(=C4C)C=C)c(C)c3CCC(=O)O)C(=C2C)CCC(=O)O)NC1=O GWZYPXHJIZCRAJ-UHFFFAOYSA-N 0.000 description 1
- RCNSAJSGRJSBKK-NSQVQWHSSA-N Biliverdin IX Chemical compound N1C(=O)C(C)=C(C=C)\C1=C\C1=C(C)C(CCC(O)=O)=C(\C=C/2C(=C(C)C(=C/C=3C(=C(C=C)C(=O)N=3)C)/N\2)CCC(O)=O)N1 RCNSAJSGRJSBKK-NSQVQWHSSA-N 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- 241000222511 Coprinus Species 0.000 description 1
- 108091000069 Cystinyl Aminopeptidase Proteins 0.000 description 1
- 102000030900 Cystinyl aminopeptidase Human genes 0.000 description 1
- 108010008488 Glycylglycine Proteins 0.000 description 1
- JWHURRLUBVMKOT-HNNXBMFYSA-N L-leucine 2-naphthylamide Chemical compound C1=CC=CC2=CC(NC(=O)[C@@H](N)CC(C)C)=CC=C21 JWHURRLUBVMKOT-HNNXBMFYSA-N 0.000 description 1
- 102100024295 Maltase-glucoamylase Human genes 0.000 description 1
- 241000223251 Myrothecium Species 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 108010028144 alpha-Glucosidases Proteins 0.000 description 1
- 150000003931 anilides Chemical class 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- QBUVFDKTZJNUPP-UHFFFAOYSA-N biliverdin-IXalpha Natural products N1C(=O)C(C)=C(C=C)C1=CC1=C(C)C(CCC(O)=O)=C(C=C2C(=C(C)C(C=C3C(=C(C=C)C(=O)N3)C)=N2)CCC(O)=O)N1 QBUVFDKTZJNUPP-UHFFFAOYSA-N 0.000 description 1
- 239000013060 biological fluid Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 239000001045 blue dye Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000003028 enzyme activity measurement method Methods 0.000 description 1
- 229940043257 glycylglycine Drugs 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- KBOPZPXVLCULAV-UHFFFAOYSA-N mesalamine Chemical compound NC1=CC=C(O)C(C(O)=O)=C1 KBOPZPXVLCULAV-UHFFFAOYSA-N 0.000 description 1
- 229960004963 mesalazine Drugs 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- CMPQUABWPXYYSH-UHFFFAOYSA-N phenyl phosphate Chemical compound OP(O)(=O)OC1=CC=CC=C1 CMPQUABWPXYYSH-UHFFFAOYSA-N 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000001052 yellow pigment Substances 0.000 description 1
Description
【発明の詳細な説明】
本発明はビリルビンオキシダーゼの存在下縮合
反応せしめることを特徴とする縮合反応方法に関
する。詳しくは、色原体と発色剤とをビリルビン
オキシダーゼの存在下で縮合せしめることを特徴
とする縮合反応方法に関し、さらに詳しくは、色
原体と結合した酵素基質を用い、これに酵素を作
用せしめ遊離した色原体と発色剤とをビリルビン
オキシダーゼの存在下で縮合せしめることを特徴
とする縮合反応方法に関する。上記反応で生成し
た色素を定量することにつて特定の酵素活性の測
定が可能である。DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a condensation reaction method characterized by carrying out the condensation reaction in the presence of bilirubin oxidase. Specifically, it relates to a condensation reaction method characterized by condensing a chromogen and a coloring agent in the presence of bilirubin oxidase, and more specifically, a condensation reaction method characterized by condensing a chromogen and a coloring agent in the presence of bilirubin oxidase. The present invention relates to a condensation reaction method characterized by condensing a liberated chromogen and a coloring agent in the presence of bilirubin oxidase. Specific enzyme activity can be measured by quantifying the dye produced in the above reaction.
