JPH029397A - Method for analyzing phosphatase - Google Patents
Method for analyzing phosphataseInfo
- Publication number
- JPH029397A JPH029397A JP16114988A JP16114988A JPH029397A JP H029397 A JPH029397 A JP H029397A JP 16114988 A JP16114988 A JP 16114988A JP 16114988 A JP16114988 A JP 16114988A JP H029397 A JPH029397 A JP H029397A
- Authority
- JP
- Japan
- Prior art keywords
- phosphatase
- acid
- reagent
- amino
- phosphoric acid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 title claims abstract description 22
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 title claims abstract description 22
- 238000000034 method Methods 0.000 title claims description 26
- -1 p-amino-substituted phenylphosphoric acid Chemical class 0.000 claims abstract description 30
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 claims description 17
- 229910019142 PO4 Inorganic materials 0.000 claims description 4
- 239000010452 phosphate Substances 0.000 claims description 4
- 102000045595 Phosphoprotein Phosphatases Human genes 0.000 claims 1
- 108700019535 Phosphoprotein Phosphatases Proteins 0.000 claims 1
- ZYPZVOKVDNSKLP-UHFFFAOYSA-N tris(4-aminophenyl) phosphate Chemical compound C1=CC(N)=CC=C1OP(=O)(OC=1C=CC(N)=CC=1)OC1=CC=C(N)C=C1 ZYPZVOKVDNSKLP-UHFFFAOYSA-N 0.000 claims 1
- 239000000758 substrate Substances 0.000 abstract description 15
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 12
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 abstract description 11
- 102000004190 Enzymes Human genes 0.000 abstract description 8
- 108090000790 Enzymes Proteins 0.000 abstract description 8
- 229910000147 aluminium phosphate Inorganic materials 0.000 abstract description 6
- 239000007822 coupling agent Substances 0.000 abstract description 6
- 235000014113 dietary fatty acids Nutrition 0.000 abstract description 6
- 229930195729 fatty acid Natural products 0.000 abstract description 6
- 239000000194 fatty acid Substances 0.000 abstract description 6
- 150000002989 phenols Chemical class 0.000 abstract description 5
- FXZCCINCKDATPA-UHFFFAOYSA-N (4-aminophenyl) dihydrogen phosphate Chemical compound NC1=CC=C(OP(O)(O)=O)C=C1 FXZCCINCKDATPA-UHFFFAOYSA-N 0.000 abstract description 4
- 239000004094 surface-active agent Substances 0.000 abstract description 4
- 125000001424 substituent group Chemical group 0.000 abstract description 3
- BTIDJAQNJLWPTI-UHFFFAOYSA-N 3-(n-ethyl-3,5-dimethoxyanilino)-2-hydroxypropane-1-sulfonic acid Chemical compound OS(=O)(=O)CC(O)CN(CC)C1=CC(OC)=CC(OC)=C1 BTIDJAQNJLWPTI-UHFFFAOYSA-N 0.000 abstract description 2
- 108010029541 Laccase Proteins 0.000 abstract description 2
- 125000000217 alkyl group Chemical group 0.000 abstract description 2
- 238000004040 coloring Methods 0.000 abstract description 2
- 230000003647 oxidation Effects 0.000 abstract description 2
- 238000007254 oxidation reaction Methods 0.000 abstract description 2
- ZBCBWPMODOFKDW-UHFFFAOYSA-N diethanolamine Chemical compound OCCNCCO ZBCBWPMODOFKDW-UHFFFAOYSA-N 0.000 abstract 1
- 239000000975 dye Substances 0.000 description 14
- 238000006243 chemical reaction Methods 0.000 description 12
- 238000012360 testing method Methods 0.000 description 12
- 230000001590 oxidative effect Effects 0.000 description 8
- CMPQUABWPXYYSH-UHFFFAOYSA-N phenyl phosphate Chemical compound OP(O)(=O)OC1=CC=CC=C1 CMPQUABWPXYYSH-UHFFFAOYSA-N 0.000 description 7
- 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 6
- 238000010521 absorption reaction Methods 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 229920003171 Poly (ethylene oxide) Polymers 0.000 description 5
- 238000002835 absorbance Methods 0.000 description 5
- 238000011088 calibration curve Methods 0.000 description 5
- 235000011007 phosphoric acid Nutrition 0.000 description 5
- RLFWWDJHLFCNIJ-UHFFFAOYSA-N 4-aminoantipyrine Chemical compound CN1C(C)=C(N)C(=O)N1C1=CC=CC=C1 RLFWWDJHLFCNIJ-UHFFFAOYSA-N 0.000 description 4
- 150000001448 anilines Chemical class 0.000 description 4
- XZKIHKMTEMTJQX-UHFFFAOYSA-N 4-Nitrophenyl Phosphate Chemical compound OP(O)(=O)OC1=CC=C([N+]([O-])=O)C=C1 XZKIHKMTEMTJQX-UHFFFAOYSA-N 0.000 description 3
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Natural products OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 3
- 108090000854 Oxidoreductases Proteins 0.000 description 3
- 102000004316 Oxidoreductases Human genes 0.000 description 3
- 239000013060 biological fluid Substances 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 210000002966 serum Anatomy 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
- PLIKAWJENQZMHA-UHFFFAOYSA-N 4-aminophenol Chemical class NC1=CC=C(O)C=C1 PLIKAWJENQZMHA-UHFFFAOYSA-N 0.000 description 2
- BTJIUGUIPKRLHP-UHFFFAOYSA-N 4-nitrophenol Chemical compound OC1=CC=C([N+]([O-])=O)C=C1 BTJIUGUIPKRLHP-UHFFFAOYSA-N 0.000 description 2
