JPS6311848A - Handy measurement of compound related to adenosine-3-phosphoric acid - Google Patents
Handy measurement of compound related to adenosine-3-phosphoric acidInfo
- Publication number
- JPS6311848A JPS6311848A JP61153969A JP15396986A JPS6311848A JP S6311848 A JPS6311848 A JP S6311848A JP 61153969 A JP61153969 A JP 61153969A JP 15396986 A JP15396986 A JP 15396986A JP S6311848 A JPS6311848 A JP S6311848A
- Authority
- JP
- Japan
- Prior art keywords
- adenosine
- atp
- adr
- imp
- compounds
- 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.)
- Pending
Links
- 150000001875 compounds Chemical class 0.000 title claims abstract description 33
- 238000005259 measurement Methods 0.000 title claims description 8
- LNQVTSROQXJCDD-KQYNXXCUSA-N 3'-AMP Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](OP(O)(O)=O)[C@H]1O LNQVTSROQXJCDD-KQYNXXCUSA-N 0.000 title abstract 4
- 238000000034 method Methods 0.000 claims abstract description 22
- GRSZFWQUAKGDAV-UHFFFAOYSA-N Inosinic acid Natural products OC1C(O)C(COP(O)(O)=O)OC1N1C(NC=NC2=O)=C2N=C1 GRSZFWQUAKGDAV-UHFFFAOYSA-N 0.000 claims abstract description 20
- 102100036286 Purine nucleoside phosphorylase Human genes 0.000 claims abstract description 20
- 235000013902 inosinic acid Nutrition 0.000 claims abstract description 20
- 108010009099 nucleoside phosphorylase Proteins 0.000 claims abstract description 20
- OQRXBXNATIHDQO-UHFFFAOYSA-N 6-chloropyridine-3,4-diamine Chemical compound NC1=CN=C(Cl)C=C1N OQRXBXNATIHDQO-UHFFFAOYSA-N 0.000 claims abstract description 19
- 102100033220 Xanthine oxidase Human genes 0.000 claims abstract description 19
- 108010093894 Xanthine oxidase Proteins 0.000 claims abstract description 19
- 102000002260 Alkaline Phosphatase Human genes 0.000 claims abstract description 17
- 108020004774 Alkaline Phosphatase Proteins 0.000 claims abstract description 17
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims abstract description 17
- 108010028584 nucleotidase Proteins 0.000 claims abstract description 17
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims abstract description 13
- 239000007788 liquid Substances 0.000 claims abstract description 13
- 229910052760 oxygen Inorganic materials 0.000 claims abstract description 12
- 239000001301 oxygen Substances 0.000 claims abstract description 12
- 101710169336 5'-deoxyadenosine deaminase Proteins 0.000 claims abstract description 11
- 230000009471 action Effects 0.000 claims abstract description 9
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 claims abstract description 8
- FDGQSTZJBFJUBT-UHFFFAOYSA-N hypoxanthine Chemical compound O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 claims abstract description 7
- UGQMRVRMYYASKQ-KQYNXXCUSA-N Inosine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C2=NC=NC(O)=C2N=C1 UGQMRVRMYYASKQ-KQYNXXCUSA-N 0.000 claims abstract description 6
- AUHDWARTFSKSAC-HEIFUQTGSA-N (2S,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)-2-(6-oxo-1H-purin-9-yl)oxolane-2-carboxylic acid Chemical compound [C@]1([C@H](O)[C@H](O)[C@@H](CO)O1)(N1C=NC=2C(O)=NC=NC12)C(=O)O AUHDWARTFSKSAC-HEIFUQTGSA-N 0.000 claims abstract description 5
- 229940028843 inosinic acid Drugs 0.000 claims abstract description 5
- 239000004245 inosinic acid Substances 0.000 claims abstract description 5
- 239000002126 C01EB10 - Adenosine Substances 0.000 claims abstract description 4
- 229960005305 adenosine Drugs 0.000 claims abstract description 4
- UGQMRVRMYYASKQ-UHFFFAOYSA-N Hypoxanthine nucleoside Natural products OC1C(O)C(CO)OC1N1C(NC=NC2=O)=C2N=C1 UGQMRVRMYYASKQ-UHFFFAOYSA-N 0.000 claims abstract description 3
- 229930010555 Inosine Natural products 0.000 claims abstract description 3
- 229960003786 inosine Drugs 0.000 claims abstract description 3
- UDMBCSSLTHHNCD-KQYNXXCUSA-N adenosine 5'-monophosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H]1O UDMBCSSLTHHNCD-KQYNXXCUSA-N 0.000 claims abstract 9
- UDMBCSSLTHHNCD-UHFFFAOYSA-N Coenzym Q(11) Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(O)=O)C(O)C1O UDMBCSSLTHHNCD-UHFFFAOYSA-N 0.000 claims abstract 8
- LNQVTSROQXJCDD-UHFFFAOYSA-N adenosine monophosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(CO)C(OP(O)(O)=O)C1O LNQVTSROQXJCDD-UHFFFAOYSA-N 0.000 claims abstract 8
- 229960003190 adenosine monophosphate Drugs 0.000 claims abstract 7
- -1 AdR Natural products 0.000 claims abstract 2
- ZKHQWZAMYRWXGA-KQYNXXCUSA-J ATP(4-) Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-J 0.000 claims description 38
- ZKHQWZAMYRWXGA-UHFFFAOYSA-N Adenosine triphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C(O)C1O ZKHQWZAMYRWXGA-UHFFFAOYSA-N 0.000 claims description 37
- 238000012360 testing method Methods 0.000 claims description 8
- 239000000126 substance Substances 0.000 claims description 4
- GZCWLCBFPRFLKL-UHFFFAOYSA-N 1-prop-2-ynoxypropan-2-ol Chemical compound CC(O)COCC#C GZCWLCBFPRFLKL-UHFFFAOYSA-N 0.000 claims description 3
- 102000013563 Acid Phosphatase Human genes 0.000 claims description 3
- 108010051457 Acid Phosphatase Proteins 0.000 claims description 3