生体液中の各種酵素の活性は種々の疾患によつ
て変動することが明らかになり、診断治療上の指
針として重要視されている。これら酵素活性の測
定のうちあるものは色原体と結合した酵素基質を
用い、これに酵素試料を作用せしめ遊離した色原
体と発色剤とを特定の存在下反応させて色素に導
き、これを比色定量する方法が採用されている。
例えば、ロイシンアミノペプチダーゼ活性の測定
は、酵素基質としてＬ−ロイシル−ｐ−ジエチル
アミノアニリドを用い、反応により遊離した色原
体ｐ−ジエチルアミノアニリンをアルカリ性下、
メタ過沃素酸ナトリウムを縮合剤として発色剤１
−ナフトール−２−スルホン酸カリウムと縮合さ
せ、生成した青色色素を比色定量することにより
測定される。しかし、従来の上記のような方法は
縮合反応が強アルカリ性または強酸性下で行われ
ること、縮合剤によつて酵素が失活することなど
により、酵素反応を行つた後に縮合反応を行わな
ければならないといつた不便があつた。 It has become clear that the activities of various enzymes in biological fluids vary depending on various diseases, and this is considered important as a guideline for diagnosis and treatment. Some of these enzyme activity measurements use an enzyme substrate bound to a chromogen, and an enzyme sample is applied to this, causing the liberated chromogen and coloring agent to react in a specific presence to form a dye. A method of colorimetric determination has been adopted.
For example, to measure leucine aminopeptidase activity, L-leucyl-p-diethylaminoanilide is used as the enzyme substrate, and the chromogen p-diethylaminoaniline released by the reaction is treated under alkaline conditions.
Color forming agent 1 using sodium metaperiodate as a condensing agent
It is measured by condensing with potassium naphthol-2-sulfonate and colorimetrically quantifying the blue dye produced. However, in the conventional methods described above, the condensation reaction is carried out under strong alkaline or acidic conditions, and the enzyme is inactivated by the condensing agent, so the condensation reaction must be carried out after the enzymatic reaction. There was a lot of inconvenience that I had to do.
本発明者らは各種酵素活性の測定法について鋭
意検討したところ、従来の縮合剤の代わりにビリ
ルビンオキシダーゼが有効であることを見いだ
し、本発明を完成した。本発明法によれば目的と
する酵素活性の測定反応と縮合反応を同時に行う
ことができ、初速度法（rate assay法）または終
点法（end point法）のいずれの方法でも測定が
可能となつた。 The inventors of the present invention have conducted intensive studies on methods for measuring various enzyme activities, and have found that bilirubin oxidase is effective in place of conventional condensing agents, thereby completing the present invention. According to the method of the present invention, the reaction for measuring the desired enzyme activity and the condensation reaction can be performed simultaneously, and measurement can be performed using either the rate assay method or the end point method. Ta.
本発明法において縮合剤として用いるビリルビ
ンオキシダーゼは、例えば特開昭57−159487号公
報および特願昭57−25613号に開示されているミ
ロセシウム（Myrothecium）属菌、またはコプ
リナス（Coprinus）属菌産生のビリルビンオキ
シダーゼが使用できる。ビリルビンオキシダーゼ
は血中の黄色色素であるビリルビンを酸化してビ
リベルジンに変える反応を触媒する酵素として知
られているが、縮合反応に有効であることは知ら
れていない。 Bilirubin oxidase used as a condensing agent in the method of the present invention is produced by, for example, Myrothecium genus bacteria or Coprinus genus bacteria disclosed in Japanese Patent Application Laid-Open No. 57-159487 and Japanese Patent Application No. 57-25613. Bilirubin oxidase can be used. Bilirubin oxidase is known as an enzyme that catalyzes the reaction of oxidizing bilirubin, a yellow pigment in blood, into biliverdin, but it is not known to be effective in condensation reactions.