- 102000013563 Acid Phosphatase Human genes 0.000 description 2
- 108010051457 Acid Phosphatase Proteins 0.000 description 2
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 2
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 2
- 101100215617 Arabidopsis thaliana ADO3 gene Proteins 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- 102000001554 Hemoglobins Human genes 0.000 description 2
- 108010054147 Hemoglobins Proteins 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- 108010044467 Isoenzymes Proteins 0.000 description 2
- MBBZMMPHUWSWHV-BDVNFPICSA-N N-methylglucamine Chemical compound CNC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO MBBZMMPHUWSWHV-BDVNFPICSA-N 0.000 description 2
- 102000003425 Tyrosinase Human genes 0.000 description 2
- 108060008724 Tyrosinase Proteins 0.000 description 2
- 125000003545 alkoxy group Chemical group 0.000 description 2
- 229960005070 ascorbic acid Drugs 0.000 description 2
- 235000010323 ascorbic acid Nutrition 0.000 description 2
- 239000011668 ascorbic acid Substances 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 125000004432 carbon atom Chemical group C* 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 230000005494 condensation Effects 0.000 description 2
- 238000009833 condensation Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 150000002440 hydroxy compounds Chemical class 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Chemical compound C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000007800 oxidant agent Substances 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- MWFOPMKUGZLPQA-UHFFFAOYSA-M sodium;3-(n-ethyl-3-methoxyanilino)propane-1-sulfonate Chemical compound [Na+].[O-]S(=O)(=O)CCCN(CC)C1=CC=CC(OC)=C1 MWFOPMKUGZLPQA-UHFFFAOYSA-M 0.000 description 2
- BJEPYKJPYRNKOW-REOHCLBHSA-N (S)-malic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O BJEPYKJPYRNKOW-REOHCLBHSA-N 0.000 description 1
- XGIKILRODBEJIL-UHFFFAOYSA-N 1-(ethylamino)ethanol Chemical compound CCNC(C)O XGIKILRODBEJIL-UHFFFAOYSA-N 0.000 description 1
- XKZQKPRCPNGNFR-UHFFFAOYSA-N 2-(3-hydroxyphenyl)phenol Chemical compound OC1=CC=CC(C=2C(=CC=CC=2)O)=C1 XKZQKPRCPNGNFR-UHFFFAOYSA-N 0.000 description 1
- SLACHNPFSGRUPD-UHFFFAOYSA-N 2-(n-ethyl-2-methylanilino)ethanol Chemical compound OCCN(CC)C1=CC=CC=C1C SLACHNPFSGRUPD-UHFFFAOYSA-N 0.000 description 1
- MENXRDLDYNLDHE-UHFFFAOYSA-N 3-(3,5-dimethoxyanilino)propane-1-sulfonic acid Chemical compound COC1=CC(NCCCS(O)(=O)=O)=CC(OC)=C1 MENXRDLDYNLDHE-UHFFFAOYSA-N 0.000 description 1
- NZAVBNVWEPQSBL-UHFFFAOYSA-N 3-(n-ethyl-3,5-dimethoxyanilino)propane-1-sulfonic acid Chemical compound OS(=O)(=O)CCCN(CC)C1=CC(OC)=CC(OC)=C1 NZAVBNVWEPQSBL-UHFFFAOYSA-N 0.000 description 1
- ZLQNJZKBJSMXCJ-UHFFFAOYSA-N 3-(n-ethyl-3-methoxyanilino)-2-hydroxypropane-1-sulfonic acid Chemical compound OS(=O)(=O)CC(O)CN(CC)C1=CC=CC(OC)=C1 ZLQNJZKBJSMXCJ-UHFFFAOYSA-N 0.000 description 1
- LHZMSRLULDAWLM-UHFFFAOYSA-N 3-(n-ethylanilino)-2-hydroxypropane-1-sulfonic acid Chemical compound OS(=O)(=O)CC(O)CN(CC)C1=CC=CC=C1 LHZMSRLULDAWLM-UHFFFAOYSA-N 0.000 description 1
- HXITYOAFXWBMLL-UHFFFAOYSA-N 3-(n-ethylanilino)propane-1-sulfonic acid Chemical compound OS(=O)(=O)CCCN(CC)C1=CC=CC=C1 HXITYOAFXWBMLL-UHFFFAOYSA-N 0.000 description 1
- ZHBXLZQQVCDGPA-UHFFFAOYSA-N 5-[(1,3-dioxo-2-benzofuran-5-yl)sulfonyl]-2-benzofuran-1,3-dione Chemical compound C1=C2C(=O)OC(=O)C2=CC(S(=O)(=O)C=2C=C3C(=O)OC(C3=CC=2)=O)=C1 ZHBXLZQQVCDGPA-UHFFFAOYSA-N 0.000 description 1
- KWIUHFFTVRNATP-UHFFFAOYSA-N Betaine Natural products C[N+](C)(C)CC([O-])=O KWIUHFFTVRNATP-UHFFFAOYSA-N 0.000 description 1
- 108010015428 Bilirubin oxidase Proteins 0.000 description 1
- WKBOTKDWSSQWDR-UHFFFAOYSA-N Bromine atom Chemical group [Br] WKBOTKDWSSQWDR-UHFFFAOYSA-N 0.000 description 1
- 101100404959 Caenorhabditis elegans dao-5 gene Proteins 0.000 description 1
- 108010031396 Catechol oxidase Proteins 0.000 description 1
- 102000030523 Catechol oxidase Human genes 0.000 description 1
- 108010075016 Ceruloplasmin Proteins 0.000 description 1
- 102100023321 Ceruloplasmin Human genes 0.000 description 1
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerol Natural products OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 1
- 108010013563 Lipoprotein Lipase Proteins 0.000 description 1
- 102100022119 Lipoprotein lipase Human genes 0.000 description 1
- XWZGINYLGNMGLJ-UHFFFAOYSA-N NC1=CC(Cl)=C(OP(O)(O)=O)C=C1 Chemical compound NC1=CC(Cl)=C(OP(O)(O)=O)C=C1 XWZGINYLGNMGLJ-UHFFFAOYSA-N 0.000 description 1
- XBCXDMXCOJVDMU-UHFFFAOYSA-N NC1=CC=C(C=C1)[P] Chemical compound NC1=CC=C(C=C1)[P] XBCXDMXCOJVDMU-UHFFFAOYSA-N 0.000 description 1
- 108700020962 Peroxidase Proteins 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 229920001214 Polysorbate 60 Polymers 0.000 description 1