- XTWYTFMLZFPYCI-KQYNXXCUSA-N 5'-adenylphosphoric acid Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O XTWYTFMLZFPYCI-KQYNXXCUSA-N 0.000 claims 2
- 102100036664 Adenosine deaminase Human genes 0.000 claims 2
- XTWYTFMLZFPYCI-UHFFFAOYSA-N Adenosine diphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(O)=O)C(O)C1O XTWYTFMLZFPYCI-UHFFFAOYSA-N 0.000 claims 2
- XKVWLLRDBHAWBL-UHFFFAOYSA-N imperatorin Natural products CC(=CCOc1c2OCCc2cc3C=CC(=O)Oc13)C XKVWLLRDBHAWBL-UHFFFAOYSA-N 0.000 claims 1
- 238000006243 chemical reaction Methods 0.000 abstract description 33
- 102000004190 Enzymes Human genes 0.000 abstract description 20
- 108090000790 Enzymes Proteins 0.000 abstract description 20
- 239000000203 mixture Substances 0.000 abstract description 12
- 102000055025 Adenosine deaminases Human genes 0.000 abstract description 10
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 abstract description 2
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 abstract description 2
- 239000002253 acid Substances 0.000 abstract 1
- 238000003556 assay Methods 0.000 abstract 1
- 229960002163 hydrogen peroxide Drugs 0.000 abstract 1
- 229940088598 enzyme Drugs 0.000 description 19
- 239000012085 test solution Substances 0.000 description 17
- 230000009467 reduction Effects 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
- 230000003247 decreasing effect Effects 0.000 description 8
- 229910052698 phosphorus Inorganic materials 0.000 description 8
- 229920006395 saturated elastomer Polymers 0.000 description 8
- 239000000243 solution Substances 0.000 description 6
- 239000003153 chemical reaction reagent Substances 0.000 description 5
- 239000012086 standard solution Substances 0.000 description 5
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 4
- 238000006911 enzymatic reaction Methods 0.000 description 4
- 239000007853 buffer solution Substances 0.000 description 3
- 238000011088 calibration curve Methods 0.000 description 3
- 238000011049 filling Methods 0.000 description 3
- 235000013305 food Nutrition 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 239000011259 mixed solution Substances 0.000 description 3
- 238000011002 quantification Methods 0.000 description 3
- 239000000523 sample Substances 0.000 description 3
- LRFVTYWOQMYALW-UHFFFAOYSA-N 9H-xanthine Chemical compound O=C1NC(=O)NC2=C1NC=N2 LRFVTYWOQMYALW-UHFFFAOYSA-N 0.000 description 2
- 102100022936 ATPase inhibitor, mitochondrial Human genes 0.000 description 2
- 101000902767 Homo sapiens ATPase inhibitor, mitochondrial Proteins 0.000 description 2
- LEHOTFFKMJEONL-UHFFFAOYSA-N Uric Acid Chemical compound N1C(=O)NC(=O)C2=C1NC(=O)N2 LEHOTFFKMJEONL-UHFFFAOYSA-N 0.000 description 2
- TVWHNULVHGKJHS-UHFFFAOYSA-N Uric acid Natural products N1C(=O)NC(=O)C2NC(=O)NC21 TVWHNULVHGKJHS-UHFFFAOYSA-N 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 238000011109 contamination Methods 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 235000013372 meat Nutrition 0.000 description 2
- 230000036284 oxygen consumption Effects 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- 229940116269 uric acid Drugs 0.000 description 2
- PHNGFPPXDJJADG-RRKCRQDMSA-N 2'-deoxyinosine-5'-monophosphate Chemical compound O1[C@H](COP(O)(O)=O)[C@@H](O)C[C@@H]1N1C(N=CNC2=O)=C2N=C1 PHNGFPPXDJJADG-RRKCRQDMSA-N 0.000 description 1
- 241000251468 Actinopterygii Species 0.000 description 1
- 102100035882 Catalase Human genes 0.000 description 1
- 108010053835 Catalase Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 101001020552 Rattus norvegicus LIM/homeobox protein Lhx1 Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- TTWYZDPBDWHJOR-IDIVVRGQSA-L adenosine triphosphate disodium Chemical group [Na+].[Na+].C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP([O-])([O-])=O)[C@@H](O)[C@H]1O TTWYZDPBDWHJOR-IDIVVRGQSA-L 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- GVPFVAHMJGGAJG-UHFFFAOYSA-L cobalt dichloride Chemical compound [Cl-].[Cl-].[Co+2] GVPFVAHMJGGAJG-UHFFFAOYSA-L 0.000 description 1
- 230000002301 combined effect Effects 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000008025 crystallization Effects 0.000 description 1
- 229940079919 digestives enzyme preparation Drugs 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000002848 electrochemical method Methods 0.000 description 1
- 230000037149 energy metabolism Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000003978 infusion fluid Substances 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000011017 operating method Methods 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 235000014102 seafood Nutrition 0.000 description 1
- GEHJYWRUCIMESM-UHFFFAOYSA-L sodium sulfite Chemical compound [Na+].[Na+].[O-]S([O-])=O GEHJYWRUCIMESM-UHFFFAOYSA-L 0.000 description 1
- 235000010265 sodium sulphite Nutrition 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 229940075420 xanthine Drugs 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t1−の矛11ノ>!I’
本発明はる水産巣、食品産業、分析機器産業、分析試薬
産業の各分野で利用される。[Detailed description of the invention] I' The present invention can be used in various fields such as aquatic nests, food industry, analytical equipment industry, and analytical reagent industry.