本発明法によつて縮合を受ける物質の一方、即
ち色原体としてはｐ−ジエチルアミノアニリン、
ｐ−ニトロアニリン、ｐ−Ｎ−エチル−Ｎ−ヒド
ロキシエチルアミノアニリン、ｐ−ニトロフエノ
ール、ｐ−ヒドロキシ安息香酸、３−カルボキシ
−４−ニトロアニリン、３−カルボキシ−４−ヒ
ドロキシアニリン、β−ナフチルアミン、フエノ
−ル、ｐ−クロルフエノール、５−アミノイソフ
タル酸などが例示され、他方発色剤としては４−
アミノアンチピリン、１−ナフトール−２−スル
ホン酸、ｐ−ジメチルアミノシンナムアルデヒ
ド、キシレノール（ジメチルフエノぶル）などが
例示される。 One of the substances subjected to condensation by the method of the present invention, ie, as a chromogen, p-diethylaminoaniline;
p-Nitroaniline, p-N-ethyl-N-hydroxyethylaminoaniline, p-nitrophenol, p-hydroxybenzoic acid, 3-carboxy-4-nitroaniline, 3-carboxy-4-hydroxyaniline, β-naphthylamine , phenol, p-chlorophenol, 5-aminoisophthalic acid, etc., while the coloring agent is 4-
Examples include aminoantipyrine, 1-naphthol-2-sulfonic acid, p-dimethylaminocinnamaldehyde, xylenol (dimethylphenol), and the like.
本発明法を利用して測定される酵素活性は、例
えばアルカリ性ホスフアターゼ、酸性ホスフアタ
ーゼ、コリンエステラーゼ、ロイシンアミノペプ
チダーゼ、シスチンアミノペプチダーゼ、γ−グ
ルタルミルトランスペプチダーゼ、α−アミラー
ゼなどである。 Enzyme activities that can be measured using the method of the present invention include, for example, alkaline phosphatase, acid phosphatase, cholinesterase, leucine aminopeptidase, cystine aminopeptidase, γ-glutamyltranspeptidase, α-amylase, and the like.
具体的には、例えばロイシンアミノペプチダー
ゼ活性の測定にはＬ−ロイシル−β−ナフチルア
ミドを基質として遊離したβ−ナフチルアミンと
ｐ−ジメチルアミノシンナムアルデヒドを縮合さ
せる方法、またはＬ−ロイシル−ｐ−ジエチルア
ミノアニリドを基質として遊離したｐ−ジエチル
アミノアニリンと１−ナフトール−２−スルホン
酸カリウムを縮合させる方法が使用できる。 Specifically, for example, leucine aminopeptidase activity can be measured by condensing liberated β-naphthylamine and p-dimethylaminocinnamaldehyde using L-leucyl-β-naphthylamide as a substrate, or by using L-leucyl-p-diethylamino. A method can be used in which liberated p-diethylaminoaniline and potassium 1-naphthol-2-sulfonate are condensed using anilide as a substrate.
γ−グルタミルトランスペプチダーゼ活性の測
定にはＬ−γ−グルタミル−３−カルボキシ−４
−ニトロアニリドを基質として遊離した３−カル
ボキシ−４−ニトロアニリンとキシレノールを縮
合させる方法、またはＬ−γ−グルタミル−ｐ−
ニトロアニリドを基質として遊離したｐ−ニトロ
アニリンとｐ−ジメチルアミノシンナムアルデヒ
ドを縮合させる方法が使用できる。 For measuring γ-glutamyl transpeptidase activity, L-γ-glutamyl-3-carboxy-4
- A method of condensing liberated 3-carboxy-4-nitroaniline and xylenol using nitroanilide as a substrate, or L-γ-glutamyl-p-
A method can be used in which p-nitroaniline released using nitroanilide as a substrate is condensed with p-dimethylaminocinnamaldehyde.
アルカリ性ホスフアターゼまたは酸性ホスフア
ターゼ活性の測定にはフエニルリン酸を基質とし
て遊離したフエノールと４−アミノアンチピリン
を縮合させる方法が使用できる。 For measuring alkaline phosphatase or acid phosphatase activity, a method can be used in which liberated phenol and 4-aminoantipyrine are condensed using phenyl phosphate as a substrate.
コリンエステラーゼ活性の測定にはｐ−ヒドロ
キシベンゾイルコリンを基質として遊離したｐ−
ヒドロキシ安息香酸と４−アミノアンチピリンを
縮合させる方法が使用できる。 To measure cholinesterase activity, free p-hydroxybenzoylcholine is used as a substrate.