- GSEJCLTVZPLZKY-UHFFFAOYSA-N Triethanolamine Chemical compound OCCN(CCO)CCO GSEJCLTVZPLZKY-UHFFFAOYSA-N 0.000 description 1
- 208000003152 Yellow Fever Diseases 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 150000003973 alkyl amines Chemical class 0.000 description 1
- 150000005215 alkyl ethers Chemical class 0.000 description 1
- BJEPYKJPYRNKOW-UHFFFAOYSA-N alpha-hydroxysuccinic acid Natural products OC(=O)C(O)CC(O)=O BJEPYKJPYRNKOW-UHFFFAOYSA-N 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 125000002490 anilino group Chemical group [H]N(*)C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 229960003237 betaine Drugs 0.000 description 1
- 229920001400 block copolymer Polymers 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 125000004106 butoxy group Chemical group [*]OC([H])([H])C([H])([H])C(C([H])([H])[H])([H])[H] 0.000 description 1
- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-N carbonic acid Chemical compound OC(O)=O BVKZGUZCCUSVTD-UHFFFAOYSA-N 0.000 description 1
- 229910052801 chlorine Inorganic materials 0.000 description 1
- 125000001309 chloro group Chemical group Cl* 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 238000001585 disappearance potential spectroscopy Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 125000006575 electron-withdrawing group Chemical group 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 125000001301 ethoxy group Chemical group [H]C([H])([H])C([H])([H])O* 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- NVVZQXQBYZPMLJ-UHFFFAOYSA-N formaldehyde;naphthalene-1-sulfonic acid Chemical compound O=C.C1=CC=C2C(S(=O)(=O)O)=CC=CC2=C1 NVVZQXQBYZPMLJ-UHFFFAOYSA-N 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 229910052736 halogen Inorganic materials 0.000 description 1
- 150000002367 halogens Chemical class 0.000 description 1
- 125000004051 hexyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 125000000040 m-tolyl group Chemical group [H]C1=C([H])C(*)=C([H])C(=C1[H])C([H])([H])[H] 0.000 description 1
- 239000001630 malic acid Substances 0.000 description 1
- 235000011090 malic acid Nutrition 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- VLAPMBHFAWRUQP-UHFFFAOYSA-L molybdic acid Chemical compound O[Mo](O)(=O)=O VLAPMBHFAWRUQP-UHFFFAOYSA-L 0.000 description 1
- HFWWEMPLBCKNNM-UHFFFAOYSA-N n-[bis(hydroxyamino)methyl]hydroxylamine Chemical compound ONC(NO)NO HFWWEMPLBCKNNM-UHFFFAOYSA-N 0.000 description 1
- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 description 1
- 238000005691 oxidative coupling reaction Methods 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 125000001147 pentyl group Chemical group C(CCCC)* 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 1
- 125000002572 propoxy group Chemical group [*]OC([H])([H])C(C([H])([H])[H])([H])[H] 0.000 description 1
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 150000003242 quaternary ammonium salts Chemical class 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 150000003871 sulfonates Chemical class 0.000 description 1
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 1
- 238000007039 two-step reaction Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
【発明の詳細な説明】
産業上の利用分野
本発明はホスファターゼの定量法に関する。さらに詳し
くは試料特に生体液中のホスファターゼを定量するに当
たって、ｐ−アミノフェノール類を遊離するような基質
を使用する方法に関する。DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to a method for quantifying phosphatases. More specifically, the present invention relates to a method of using a substrate that liberates p-aminophenols when quantifying phosphatase in a sample, particularly a biological fluid.
生成したｐ−アミノフェノール類は公知の方法例えば他
のアニリン誘導体やフェノール誘導体と酸化縮合させて
定ｌ的に色素を生成させ、比色定量することができる。The produced p-aminophenols can be subjected to oxidative condensation using known methods such as other aniline derivatives or phenol derivatives to quantitatively produce a dye, which can be determined colorimetrically.
従来の技術
従来ホスファターゼの定量法として、次の方法が知られ
ている。2. Description of the Related Art Conventionally, the following method is known as a method for quantifying phosphatase.
（Ａ）β−グリ七クロリン酸基質として用いホスファタ
ーゼを作用させ、生成するリン酸をモリブデン酸と反応
させてリンモリブデン酸ブルーとして比色定量する方法
。この反応は生体液中の成分であるタンパク質によって
阻害されるため、トリクロル酢酸などで前もって蛋白を
除く必要があり、操作が面倒である。〔吉田孝光。(A) A method in which β-glypeptachlorophosphate is used as a substrate, phosphatase is allowed to act, and the resulting phosphoric acid is reacted with molybdic acid to produce phosphomolybdate blue, which is determined colorimetrically. Since this reaction is inhibited by proteins, which are components in biological fluids, it is necessary to remove the proteins with trichloroacetic acid or the like beforehand, which makes the procedure cumbersome. [Takamitsu Yoshida.
北吋元仕、臨床化学分析■、酵素 ８８−１１６（＋９
７０） ］。Motoshi Kitago, Clinical chemistry analysis■, Enzyme 88-116 (+9
70) ].