え米立且遺
従来ATP (アデノシン3リン酸)関連化合物すなわ
ちATP分解物の組成分析にはクロマトグラフ法や酵素
法が行われていたが、その操作には数十分ないし数時間
を要し、しかも高価な液体クロマトグラフ装置や紫外分
光光度計を要していたが、先に木発明者等はATP分解
物の組成1分析を数分間程度で迅速にかつ簡便な溶存酸
素(DO)測定装置によって行い、魚、肉類の鮮度を判
定する方法を見出した(特開昭59−232097およ
び特開昭60−4?H5)。Traditionally, chromatographic methods and enzymatic methods have been used to analyze the composition of ATP (adenosine triphosphate)-related compounds, that is, ATP decomposed products, but these procedures require tens of minutes to several hours. , and required an expensive liquid chromatography device and ultraviolet spectrophotometer, but the inventors of the tree have developed a method to quickly and easily measure dissolved oxygen (DO) to analyze the composition of ATP decomposed products in just a few minutes. We have found a method for determining the freshness of fish and meat using a device (Japanese Patent Application Laid-open No. 59-232097 and Japanese Patent Application Laid-Open No. 60-4-H5).
特開昭59−232097においてはキサンチンオキシ
ダーゼ(XO) 、ヌクレオシドホスフォリラーゼ(N
P)+キサンチンオキシダーゼ(XO) 、アルカリホ
スファターゼ(AP)+ヌクレオシドホスフォリラーゼ
(NP)+キサンチンオキシダーゼ(XO)の各酵素の
作用の下に溶存酸素(DO)減少量を用いて、それぞれ
ヒポキサンチン (H11)、イノシン(HX R)
、およびイノシン酸(IMP)の量を求めた。In JP-A-59-232097, xanthine oxidase (XO) and nucleoside phosphorylase (N
P) + xanthine oxidase (XO), alkaline phosphatase (AP) + nucleoside phosphorylase (NP) + xanthine oxidase (XO) using the reduced amount of dissolved oxygen (DO) under the action of each enzyme, hypoxanthine (H11), inosine (HX R)
, and the amount of inosinic acid (IMP) were determined.
特開昭60−471395においては、高純度のアルカ
リホスファターゼ(A P)の代わりに粗アルカリホス
ファターゼ(粗AP)を用いることによって、粗AP+
NP+XOの複合作用によって、前記出願の適用範囲を
拡げて、ATP+ADP本A I’vl P+ I M
P+H! R+H!の測定を可能とした。In JP-A-60-471395, by using crude alkaline phosphatase (crude AP) instead of highly purified alkaline phosphatase (AP), crude AP+
Due to the combined effect of NP+XO, the scope of application of the above application is expanded and ATP+ADP book A I'vl P+ I M
P+H! R+H! This made it possible to measure
ネ11が ・ しようと干るIjtIInへATPは動
物のエネルギー代謝の鍵物質(Keysubstanc
e )であることからATP関連化合物の411定は玉
要である。前述したように、本発明者等は既に特定の酵
素の作用下にDoの減少によりATP関連化合物を測定
したが、本発明では更に酵素の使用種類を拡張して1本
発明者等のこれまで開発した技術を更に発展せしめよう
とするもので、特にAMP、Ad Rの分別定量を可能
にし、本発明者等のall定法の利用範囲を拡げること
を目的としている。ATP is a key substance in the energy metabolism of animals.
e), the 411 constant of ATP-related compounds is important. As mentioned above, the present inventors have already measured ATP-related compounds by decreasing Do under the action of a specific enzyme, but in the present invention, the types of enzymes used are further expanded. This is an attempt to further develop the developed technology, and in particular, the purpose is to enable the separate quantification of AMP and Ad R, and to expand the scope of use of the all standard method developed by the present inventors.
IELjm占 L るための一
本発明はATP関連化合物を定量する際、酵素ヌクレオ
シドホスホリラーゼ(N P)とキサンチンオキシダー
ゼ(XO)の複合作用の下に溶存酸素減少量又は過酸化
水素生成量の電気化学的信号によりall定する方法に
おいて、被検液にNPとxoの複合作用をさせる前に、
(i)酸ホスファターゼ(Ac P)もしくはヌクレオ
チダーゼ(NT)を作用させるか、(ii)アデノシン
デアミナーゼ(ADA)を作用させるが、 (iii)
ヌクレオチダーゼ(NT)を作用させたのちに、ADA
を作用させることによって、それぞれIMP、Ad R
,AMPを測定し、かくしてこれらの化合物の少くとも
1つを測定することよりなるATP関連化合物の測定方
法を提供するものであって、又この方法を使用する際に
、被検液に前景って、粗アルカリホスファターゼ(11
AP)を添加し予備反応させることによって、ATP関
連化合物の混合液よりATP+ADP+AMP+Ad
R+I MF+Hz R+Hx (以後、総ATP関
連化合物と呼ぶ)、AMP、Ad R,IMP、HX
R。The present invention is an electrochemical method for determining the amount of dissolved oxygen decreased or the amount of hydrogen peroxide produced under the combined action of the enzymes nucleoside phosphorylase (NP) and xanthine oxidase (XO) when quantifying ATP-related compounds. In the method of determining all by the target signal, before subjecting the test liquid to the combined action of NP and xo,
(i) act with acid phosphatase (Ac P) or nucleotidase (NT), or (ii) act with adenosine deaminase (ADA), but (iii) act with adenosine deaminase (ADA).