A method of condensing hydroxybenzoic acid and 4-aminoantipyrine can be used.
本発明法により特定の酵素活性を測定するに
は、その酵素が触媒することができる色原体と結
合した前述のような酵素基質を用い、酵素基質、
色原体と縮合可能な発色剤、ビリルビンオキシダ
ーゼ、被測定酵素試料および必要に応じ緩衝液を
混合し一定温度で反応させる。反応により生じた
色素を比色またはその他の方法により定量するこ
とにより酵素活性が測定される。なお、発色剤を
結合せしめた酵素基質を使用し、しかるのち発色
剤と色原体を縮合させた酵素活性を測定すること
もできる。 In order to measure a specific enzyme activity by the method of the present invention, an enzyme substrate as described above coupled to a chromogen that can be catalyzed by the enzyme is used.
A coloring agent capable of condensing with a chromogen, bilirubin oxidase, an enzyme sample to be measured, and a buffer solution if necessary are mixed and reacted at a constant temperature. Enzyme activity is measured by quantifying the dye produced by the reaction by colorimetry or other methods. Note that it is also possible to use an enzyme substrate to which a color former is bound, and then measure the enzyme activity by condensing the color former and the chromogen.
ビリルビンオキシダーゼの使用量は、反応液中
で約0.5〜50u／mlである。また、反応のPHは約
4.5〜10.5、温度は約20〜40℃の範囲であるが、
上記ビリルビンオキシダーゼまたはラツカーゼの
使用量並びに反応条件は、測定しようとする酵素
の反応条件に応じ適宜変化させることができる。
なおビリルビンオキシダーゼの活性は、フエノー
ルと４−アミノアンチピリンを基質としてPH7.0、
37℃で反応させたとき、１分間に505nmにおける
吸光度を0.01変化させる酵素量を１単位（ｕ）と
した。 The amount of bilirubin oxidase used is about 0.5 to 50 u/ml in the reaction solution. Also, the pH of the reaction is approximately
4.5-10.5, the temperature ranges from about 20-40℃,
The amount of bilirubin oxidase or latcase used and the reaction conditions can be changed as appropriate depending on the reaction conditions of the enzyme to be measured.
The activity of bilirubin oxidase is PH7.0 using phenol and 4-aminoantipyrine as substrates.
One unit (u) was defined as the amount of enzyme that caused a 0.01 change in absorbance at 505 nm per minute when reacted at 37°C.
以下実施例により本発明を具体的に説明する。 The present invention will be specifically explained below using Examples.
実施例 １
1.0mML−ロイシル−ｐ−ジエチルアミノアニ
リド、0.5mM1−ナフトール−２−スルホン酸カ
リウムおよび10.0u／mlビリルビンオキシダーゼ
を含む、0.1Mリン酸緩衝液（PH7.0）1.0mlと
155u／mlのロイシンアミノペプチダーゼ活性を
含む標準血清（コンセーラ：日水製薬社商標）０
〜80μの各容量を混合して、37℃で反応し
670nmにおける吸光度の増加を測定した（吸光度
は反応液1.02ml当りに補正した）。１分間当りの
吸光度の増加とロイシンアミノペプチダーゼ活性
の関係（検量線）は直線的であつた（図示に示
ず）。Example 1 1.0 ml of 0.1M phosphate buffer (PH7.0) containing 1.0mM leucyl-p-diethylaminoanilide, 0.5mM potassium 1-naphthol-2-sulfonate and 10.0u/ml bilirubin oxidase.
Standard serum containing 155u/ml leucine aminopeptidase activity (Concera: Nissui Pharmaceutical Co., Ltd. trademark) 0
Mix each volume of ~80μ and react at 37 °C.
The increase in absorbance at 670 nm was measured (absorbance was corrected per 1.02 ml of reaction solution). The relationship (calibration curve) between the increase in absorbance per minute and leucine aminopeptidase activity was linear (not shown).