（Ｂ）基質としてフェニルリン酸を用い生成するフェノ
ールを酸化剤例えばフェリシアン化カリウムを使って４
アミノアンチピリンと縮合させ、５００止付近に極大吸
収を持つ色素を生成させ、これを比色定量する方法。こ
の方法では５００ｎｍ付近は血液中のヘモグロビンやビ
リルビンなどの影響を受けるほか、フェリシアン化カリ
ウムなどの酸化剤はホスファターゼそのものを阻害する
ため、同時反応が出来ず、２段反応（ホスファターゼの
反応と酸化呈色反応）を行わねばならない。(B) The phenol produced using phenyl phosphate as a substrate is oxidized using an oxidizing agent such as potassium ferricyanide.
A method of condensing with aminoantipyrine to produce a dye with maximum absorption around 500, and quantifying it colorimetrically. In this method, wavelengths around 500 nm are affected by hemoglobin and bilirubin in the blood, and oxidizing agents such as potassium ferricyanide inhibit phosphatase itself, so simultaneous reactions cannot be carried out, resulting in a two-step reaction (phosphatase reaction and oxidation coloration). reaction) must be carried out.
（Ｃ）基質としてｐ−ニトロフェニルリン酸を用い、遊
離するｐ−ニトロフェノールが４０５０ｍに吸収極大を
示し、これによる吸収を希アルカリで反応を止めた後比
色定量する。この方法はヘモグロビン、ビリルビンによ
る阻害の問題があり、必ず試薬盲検（基質を用いない盲
検と検体を用いない盲検０２種類）を必要とするので模
作が煩雑である。(C) Using p-nitrophenyl phosphoric acid as a substrate, liberated p-nitrophenol shows an absorption maximum at 4050 m, and the resulting absorption is determined colorimetrically after stopping the reaction with a dilute alkali. This method has the problem of inhibition by hemoglobin and bilirubin, and requires reagent blind testing (blind testing without substrate and blind testing without sample), making it complicated to replicate.
又、ｐ−二トロフェノールの生成を４０５ｎｍでレート
アッセイする方法が知られている（）＼ウヂー７７．
Ｃｌ１ｎ、 Ｃｈｉｎ、＾ｃｔａ １５２４１−２４５
頁。In addition, a method of rate assaying the production of p-nitrophenol at 405 nm is known ()\Uzzi 77.
Cl1n, Chin, ^cta 15241-245
page.
１９６７）。1967).
この方法は試薬盲検を必要としなくなったが、やはり黄
療血清では誤差が大きくなる他、基質がフェニルリン酸
より酸性度の強いｐ−ニトロフェニルリン酸であるため
、イソ酵素の由来によってその反応性が異なっている（
日本臨床３４巻秋期増刊号７６８−７７７頁、 １９７
６）。Although this method no longer requires reagent blinding, there is still a large error when using yellow serum, and since the substrate is p-nitrophenyl phosphate, which is more acidic than phenyl phosphate, it depends on the origin of the isoenzyme. The reactivity is different (
Japan Clinical Volume 34 Autumn Special Issue pp. 768-777, 197
6).
発明が解決しようとする課題
（Ｃ）法におけるｐ−ニトロフェニルリン酸では、ニト
ロ基は強い電子吸引基として働きその結果リン酸基の酸
性度を上げている。このニド四基の代わりにむしろ電子
を供与する様な基、例えばアミノ基、ヒドロキシ基、ア
ルコキシ基を付ければ酸性度を下げてフェニルリン酸の
基質に近いイソ酵素の挙動を示すことが期待されまた、
かかる基質を用いてホスファターゼによりリン酸基が取
れて生成するフェノール類が酸化酵素の存在下に安定な
色素を生ずるような基質は開発されていない。Problems to be Solved by the Invention In the p-nitrophenyl phosphoric acid used in method (C), the nitro group acts as a strong electron-withdrawing group, thereby increasing the acidity of the phosphoric acid group. It is expected that if a group that donates electrons, such as an amino group, hydroxyl group, or alkoxy group, is attached instead of the four nido groups, the acidity will be lowered and the behavior of an isoenzyme similar to that of the substrate of phenylphosphate will be exhibited. Also,
No substrate has been developed in which the phosphate group is removed by phosphatase using such a substrate, and the resulting phenols produce stable dyes in the presence of oxidizing enzymes.
課題を解決するための手段
本発明によれば、ｐ−アミ／置換（もしくは非置換）フ
ェニルリン酸に試料中のホスファターゼを作用させ、生
成するｐ−アミン置換（もしくは非置換）フェノールを
定量することによってホスファターゼを定量できる。Means for Solving the Problems According to the present invention, phosphatase in a sample is allowed to act on p-amine/substituted (or unsubstituted) phenyl phosphate, and the produced p-amine substituted (or unsubstituted) phenol is quantified. Phosphatase can be quantified by this method.
生成したｐ−アミノ置換（もしくは非置換）フェノール
は酸化酵素の存在下アニリン類もしくはフェノール類と
酸化カップリングして色素を生成するのでこの色素を定
量することによってホスファターゼを定量できる。The produced p-amino substituted (or unsubstituted) phenol undergoes oxidative coupling with anilines or phenols in the presence of an oxidizing enzyme to produce a dye, and by quantifying this dye, phosphatase can be determined.
上記反応の反応式が示される。The reaction formula for the above reaction is shown.
Ｒ，１
式中、Ｒは置換基を示し、ｎは０又は１〜４の正ご文を
伯し、ｎ≧２においてＲは同一もしくは異なってよい。R,1 In the formula, R represents a substituent, n represents 0 or 1 to 4, and when n≧2, R may be the same or different.
本発明で基質として用いられるｐ−アミノ置換（もしく
は非置換）フェニルリン酸はホスファターゼを作用させ
ることによって生成するｐ−アミノ置換（もしくは非置
換）フェノールを酸化酵素の作用によってカップリング
剤と反応させ色素を生成しうる化合物であればいずれも
用いうる。The p-amino substituted (or unsubstituted) phenyl phosphate used as a substrate in the present invention is obtained by reacting p-amino substituted (or unsubstituted) phenol produced by the action of a phosphatase with a coupling agent by the action of an oxidase. Any compound capable of producing a dye can be used.