After acting with nucleotidase (NT), ADA
By acting on IMP and Ad R, respectively.
, AMP, and thus at least one of these compounds; and crude alkaline phosphatase (11
By adding AP) and pre-reacting, ATP+ADP+AMP+Ad is obtained from the mixture of ATP-related compounds.
R+I MF+Hz R+Hx (hereinafter referred to as total ATP-related compounds), AMP, Ad R, IMP, HX
R.
HXを溶存酸素減少量又は過酸化水素生成量の電気化学
的信号によりΔIII定する方法を提供するものである
。The present invention provides a method for determining HX by ΔIII based on an electrochemical signal of the amount of dissolved oxygen decreased or the amount of hydrogen peroxide produced.
本発明はATP関連化合物の測定において、下記フロー
シート(1)に示される酵素反応にもとづき、各成分を
順次酵素化学的に変化させ、最終的にキサンチンオキシ
ダーゼ(XO)によりヒボキサンチン(Hz)がキサン
チン(X)を経て、尿酸(UA)に酸化される過程で、
HX 1分子につき吸収される2分子の02又は生成さ
れる2分子のH2O2の漬を電気化学的センサーでとら
えることを骨子とするものである。In the measurement of ATP-related compounds, the present invention sequentially enzymatically changes each component based on the enzymatic reaction shown in the flow sheet (1) below, and finally converts hyboxanthin (Hz) to xanthine using xanthine oxidase (XO). In the process of being oxidized to uric acid (UA) through (X),
The main idea is to use an electrochemical sensor to detect two molecules of 02 absorbed or two molecules of H2O2 produced per molecule of HX.
フローシーh(1)中で粗AP(粗アルカリホスファタ
ーゼ)とあるのは、本発明者等が先の出願(特開昭Go
−47895)で述べた如<、ATP、ADP、AMP
にも作用を及ぼすAP酵素液で、この粗APはざらにA
d Rも分解することがわかっている。なおAPには高
純度のものもあるが、このものはIMFをHxRに変換
するのみで、ATP、ADP、AMPには有効に作用し
ない。The term crude AP (crude alkaline phosphatase) in Flow Sheet h(1) was originally published by the present inventors in the earlier application (Japanese Patent Laid-Open Publication No.
-47895), ATP, ADP, AMP
This is an AP enzyme solution that also acts on
It is known that dR is also decomposed. Although some APs are of high purity, they only convert IMF to HxR and do not effectively act on ATP, ADP, and AMP.
本発明に用いるセンサーにはポーラログラフ式あるいは
ガルへ二電池式酸素センサー及びポーラログラフ式過酸
化水素センサー等公知のものが用いられる。これらのセ
ンサーの出力信号はAll!ll分定モル濃度の2倍に
相当する電流であるため、酸素濃度又は過酸化水素濃度
既知の純水を基準として、測定成分の標準液なしで定量
することができる。さらに試料中に紫外線吸収性の物質
や色や薊りがあってもalll 疋に何等支障を生じな
い利点がある。As the sensor used in the present invention, a known sensor such as a polarographic type, a two-cell galvanic oxygen sensor, or a polarographic hydrogen peroxide sensor can be used. The output signals of these sensors are All! Since the current corresponds to twice the fixed molar concentration, it is possible to quantify the component to be measured without using a standard solution of the component to be measured, using pure water with a known concentration of oxygen or hydrogen peroxide as a reference. Furthermore, there is an advantage that even if there is an ultraviolet absorbing substance, color, or tinge in the sample, there will be no problem in all cases.
;(発明に用いる装置は、小型軽量で取扱いも容易なた
め、実験室以外の生産現場、農場、野外で使用すること
も可能である。(The device used in the invention is small, lightweight, and easy to handle, so it can be used at production sites other than laboratories, on farms, and outdoors.
第1図は1本発明に用いる反応槽及び装置系統図である
。すなわち図中1は反応槽で、その容積は1〜21程度
の小さいものが酵素、試薬の節約ト有利である。2は反
応槽の密栓でその中心に液注入用の例えば直径1mm程
度の細い穴3が備えられている。この細穴に入った液は
水封効果つまりクスターラーの攪拌子、6は温度制御用
ジャケットで外部の恒温水7を循環させるためのもので
ある0反応槽の形状は特に限定はないが、反応液の定常
的混合攪拌ができて反応温度の制御、試薬の注入に便利
で、しかも外界からの酸素が反応液に溶は込まない構造
+あることが必須条件である。FIG. 1 is a system diagram of a reaction tank and equipment used in the present invention. That is, 1 in the figure is a reaction tank, and a small one having a volume of about 1 to 21 is advantageous in terms of saving enzymes and reagents. Reference numeral 2 denotes a sealing plug for the reaction tank, and a thin hole 3 having a diameter of about 1 mm, for example, is provided in the center for liquid injection. The liquid entering this small hole has a water seal effect, that is, a Kusterer stirrer, and 6 is a temperature control jacket for circulating constant temperature water 7 from the outside.The shape of the reaction tank is not particularly limited, but It is essential to have a structure that allows constant mixing and stirring of the reaction solution, which is convenient for controlling the reaction temperature and injection of reagents, and which prevents oxygen from the outside world from dissolving into the reaction solution.