上記標準血清の代わりに血清試料20μを用い
同様に操作し、得られた吸光度を上記検量線と対
比したところ、ロイシンアミノペプチダーゼ活性
は320u／mlであつた。 The same procedure was carried out using 20μ of a serum sample instead of the above standard serum, and the obtained absorbance was compared with the above calibration curve, and the leucine aminopeptidase activity was found to be 320μ/ml.
実施例 ２
2.0mML−γ−グルタミル−３−カルボキシ−
４−ヒドロキシアニリド、10.0mMグルシルグリ
シン、7.0mMキシレノールおよび10.0u／mlビリ
ルビンオキシダーゼを含む0.1Mトリス塩酸緩衝
液（PH8.45）3.0mlと血清試料50μを混合して、
37℃、５分間反応し、635nmにおける吸光度の増
加を測定した。得られた吸光度とあらかじめ標準
血清を用いて作成した検量線とを対比してγ−グ
ルタミルトランスペプチダーゼ活性を求めたとこ
ろ、195IU／であつた。Example 2 2.0mML-γ-glutamyl-3-carboxy-
Mix 50μ of the serum sample with 3.0ml of 0.1M Tris-HCl buffer (PH8.45) containing 4-hydroxyanilide, 10.0mM glycylglycine, 7.0mM xylenol and 10.0u/ml bilirubin oxidase.
The reaction was carried out at 37°C for 5 minutes, and the increase in absorbance at 635 nm was measured. The γ-glutamyl transpeptidase activity was determined by comparing the obtained absorbance with a calibration curve prepared in advance using standard serum, and found to be 195 IU/.
実施例 ３
1.0mMフエニルリン酸−２−ナトリウム、
0.4mM4−アミノアンチピリンおよび10.0u／mlビ
リルビンオキシダーゼを含む0.1M炭酸塩緩衝液
（PH10.3）3.0mlと血清試料100μを混合して、37
℃で５分間反応し505nmにおける吸光度の増加を
測定した。得られた吸光度とあらかじめ標準血清
を用いて作成した検量線とを対比してアルカリ性
ホスフアターゼ活性を求めたところ、100.2ミリ
ｕ／mlであつた。Example 3 1.0mM phenyl phosphate-2-sodium,
Mix 100μ of serum sample with 3.0ml of 0.1M carbonate buffer (PH10.3) containing 0.4mM 4-aminoantipyrine and 10.0u/ml bilirubin oxidase to
The reaction was carried out at ℃ for 5 minutes and the increase in absorbance at 505 nm was measured. The alkaline phosphatase activity was determined by comparing the obtained absorbance with a calibration curve prepared in advance using standard serum and found to be 100.2 milliu/ml.
実施例 ４
2.0mMフエニルテトラマルトシド、0.4mM4−
アミノアンチピリン、α−グルコシダーゼ
10.0u／mlおよび0.0u／mlビリルビンオキシダー
ゼを含む0.1Mリン酸緩衝液（PH7.0）3.0mlと血清
試料20μを混合して、37℃で５分間反応し
505nmにおける吸光度の増加を測定した。得られ
た吸光度とあらかじめ標準血清を用いて作成した
検量線とを対比してα−アミラーゼ活性を求めた
ところ、250u／dlであつた。Example 4 2.0mM phenyltetramaltoside, 0.4mM4-
Aminoantipyrine, α-glucosidase
Mix 3.0ml of 0.1M phosphate buffer (PH7.0) containing 10.0u/ml and 0.0u/ml bilirubin oxidase with 20μ of serum sample and react at 37°C for 5 minutes.
The increase in absorbance at 505 nm was measured. The α-amylase activity was determined by comparing the obtained absorbance with a calibration curve prepared in advance using standard serum, and it was found to be 250 u/dl.
図面は本発明法によるロイシンアミノペプチダ
ーゼ活性測定の検量線を表す図である。
The drawing shows a calibration curve for measuring leucine aminopeptidase activity according to the method of the present invention.