かかる化合物としてベンゼン環上の置換基は１〜４個有
してよく、ハロゲン例えば塩素原子、臭素原子等、炭素
数１〜６のアルキル例えばメチル。Such a compound may have 1 to 4 substituents on the benzene ring, including halogen such as chlorine atom and bromine atom, and alkyl having 1 to 6 carbon atoms, such as methyl.
エチル、プロピル、ブチル、ペンチル、ヘキシル等、炭
素数１〜６のアルコキシ例えばメトキシ。Alkoxy having 1 to 6 carbon atoms, such as ethyl, propyl, butyl, pentyl, hexyl, etc., such as methoxy.
エトキ／、プロポキシ、ブトキシ等が例示される。Examples include ethoxy/, propoxy, and butoxy.
本発明で用いられる具体的ｐ−アミン置換（もしくは非
置換）フェニルリン酸としてはｐ−アミノフェニルリン
酸、２．６ジブロムー４−アミノフェニルリン酸、２．
６ジクロルー４−アミノフェニルリン酸、２．５ツメチ
ル−４−アミノフェニルリン酸、３．４ツメチル−４−
アミノフェニルリン酸２．６ジメチルー４−アミノフェ
ニルリン酸、２．５ジメトキシ−４−アミノフェニルリ
ン酸、２−クロル−４−アミノフェニルリン酸、２−カ
ルボキシ−４−アミノフェニルリン酸、３．５ジクロル
−４−アミノフェニルリン酸、３，５ジブロム−４−ア
ミノフェニルリン酸等があげられる。これらは０．１ｍ
Ｍ−５０ｍＭで用いられる。Specific p-amine substituted (or unsubstituted) phenyl phosphoric acids used in the present invention include p-aminophenyl phosphoric acid, 2.6 dibromo-4-aminophenyl phosphoric acid, 2.
6-dichloro-4-aminophenyl phosphate, 2.5-methyl-4-aminophenyl phosphate, 3.4-methyl-4-
Aminophenyl phosphoric acid 2.6 dimethyl-4-aminophenyl phosphoric acid, 2.5 dimethoxy-4-aminophenyl phosphoric acid, 2-chloro-4-aminophenyl phosphoric acid, 2-carboxy-4-aminophenyl phosphoric acid, 3 Examples include .5 dichloro-4-aminophenyl phosphoric acid and 3,5 dibromo-4-aminophenyl phosphoric acid. These are 0.1m
M-used at 50mM.
反応は適当な緩衝液中で行われ、例えばトリスヒドロキ
シアミノメタノン、バルビッール、クエン酸、リンゴ酸
、炭酸、の他、グツドの緩衝剤（同位化学研究所 第１
５版上合カタログ）が用いられる。The reaction is carried out in an appropriate buffer, such as trishydroxyaminomethane, barbyl, citric acid, malic acid, carbonic acid, and Gud's buffer (Isotope Research Institute No. 1).
The 5th edition Joai Catalog) is used.
反応によって生成したｐ−アミノ首喚（もしくハ非Ｉ
Ｉｓ ）フェノールの定！１はそれ自体公知の方法もし
くは新規に開発される方法によって定量することができ
る。The p-amino compound (or p-amino compound) produced by the reaction
Is ) Determination of phenol! 1 can be quantified by a method known per se or a newly developed method.
簡単な方法の１つとして生成したｐ−アミノ置換（もし
くは非置換）フェノールとカップリグ剤とを酸化酵素の
存在下に反応さセて色素を生成させ生成色素を定量する
方法があげられる。One simple method is to react the produced p-amino substituted (or unsubstituted) phenol with a coupling agent in the presence of an oxidizing enzyme to produce a dye, and quantify the produced dye.
色素の定量は色素の吸収極大値における反応液の着色に
よる吸収の変化を測定することによって達成できる。Quantification of the dye can be achieved by measuring the change in absorption due to coloring of the reaction solution at the maximum absorption value of the dye.
用いられる酸化酵素としては酸素を一方の基質にするも
のが望ましく、モノフェノールモノオキシゲナーゼ（Ｍ
ＰＯ）、 ジフェノールオキンダーゼ、ビリルビンオ
キシダーゼ（ＢＬＯＤ＞、 ラフカーゼ（ＬへＣ）、
アスコルビン酸オキシダーゼ（ＡＯＤ）、セルロプラス
ミン等が好適であり、０、０１−１００ Ｕ／ｍｌの濃
度テ用イラレル。The oxidizing enzyme used is preferably one that uses oxygen as one of its substrates, and monophenol monooxygenase (M
PO), diphenol okindase, bilirubin oxidase (BLOD>, rough case (L to C),
Ascorbic acid oxidase (AOD), ceruloplasmin, etc. are preferred, with concentrations of 0.01-100 U/ml.
カップリング剤としては４−アミノアンチピリンと酸化
縮合して色素を生成するアニリン誘導体。The coupling agent is an aniline derivative that undergoes oxidative condensation with 4-aminoantipyrine to produce a dye.