センサー8は前記したもののうちの任意のものが用いら
れる。9は増幅器である。10は市阪の任意のmVレコ
ーダでIOmVフルスケールで分’E位のスピードでの
記録可能なものが用いられる。11は測定結果を自動も
ガ算する電算機であるが必ずしも必要ではない。As the sensor 8, any one of those described above can be used. 9 is an amplifier. Reference numeral 10 indicates an arbitrary mV recorder manufactured by Ichisaka, which is capable of recording at a speed of approximately 100 mV at a full scale of 10 mV. Reference numeral 11 is a computer that automatically calculates the measurement results, but this is not necessary.
次に本発明に用いる酵素について説明する。Next, the enzyme used in the present invention will be explained.
第1表に水沈で用いる酵素とその反応条件を示す。組ア
ルカリホスファターゼ(粗AP)、l’dホスファター
ゼ(Ac P)、ヌクレオチダーゼ(N T )の3酵
素は試料液を別容器で予備反応させる。また酵素製剤に
おこりがちなカタラーゼの混入による02発生現象は水
沈の実施上の大きな妨げとなるので、これを防ぐためリ
ン酸緩衝液にアジ化ナトリウム(NaN3)をlIII
M程添加することが望ましい。Table 1 shows the enzymes used in water precipitation and their reaction conditions. The sample solution is preliminarily reacted with the three enzymes, alkaline phosphatase (crude AP), l'd phosphatase (Ac P), and nucleotidase (N T ) in a separate container. In addition, the occurrence of 02 due to the contamination of catalase, which tends to occur in enzyme preparations, is a major hindrance to the implementation of water precipitation, so to prevent this, sodium azide (NaN3) is added to the phosphate buffer.
It is desirable to add about M.
/′
7、/
/′
/−m−−、−−
なお、以下の実施例では第2表に示したATP関如化合
物の純品を混合して、各種のATPI″A連化合物の混
在する液を:A製して用いた。/' 7, / /' /-m--, -- In the following examples, the pure products of the ATP compounds shown in Table 2 are mixed, and various ATPI''A-related compounds are mixed. The liquid was prepared from A and used.
注二 よ 結晶水を考慮しないで純度を算出した。(こ
の試薬のロフトは
Cl0H1IN4 N a20.lP ・7.5 Hz
0の分子式を示した)
1−記名試薬単独のIQ mMの水溶液を原液として調
製し1次に各原液を適宜混合し、更に0.1Mのリン酸
緩衝液で希釈して、各成分の所定の濃度を有する被検液
をrA製した。Note 2: Purity was calculated without considering water of crystallization. (The loft of this reagent is Cl0H1IN4 N a20.lP ・7.5 Hz
0) 1- Prepare an IQ mM aqueous solution of the named reagent alone as a stock solution. First, mix each stock solution appropriately, and further dilute with 0.1M phosphate buffer to obtain the specified concentration of each component. A test solution having a concentration of was prepared by rA.
実施例
(a)イノシン酸(IMP)の定量
以下酸素センサーを用いる場合について操作手順を述べ
るが、過酸化水素センサーの場合もこれに準する。Example (a) Quantification of inosinic acid (IMP) The operating procedure will be described for the case where an oxygen sensor is used, but the same applies to the case where a hydrogen peroxide sensor is used.
被検液:
L通したようにして、IMF、HK R,Hzの3成分
をそれぞれ0.2mMの濃度で含有する被検液を調製し
た。Test solution: A test solution containing three components, IMF, HK R, and Hz, each at a concentration of 0.2 mM, was prepared by passing the sample through L.
予備反応条件:
被検液中のIMFを予めHz Rに変化するために、被
検液の一部に、酵素AcPもしくはNTを添加して予備
反応させた。この被検液を52とする。その予備反応の
条件は第3表の通りである。Preliminary reaction conditions: In order to change the IMF in the test solution to Hz R in advance, the enzyme AcP or NT was added to a portion of the test solution for a preliminary reaction. This test liquid is designated as 52. The conditions for the preliminary reaction are shown in Table 3.
第3表予備反応条件
定111操作:
2000μ容積の反応槽(第1図(1))に37℃で空
気飽和させたP、B、緩衝液を充填し、密栓(2)をし
、その細穴(3)よりマイクロシリンジで、被検液S+
(予備反応させない) 50ILjを注入する。そ
してDOレコーダーの指示が安定しタラ、 X OトN
P If) IA合酵素液25gj(MO=20μ、
NP=5μ)を注入する。ただちに酵素反応が起こり、
第2図のようにHz +Hx Rの酸化に由来するDO
減少dTo+)!xRがレコーダー1−に記録される。Table 3 Preliminary Reaction Condition Setting 111 Operation: A 2000 μ volume reaction tank (Figure 1 (1)) was filled with air-saturated P, B, and buffer solutions at 37°C, sealed tightly (2), and the tube was sealed. Inject test liquid S+ with a microsyringe through hole (3).
(Do not pre-react) Inject 50 ILj. And the instructions on the DO recorder became stable.
P If) IA synthetic enzyme solution 25gj (MO=20μ,
NP=5μ). An enzymatic reaction occurs immediately,
As shown in Figure 2, DO derived from the oxidation of Hz +Hx R
decrease dTo+)! xR is recorded on recorder 1-.
DO減少の停止トを確認したのち、L記の如く予備反応
させた混合液S2100μ(この量は5150μに相当
する)を注入すると、今度はHz + HX R+ I
Mpt:由来するDO減少di(we)lxR+l1
IPが記録される。After confirming that the DO decrease has stopped, inject 2100μ of the mixed solution S2 (this amount corresponds to 5150μ) pre-reacted as described in L, this time Hz + HX R + I
Mpt: derived DO decrease di(we)lxR+l1
IP is recorded.
d IMP : dH!◆H!R−IMP ’
H!+H!Rこの式で示されるように第2段と第1段
のり。dIMP: dH! ◆H! R-IMP'
H! +H! RThe second stage and the first stage glue as shown in this formula.