Claims (1)
（）で表わされる色原体とビリルビンオキシダ
ーゼによつて該色原体と酸化縮合して色素を形成
する発色剤とを反応せしめることを特徴とする縮
合反応方法。 （ただし式中、R1は水酸基又はアミノ基を示し、
R2、R3、R4、R5又はR6は水素原子、ハロゲン原
子、ニトロ基、置換アミノ基、水酸基又はカルボ
キシル基を示し、R4とR5は環を形成する基でも
よい。） ２ 一般式（）で表わされる色原体が酵素反応
により基質より遊離されたものである特許請求の
範囲第１項記載の縮合反応方法。[Claims] 1. In the presence of bilirubin oxidase, a chromogen represented by the general formula () is caused to react with a coloring agent that forms a pigment through oxidative condensation with the chromogen by bilirubin oxidase. Characteristic condensation reaction method. (However, in the formula, R1 represents a hydroxyl group or an amino group,
R 2 , R 3 , R 4 , R 5 or R 6 represents a hydrogen atom, a halogen atom, a nitro group, a substituted amino group, a hydroxyl group or a carboxyl group, and R 4 and R 5 may be a group forming a ring. ) 2 The condensation reaction method according to claim 1, wherein the chromogen represented by the general formula () is released from the substrate by an enzymatic reaction.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP10331783A JPS59227300A (en) | 1983-06-09 | 1983-06-09 | Condensation reaction |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP10331783A JPS59227300A (en) | 1983-06-09 | 1983-06-09 | Condensation reaction |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS59227300A JPS59227300A (en) | 1984-12-20 |
| JPH0243479B2 true JPH0243479B2 (en) | 1990-09-28 |
Family
ID=14350819
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP10331783A Granted JPS59227300A (en) | 1983-06-09 | 1983-06-09 | Condensation reaction |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS59227300A (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5963198A (en) * | 1982-10-01 | 1984-04-10 | Toyo Jozo Co Ltd | Quantitative assay using enzyme |
-
1983
- 1983-06-09 JP JP10331783A patent/JPS59227300A/en active Granted
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5963198A (en) * | 1982-10-01 | 1984-04-10 | Toyo Jozo Co Ltd | Quantitative assay using enzyme |
Also Published As
| Publication number | Publication date |
|---|---|
| JPS59227300A (en) | 1984-12-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Fossati et al. | Enzymic creatinine assay: a new colorimetric method based on hydrogen peroxide measurement. | |
| JP3080770B2 (en) | Method for measuring ions in liquid and composition therefor | |
| JPH0470000B2 (en) | ||
| Lorentz et al. | Arylesterase in serum: elaboration and clinical application of a fixed-incubation method. | |
| JPS6049477B2 (en) | Glycerol oxidase and its production method, and method for quantifying glycerol using glycerol oxidase | |
| US5047329A (en) | Method for the measurement of creatine or creatinine and reagents for these measurements | |
| EP0116307B1 (en) | Composition, analytical element and method for the quantification of creatine kinase | |
| Rick et al. | Pepsin (pepsin, gastricsin, pepsinogen, uropepsinogen) | |
| JPS5819279B2 (en) | Method and reagent for measuring NAD(P)H or salicylate | |
| Mamoru et al. | A new colorimetric method for determination of serum glucose | |
| EP0133681B1 (en) | Enzymatic urea assay | |
| EP0135092A2 (en) | Method for determination of ammonia | |
| US4695539A (en) | Process for quantitative determination of substrate treated with oxidase | |
| JPH0571239B2 (en) | ||
| JPH0243479B2 (en) | ||
| JPH1014596A (en) | Measurement of composition in humor and reagent therefor | |
| US5474908A (en) | Method of enzymatically measuring hydrogen peroxide and reagent therefor | |
| JPH0131880B2 (en) | ||
| US5350678A (en) | Method of differential assay for α-amylase isozymes and a kit for the same | |
| JPH026520B2 (en) | ||
| CS227331B2 (en) | Method of glycerol determination | |
| US5204242A (en) | Substituted phenols | |
| JP4544598B2 (en) | Liquid reagent and storage method | |
| Maguire | Elimination of the" chromogen oxidase" activity of bilirubin oxidase added to obviate bilirubin interference in hydrogen peroxide/peroxidase detecting systems. | |
| JP2643205B2 (en) | Highly sensitive colorimetric method |