フェノール誘導体が用いられ、アニリン類として、公知
のパーオキシダーゼの存在下過酸化水素を定量するため
に使用される色源体例えばＮ−エチルＮ （３−メチ
ルフェニル）−Ｎ′−サクンニルエチレンジアミン（Ｅ
ＭＳＥ）、Ｎ−エチルＮ−（２−ヒドロキシ−３−スル
ホプロピル）−３，５−ジメトキシアニリン（ＤＡＯ３
）、Ｎ−エチル−Ｎ−ヒドロキシエチルトルイジン、Ｎ
−エチル−Ｎ−（２−ヒドロキシ−３−スルホプロピル
）−ｍ−アニシジン（ＡＤＯ３）、Ｎ−エチルＮ−スル
イブロピルーｍ−アニシジン（ＡＤＰＳ）Ｎ−エチル−
Ｎ−（２−ヒドロキシ−３−スルホプロピル）アニリン
、Ｎ−エチル−Ｎ−スルホプロピルアニリン、Ｎ−エチ
ル−Ｎ−スルホプロピル−３，５−ジメトキシアニリン
（ＤＡＰＳ）、Ｎ−スルホプロピル−３，５−ジメトキ
シアニリン。Phenol derivatives are used, as anilines, and chromogens used to quantify hydrogen peroxide in the presence of known peroxidases, such as N-ethyl N (3-methylphenyl)-N'-sacunylethylenediamine ( E
MSE), N-ethyl N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline (DAO3
), N-ethyl-N-hydroxyethyltoluidine, N
-Ethyl-N-(2-hydroxy-3-sulfopropyl)-m-anisidine (ADO3), N-ethyl N-suluibropyru-m-anisidine (ADPS) N-ethyl-
N-(2-hydroxy-3-sulfopropyl)aniline, N-ethyl-N-sulfopropylaniline, N-ethyl-N-sulfopropyl-3,5-dimethoxyaniline (DAPS), N-sulfopropyl-3, 5-dimethoxyaniline.
Ｎ （２−ヒドロキシ−３−スルホプロピル）−３
．５−ジフトキンアニリン。Ｎ−エチル−Ｎ−（２−ヒ
ドロキン−３−スルホプロピル）−ｍトルイジン（ＴＯ
Ｏ５）、Ｎ−エチル−Ｎ−スルホプロピル−ｍ）ルイジ
ン（ＴＯＰＳ）、ＮＮ＝Ｎニジスルホプロピルアニリン
ＳＡ）、ＮＮ−ジスルホプロピル−ｍ−トルイジン（Ｄ
ＳＴ）。N (2-hydroxy-3-sulfopropyl)-3
．． 5-diftquinaniline. N-ethyl-N-(2-hydroquine-3-sulfopropyl)-mtoluidine (TO
O5), N-ethyl-N-sulfopropyl-m)luidine (TOPS), NN=Nnidisulfopropylaniline SA), NN-disulfopropyl-m-toluidine (D
ST).
ＮＮ−ジスルホプロピル−ｍ−アニンジン（ＤＳＡＮ）
、ＮＮ−ジスルホプロピル−３，５−ジメトキシアニリ
ン（ＤＳＯＡ）、ＮＮ−ジスルホプロピル−３，５−ジ
メチルアニリン（ＤＳＤＡ）等が［計ハふれ、フェノー
ル類としては、フェア′−ルのほか、ｐ−キンレノール
（Ｐ Ａ ） ＋ ２．４−ノグロルフェノール等が好
適で０．１〜ｌ Ｑ ｍｇ７’ｍｌで用いられる。NN-disulfopropyl-m-aningine (DSAN)
, NN-disulfopropyl-3,5-dimethoxyaniline (DSOA), NN-disulfopropyl-3,5-dimethylaniline (DSDA), etc.; , p-quinlenol (PA) + 2.4-nogrolphenol, etc. are preferred and used in an amount of 0.1 to 1 Q mg 7'ml.
酵素反応を順次行ってもよいが、反応に必要な試薬を一
度に試料に加えて生成色素の定１を行うことができる。Although the enzymatic reactions may be carried out sequentially, the determination of the produced dye can be carried out by adding the reagents necessary for the reaction to the sample all at once.
かかる際には、基質、酸化酵素、カップリング剤、その
他必要な成分を適哨含有するように緩衝液に溶解して試
薬液を調製し、これを用いればよい。In such a case, a reagent solution may be prepared by dissolving the substrate, oxidase, coupling agent, and other necessary components in a buffer solution, and then used.
色素の定量を行うには、生成色素の吸光の変化を反応開
始後の適当な２点で測定し、予め標準試料を用いて検量
線を作成しておき、この検量線からホスファターゼ活性
をｒｖＩ単に求めることができる。To quantify the dye, measure the change in absorbance of the produced dye at two appropriate points after the start of the reaction, create a calibration curve in advance using standard samples, and use this calibration curve to determine the phosphatase activity by simply rvI. You can ask for it.
ホスファターゼは基質をリン酸に分解するばかりでなく
、生成したリン酸を他のヒドロキシ化合物に変換する作
用も有するので、この受容体として種々のヒドロキシ化
合物を用いることが望ましく、例えば、トリスヒドキシ
アミノメタン、プロパツール、２−アミノ−２−メチル
−１−プロパツール エチルアミノエタノール（Ｅ、’
ｌ、Ｅ）、Ｎ−メチル−Ｄ−グルカミン（ＭＥＧ）、
ジェタノールアミン（ＤＥＡ）、トリエタノールアミ
ンなどが利用でき、これらは１０−５０００ｍＭで用い
られる。Since phosphatase not only decomposes the substrate into phosphoric acid but also converts the generated phosphoric acid into other hydroxy compounds, it is desirable to use various hydroxy compounds as this receptor. Methane, propatool, 2-amino-2-methyl-1-proptool ethylaminoethanol (E,'
l, E), N-methyl-D-glucamine (MEG),
Jetanolamine (DEA), triethanolamine, etc. are available and are used at 10-5000mM.