減少値の差がIMFの濃度に対応するDo減少幅に相当
する。勿論、酵素xOとNPを混合液の形でなく、先ず
xoを添加して、それに相当するDO減少を記録した上
で、NPを添加してその際のDO減少を記録することも
できる。この場合はdI(!+I(IR= d )It
十d 1(tRとなる。このIMFの定量の方法におい
ては、予備反応において用いられる酵素Ac P又はN
Tが、S2として反応槽に注入される際に、S1中のI
MF成分が化学変化しないことを利用している。即ちA
c Pの作用はP、B、 l衝液の燐酸イオンによって
阻害されるために1反応槽内のP、B、緩衝液中にS2
を注入してもS1中のIMF成分は変化せず、又NTを
用いた場合は、アルカリ性のS2液を注入しても、反応
槽のpHはP、B、緩挿l液にもとづいて殆ど変化せず
、中性付近に保たれるために、NTがS1中のIMF成
分に作用しないからである。The difference between the decrease values corresponds to the Do decrease width corresponding to the IMF concentration. Of course, instead of using the enzyme xO and NP in the form of a mixed solution, it is also possible to first add xo and record the corresponding DO decrease, and then add NP and record the DO decrease at that time. In this case, dI(!+I(IR=d)It
10d 1 (tR). In this method for quantifying IMF, the enzyme Ac P or N used in the preliminary reaction
When T is injected into the reaction tank as S2, I in S1
It takes advantage of the fact that the MF component does not undergo chemical changes. That is, A
c Since the action of P is inhibited by phosphate ions in the P, B, and l buffer solutions, S2 is present in the P, B, and buffer solutions in one reaction tank.
Even if NT is injected, the IMF component in S1 does not change, and when NT is used, even if alkaline S2 solution is injected, the pH of the reaction tank remains almost the same based on P, B, and the slow infusion solution. This is because NT does not affect the IMF component in S1 because it remains unchanged and remains near neutrality.
次に濃度の算出法を述べる。37℃の空気飽和水を満た
した密栓をした反応槽に微量の塩化コバルトを添加した
0、5M Na2SO3溶液 10044を注入し、
DO飽和からDoゼロに至る減少曲線を得て、減少幅d
oを測定する。この長さが37℃における02飽和10
.214 g mat/ajに対応するので1次の(2
)式によりIMFの濃度が求められる。Next, we will explain how to calculate the concentration. A 0.5M Na2SO3 solution 10044 to which a trace amount of cobalt chloride was added was injected into a tightly closed reaction tank filled with air-saturated water at 37°C.
A decreasing curve from DO saturation to Do zero is obtained, and the decreasing width d
Measure o. This length is 02 saturation 10 at 37℃
.. Since it corresponds to 214 g mat/aj, the linear (2
) The concentration of IMF is determined by the formula.
但し (2)式において
C:定量成分の濃度(u ll1o l/m1)d :
定量成分についてのDo減少幅(cm)do:空気飽和
水についてのDo減少幅(CII)Go’2 :空気
飽和水の酸素濃度(pmol/m1)37℃では0.2
14 (gmol/m1)2 :酸素当付数
V 二反応槽の容積(IIJ)
vs:被検液(Sl)の容積(μ)
かくシテ求められたIMP、Hz R,Hz各成分の濃
度は定量的であり、最初の0.20Mに近い値を示した
。However, in formula (2), C: concentration of quantitative component (u ll1o l/m1) d:
Do reduction width for quantitative components (cm) do: Do reduction width for air-saturated water (CII) Go'2: Oxygen concentration in air-saturated water (pmol/ml) 0.2 at 37°C
14 (gmol/m1) 2: Number of oxygen units V Volume of two reaction vessels (IIJ) vs: Volume of test liquid (Sl) (μ) Thus, the determined IMP, Hz R, Hz The concentration of each component is It was quantitative and showed a value close to the initial 0.20M.
第6図(1)、(2)はそれぞれAc P、NTを用い
てIMF濃度既知の標準液を用いて、IMF濃度と酸素
消費量(この消費量は標準液を用いたときのDo減少値
と前記do値との関係より算出できる)をプロットした
検量線である0両図によってどちらの酵素を用いてもI
MF1モルに対して0□2モルが消費されることが定量
的に示され、IMPc度を02消費による電気化学的信
号によって定量する本発明方法の意義がある。Figures 6 (1) and (2) show the IMF concentration and oxygen consumption (this consumption is the Do reduction value when using the standard solution) using a standard solution with a known IMF concentration using Ac P and NT, respectively. According to the calibration curve, which is calculated from the relationship between the
It has been quantitatively shown that 0□2 mol is consumed per 1 mol of MF, and there is significance in the method of the present invention in which the degree of IMPc is determined by an electrochemical signal based on 02 consumption.
なお、測定誤差を生じないためには1反応(測定)終了
時に残存する溶存酸素(DO)が、位相時の少くとも1
0〜20%残存するようにすることが必要である。その
ためには、反応槽に注入する被検液の4を予め十分調整
する。In addition, in order to avoid measurement errors, the dissolved oxygen (DO) remaining at the end of one reaction (measurement) must be at least 1
It is necessary that 0 to 20% remain. For this purpose, the amount of the test liquid to be injected into the reaction tank is sufficiently adjusted in advance.