また生体液には脂肪成分などによる濁りがある場合、界
面活性剤やリポプロティンリパーゼ等を用いて可溶化す
ることができる。界面活性剤としては、アルキル硫酸エ
ステル、脂肪酸塩、アルキルベンゼンスルホン酸塩、ア
ルキルナフタレンスルホン酸塩、ジアルキルスルホコハ
ク酸エステル塩、アルキルリン酸エステル塩、ナフタレ
ンスルホン酸ホルマリン縮合物、ポリオキシエチレンア
ルキルＭ酸エステル塩、ポリオキシエチレンアルキルエ
ーテル、ポリオキシエチレンアルキルフェノールエーテ
ル、ポリオキシエチレン脂肪酸エステル、ソルビタン脂
肪酸エステル、ポリオキシエチレンソルビタン脂肪酸エ
ステル、グリセリン脂肪酸エステル、オキシエチレンオ
キシプロピレンブロックコポリマー、アルキルアミン塩
、第４級アンモニウム塩、ポリオキシエチレンアルキル
アミン、アルキルベタインなどが０．１−１００ｍｇ／
ｍ１で用いられる。Furthermore, if the biological fluid has turbidity due to fat components, etc., it can be solubilized using a surfactant, lipoprotein lipase, or the like. As surfactants, alkyl sulfate esters, fatty acid salts, alkylbenzene sulfonates, alkylnaphthalene sulfonates, dialkyl sulfosuccinate ester salts, alkyl phosphate ester salts, naphthalene sulfonic acid formalin condensates, polyoxyethylene alkyl M acid esters Salt, polyoxyethylene alkyl ether, polyoxyethylene alkylphenol ether, polyoxyethylene fatty acid ester, sorbitan fatty acid ester, polyoxyethylene sorbitan fatty acid ester, glycerin fatty acid ester, oxyethylene oxypropylene block copolymer, alkylamine salt, quaternary ammonium Salt, polyoxyethylene alkylamine, alkyl betaine, etc. 0.1-100mg/
Used in m1.
本発明によればｐ−アミノ−置換（もしくは非置換）フ
ェニルリン酸酸化、酸化酵素及びカップリング剤からな
るホスファターゼ定量用組成物が提供される。According to the present invention, a composition for quantifying phosphatase is provided, which comprises a p-amino-substituted (or unsubstituted) phenyl phosphate oxidizing enzyme, an oxidizing enzyme, and a coupling agent.
これらの成分は前述の使用濃度の量比からなる。These components consist of the quantitative ratios of concentrations used above.
この組成物にリン酸受容体５界面活性剤を加えることが
できる。A phosphate acceptor 5 surfactant can be added to this composition.
以下に本発明の態様を実施例によって示す。Aspects of the present invention will be illustrated below by way of examples.
実施例１゜
アルカリホスファターゼの定量
試薬液（ｐ Ｈ＝ ９．５ ）
ＤＥＡ ＩＭｐ−アミノフェ
ニルリン酸 ２ｍｇ／ｍ１ＤＡＯ３１ｍｇ／ｍ１
ＢＬＯＤ Ｏ，５Ｕ／ｍｌト
リ トンＸ −１００１ｍｇ／ｍｌテスト１゜
試料として下記Ａ−Ｅを用いた
一Δ：蒸留水０．０５ｍ１
Ｂ：アルカリホスファターゼ０．０５［１／ｍｌ ０．
０５ｍ１Ｃ： 〃０．１ 〃０．０５１１Ｄ
： ” ０２ 〃 ００５”
Ｅ： 〃０．５ 〃０．０５１１各試料に３
．Ｑｍｌの試薬液を加え３７℃で１０分間保持した後、
６７８止における吸光の変化を測定したときの吸光度は
Ａ−Ｅについてそれぞれ０゜０．２００，０．４０９，
０．８１５．２．０４２であり第１図に検１線として示
される。Example 1 Alkaline phosphatase quantitative reagent solution (pH = 9.5) DEA IMp-aminophenyl phosphate 2 mg/ml DAO 31 mg/ml BLOD O, 5 U/ml
Liton
05m1C: 〃0.1 〃0.0511D
: ” 02 〃 005”
E:〃0.5〃0.05113 for each sample
．． After adding Qml of reagent solution and holding at 37°C for 10 minutes,
When measuring the change in absorbance at the 678 stop, the absorbance was 0°0.200, 0.409, and 0.409 for A-E, respectively.
0.815.2.042 and is shown as the first line in FIG.
テスト２゜
また別途不法の他、ベツンーローリー法（Ｂｅｓｓｙ−
Ｌｏｗｒｙ）に準拠した方法の試薬として（ａ法）アル
カリ性ホスファＢ−テストヮコ−（和光補薬）。Test 2゜Bessy-Lowry method (Bessy-Lawley method)
(Method a) Alkaline Phospha B-Test Wako (Wako Hyakuyaku).
カイノドキング法（Ｋｉｎｄ−Ｋｉｎｇ）に準拠した方
法の試薬として（ｂ法）アルカリ性ホスファに一テスト
ヮコー（和光補薬）の３種層の方法で■アルカリ性ホス
ファターゼ０．ＩＵ／ｍｌを標準にして■黄厄患者血清
をそれぞれ検体として用い、ホスファターゼ活性を１０
回ずつ測定したところ第１表の結果を得た。As a reagent for a method based on the Kind-King method (method b), one test of alkaline phosphatase and one test Wako (Wako Hyakuyaku) are used in a three-layer method. Using IU/ml as the standard, using serum from a patient with yellow fever as a specimen, the phosphatase activity was adjusted to 10
The results shown in Table 1 were obtained by measuring each time.
第
表
本状による測定値は黄痩血清の如何に関わらず再現性の
指標であるＣｖ％に於いて格段によい結果を示している
。The measured values according to Table 1 show extremely good results in terms of Cv%, which is an index of reproducibility, regardless of the type of yellow serum.
実施例２゜
実施例１のＤＡＯ５のかわりにＥＭＳＥ、ＡＤＯ３，Ｔ
ＯＰＳ、Ｍ、ＡＩＰＳ、ＡＤＰＳ、ＤＡＰＳＴＯＯ３，
ＤＳＯＡ、ＰＸを使用してテスト１と同様なテストを行
った。第２表にｐ−アミンフェノールとこれらアニリン
誘導体、フェノールＨ体とがカップリングして生成する
色斗の極大吸収波長を示す。また、これらの波長で測定
した結果の検量線を第２図に示す。Example 2 EMSE, ADO3, T instead of DAO5 in Example 1
OPS, M, AIPS, ADPS, DAPSTOO3,
A test similar to Test 1 was conducted using DSOA and PX. Table 2 shows the maximum absorption wavelengths of Ato produced by the coupling of p-amine phenol, these aniline derivatives, and the H form of phenol. Moreover, the calibration curve of the results of measurements at these wavelengths is shown in FIG.