(b)アデノシン(Ad R)の定着
反応槽に37℃で十分に空気飽和したP、B 、を充填
し密栓後、H! 、Hz R及びAd Rをそれぞれ0
.2mMを含む被検液50μを注入し、次に酵素xO+
NP、25gjを注入する。第3図のように、初めのD
Oの減少がレコーダーに記録されるので、その反応の停
止トを確認してから、酵素ADA5μをさらに注入する
と再びDO減少(dAdR)が記録される。かくして求
められたAd R,H! RlHzの濃度はそれぞれ最
初の0.2mMに近い値を示した。第7図は、Ad R
の標準液について上記方法で求めた酵素消費量の検量線
であり、AdR1モルに対し2モルの02が吸収される
ことが分る。(b) Fill an adenosine (Ad R) fixing reaction tank with P, B, sufficiently saturated with air at 37°C, and after sealing, H! , Hz R and Ad R are respectively 0.
.. Inject 50μ of test solution containing 2mM, then enzyme xO+
Inject 25gj of NP. As shown in Figure 3, the first D
Since the decrease in O is recorded on the recorder, after confirming that the reaction has stopped, when the enzyme ADA5μ is further injected, the decrease in DO (dAdR) is recorded again. Thus obtained Ad R,H! The RlHz concentration each showed a value close to the initial 0.2mM. Figure 7 shows Ad R
This is a calibration curve of the enzyme consumption amount determined by the above method for the standard solution of AdR, and it can be seen that 2 moles of 02 are absorbed per 1 mole of AdR.
したがって実用的には、特にこのような検量線を作製せ
ず、(2)式を用いてAd Rを直ちに定量することが
できる。Therefore, practically, Ad R can be immediately quantified using equation (2) without particularly preparing such a calibration curve.
(c)AMPの定量
別容器にG、B、 180ILj、 NT20gj、
H! 、 HxR,AMP、IMF、Ad Rいずれも
0.2mMを含む検液200ILjをとり、37℃で約
5分間予備反応させる0反応槽に37℃で空気飽和した
P、B、を充填した後、密栓をし、上記の如く予備反応
させた混合液S、 100μ(この量は原検液50g
jに相当する)を注入し、次にXO+NP25μを注入
すると、(a)で述べたように、Hx+HxR+IMP
に由来するDO減少が得られる。レコーダーでDO減少
の停止を確認してからADA5μを注入すると、AMP
+Ad Rに由来するDo減少d AMPやAdRが得
られる(第4図(1))、これはNTがIMPをHXR
にするだけでなく、AMPをAd Rにする特性をもっ
ためである。次に、新たに反応槽に37°Cで空気飽和
したP、B、を充填した後、v:栓をし、原検液50μ
を注入して、(b)で述べたと全く同様な操作を繰返し
てd AdRを得る(第4図(2) ) −dAMp
= dAMp+AaR−claaRによってAMPの量
に相当するDo減少’[d AMPを得るが、この際第
4図(1)、(2)に示す2本のり。(c) G, B, 180ILj, NT20gj, in AMP quantitative containers.
H! Take 200 ILj of test solution containing 0.2mM of each of HxR, AMP, IMF, and Ad R, and pre-react at 37°C for about 5 minutes.After filling the reaction tank with P and B saturated with air at 37°C, Seal tightly and add 100μ of the mixed solution S pre-reacted as above (this amount is equivalent to 50g of the original test solution).
j) and then XO+NP25μ, Hx+HxR+IMP as mentioned in (a)
A DO reduction derived from the above is obtained. When ADA5μ is injected after confirming the stoppage of DO decrease with a recorder, AMP
+Do decrease derived from AdR AMP and AdR are obtained (Figure 4 (1)), this is because NT converts IMP into HXR.
This is because it has the property of not only converting AMP to Ad R, but also converting AMP to Ad R. Next, after newly filling the reaction tank with P and B saturated with air at 37°C, the v: is stoppered, and the original test solution is 50μ
and repeating the same operation as described in (b) to obtain dAdR (Figure 4 (2)) -dAMp
= dAMp + AaR - Do decrease corresponding to the amount of AMP by claaR' [d AMP is obtained, but at this time, the two bonds shown in Fig. 4 (1) and (2).
減少曲線を利用することができる。かくして得られたd
AMPを(2)式に代入してAMP濃度を算出する。A decreasing curve can be used. Thus obtained d
The AMP concentration is calculated by substituting AMP into equation (2).
かくして求められたAMPおよびその他の成分の1=度
はそれぞれ最初の0.2mMに近い値奢示した。−[−
記の方法においてXO+NPの混合液の代りにxOおよ
びNPをそれぞれ順次注入してHXおよびHXRの含量
に相当するD O減少幅を記録して行うこともできる。The thus determined concentrations of AMP and other components were close to the initial value of 0.2 mM, respectively. −[−
The method described above can also be carried out by sequentially injecting xO and NP instead of the XO+NP mixture and recording the DO reduction width corresponding to the HX and HXR contents.
第8図は標準液を用いて得たAMP濃度と酸素消費量と
の関係を示す検に線である。FIG. 8 is a line showing the relationship between AMP concentration and oxygen consumption obtained using a standard solution.
(d)ATP関連化合物の混合液中のH! % HX
RX、l/IMF、Ad R,AMPおよび総ATP関
連化合物の定量法。(d) H! in a mixture of ATP-related compounds! %HX
Methods for determination of RX, l/IMF, Ad R, AMP and total ATP related compounds.
本発明者等が先に出願した特開昭80−471395の
中で述べたHX 、Hz R,1ATP関連化合物のJ
11定法に前記(a)(b) (c)を組み合わせると
ATP関連化合物の混合液よりHX 、HX RlIM
F、Ad RおよびAMPの5次分を短時間で定量する
ことができる。J of HX, Hz R, 1ATP related compounds described in JP-A-80-471395 previously filed by the present inventors.