第 ２ 表 のテスト１を繰返して第１図と同じ検量線が得られた。Table 2 By repeating Test 1, the same calibration curve as shown in FIG. 1 was obtained.
実施例４゜
実施例１のｐ−アミノフェニルリン酸のかわりに■２．
６−ジプロムー４−アミノフェニルリン酸■２．６−
シクロロー４−アミノフェニルリン酸■３．５−ジメチ
ル−４−アミノフェニルリン酸■２カルボキシ−４−ア
ミノフェニルリン酸を使用してテスト１と同じ操作をし
て第３図に示す検１線を得た。但し測定波長は第３表に
示す。Example 4゜Instead of p-aminophenyl phosphoric acid in Example 1, ■2.
6-dipromo-4-aminophenyl phosphate■2.6-
Cycloro-4-aminophenyl phosphate ■ 3.5-dimethyl-4-aminophenyl phosphate ■ Perform the same operation as Test 1 using 2-carboxy-4-aminophenyl phosphoric acid and test line 1 shown in Figure 3. I got it. However, the measurement wavelengths are shown in Table 3.
第 ３ 表
実施例３゜
１例１のビリルビンオキンダーゼの代わりにアスコルビ
ン酸オキシターｔ’ （ｌ ＯＯＵ／ｍｌ） 。Table 3 Example 3゜1 Instead of bilirubin okindase in Example 1, ascorbic acid oxiter t' (l OOU/ml) was used.
ジフェノールオキシダーゼ（１，５Ｕ／ｍｌ） 、モノ
フェノールモノオキシゲナーゼ（１，０／＋１１１）、
又はラフカーゼ（５，ＯＵ／ｍｌ）を使用して実施例１
実施例５゜
酸性ホスファターゼの定量
試薬液（ｐＨ＝４．９）
クエン酸ナトリウム
１Ｍ
ｐ−アミノフェニルリンＩＩ ２ｍｇ／ｍｌＤ
、へ Ｏ５ｌ ■／ ｍ Ｉ
ＢＬＯＤ ０．１単位／ ｍ
Ｉト リ ト ンＸ −１００１ｑ／ｍ１２Ｓ留水、又
は酸性ホスファターゼ（シグマ製）を０．０５． ０．
１． ０．２又は０．５ Ｕ／ｍｌ含有する試侵各Ｑ、
ｆＪ５ｍｌに試薬液３．Ｑ＋ｎｌを加え３７℃で１０分
間の６７８ｎｍにおける吸光の変化を測定した結果第４
図に示す検量線を得た。Diphenol oxidase (1,5 U/ml), monophenol monooxygenase (1,0/+111),
or Example 1 using rough case (5, OU/ml)
Example 5 Acid phosphatase quantitative reagent solution (pH=4.9) Sodium citrate 1M p-aminophenyl phosphorus II 2mg/mlD
, to O5l ■/m I BLOD 0.1 unit/m
I Triton X-1001q/m12S distilled water or acid phosphatase (Sigma) at 0.05%. 0.
1. Each trial Q containing 0.2 or 0.5 U/ml,
Add reagent solution 3 to fJ5ml. The fourth result of adding Q+nl and measuring the change in absorbance at 678 nm at 37°C for 10 minutes.
A calibration curve shown in the figure was obtained.
１１図−第・１図はホスファターセ含量と吸光度、）関
係についてｙ）ｔＱＩ線を示している。
第２図又は第３図における番号は実施例２における色源
体の番号又は実施例４における基質の番号に対応する。
１）、１ ０．λ ｏ、３０，４ ０５七Ｘわ科１（鳴
υFigure 11 - Figure 1 shows the tQI line for the relationship between phosphatase content and absorbance. The numbers in FIG. 2 or 3 correspond to the chromogen numbers in Example 2 or the substrate numbers in Example 4. 1), 1 0. λ o, 30, 4 057
Claims (1)
ェニルリン酸にホスファターゼを作用させ、生成するｐ
−アミノ−置換（もしくは非置換）フェノールを定量す
ることを特徴とするホスファターゼの定量法。p-aminophenyl phosphate or p-amino-substituted phenyl phosphate is reacted with phosphatase to produce p.
A method for quantifying phosphatase, which comprises quantifying -amino-substituted (or unsubstituted) phenol.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63161149A JP2713425B2 (en) | 1988-06-29 | 1988-06-29 | Determination of phosphatase |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63161149A JP2713425B2 (en) | 1988-06-29 | 1988-06-29 | Determination of phosphatase |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH029397A true JPH029397A (en) | 1990-01-12 |
| JP2713425B2 JP2713425B2 (en) | 1998-02-16 |
Family
ID=15729526
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP63161149A Expired - Fee Related JP2713425B2 (en) | 1988-06-29 | 1988-06-29 | Determination of phosphatase |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2713425B2 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114624205A (en) * | 2022-02-28 | 2022-06-14 | 海南绿峰资源开发有限公司 | Detection method of alkaline phosphatase |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS60232099A (en) * | 1984-05-01 | 1985-11-18 | Nitsusui Seiyaku Kk | Method for measuring activity of alkaline phosphatase |
-
1988
- 1988-06-29 JP JP63161149A patent/JP2713425B2/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS60232099A (en) * | 1984-05-01 | 1985-11-18 | Nitsusui Seiyaku Kk | Method for measuring activity of alkaline phosphatase |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114624205A (en) * | 2022-02-28 | 2022-06-14 | 海南绿峰资源开发有限公司 | Detection method of alkaline phosphatase |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2713425B2 (en) | 1998-02-16 |
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