11 When the above (a), (b) and (c) are combined with the conventional method, HX, HX RlIM can be obtained from a mixture of ATP-related compounds.
The fifth-order components of F, Ad R, and AMP can be quantified in a short time.
(d−1)
別容器Aに、G、B、 180gj、 NT20tLj
、第2表注に記した要領で調製したHX 、HX R,
IMP、AdR,AMP、ADP、ATPの0.2mM
を舎む被検液200μを採り、37°Cで約5分間予備
反応させておく(これを被検液S2とする)。又、別容
器BにG、8.18Qμ、粗AP2Qμ、上記と同じ検
液200μをとり、37°Cで約5分間予備反応させて
おく(これを被検液S3とする)0反応槽に37°Cで
空気飽和したP、B、を充填した後、密栓をし、第5図
(1)に従って検液S、50μを注入し。(d-1) In another container A, G, B, 180gj, NT20tLj
, HX , HX R, prepared as described in the notes to Table 2
0.2mM of IMP, AdR, AMP, ADP, ATP
Take 200μ of the test solution containing the sample and pre-react it at 37°C for about 5 minutes (this will be referred to as test solution S2). Also, in a separate container B, take G, 8.18Qμ, crude AP2Qμ, and 200μ of the same test solution as above, and pre-react at 37°C for about 5 minutes (this will be the test solution S3). After filling the tube with air-saturated P and B at 37°C, seal the tube tightly and inject 50μ of test solution S according to Fig. 5 (1).
レコーダーの指示が安定したら、X02Qμを注入しH
Xの量に相当するDO減少値diを読みとり、次にNP
5gjを注入しHxRの量に相当するdlを読みとる0
次に、別容器Aで予備反応させた液52100μ(この
量はS、 50μの内容と同等である)を注入してHx
+Hx R+IMFの量に相当するd3を記録する0
次にADA5μを注入してAMP+2Ad Rの量に相
当するd、を記録する(2AdRはSl とS2中の含
量の合計を示す)0次に別器Bで予備反応させた検液5
3100μ(この量はS、 50μや52100μに相
当する)を反応槽に注入して総ATPrA連化合物に相
当するd5を記録する。旧述したように、別容器Aおよ
びBで予備反応をPHアルカリ側で行い、反応槽内の反
応を中性付近で行うことによって、最適pHの差異によ
る各反応を使い分けることができる。When the recorder indication is stable, inject X02Qμ and H
Read the DO reduction value di corresponding to the amount of X, then NP
Inject 5gj and read dl corresponding to the amount of HxR0
Next, inject 52,100μ of the pre-reacted liquid in another container A (this amount is equivalent to the content of S, 50μ) and add Hx.
+Hx R+Record d3 corresponding to the amount of IMF0
Next, inject 5μ of ADA and record d, which corresponds to the amount of AMP + 2AdR (2AdR indicates the sum of the contents in Sl and S2).
Inject 3100μ (this amount corresponds to S, 50μ or 52100μ) into the reaction vessel and record d5, which corresponds to the total ATPrA conjugate. As previously mentioned, by performing preliminary reactions in separate containers A and B on the alkaline pH side and performing reactions in the reaction vessels near neutrality, it is possible to use each reaction depending on the difference in optimum pH.
[−記の結果より直接AMPとAd Rを定量すること
がでさないために1次のような実験を続ける。上記の実
験と同じ条件であるが、注入する順序を4g 、XO,
NP、ADA、S2 、Slとして第5図(2)のよう
なりo減少曲線を得る。[-Since it is not possible to directly quantify AMP and AdR from the results described above, the following experiment is continued. The same conditions as in the above experiment, but the injection order was changed to 4g, XO,
For NP, ADA, S2, and Sl, a decreasing curve as shown in FIG. 5 (2) is obtained.
この時、各減少幅は、 dlがHXの量に相当し、
dlがHz Rの量に、d3′がAd Rの量に、d4
′がHX +HX R+IMF+Ad R+AMPのか
に、 d、が総ATP関連化合物の量に相当する。よっ
て dHx=d+ 、dHxR= dl、d Iにp
= d3 (dl + dl )
、 d AdR= d:+′ 、d AM
P = +L −2d3’ = dn’ (d3+
d3’ )となる。これらの値を(2)式に代入して
各成分濃度が求められる。又d5やa、、 −d、;の
偵を(2)式に代入して夫々総ATP関連化合物および
ADP+ATPの濃度を求めることができる。かくして
求められた各成分の濃度は定量的で、最初の0.2mM
に近い値を示した。At this time, each decrease width is as follows: dl corresponds to the amount of HX,
dl is the amount of Hz R, d3' is the amount of Ad R, d4
' is HX + HX R + IMF + Ad R + AMP, and d corresponds to the amount of total ATP-related compounds. Therefore, dHx=d+, dHxR= dl, p in dI
= d3 (dl + dl)
, d AdR= d:+' , d AM
P = +L -2d3' = dn' (d3+
d3'). By substituting these values into equation (2), the concentration of each component is determined. Also, by substituting d5, a, -d, ; into equation (2), the concentrations of total ATP-related compounds and ADP+ATP can be determined, respectively. The concentration of each component determined in this way is quantitative, and the initial concentration of 0.2mM
It showed a value close to .
(d−2)
Hx O,1mM
HX R,IMF、Ad R,AMP、A D P 、
A T P 0.2mM(総ATP関連化合物 1
.3mM)
を用いて(d−1)と同様な測定を2回行った。求めら
れた各成分の濃度は下記の通りであるが、略定量的であ
った。(d-2) HxO, 1mM HXR, IMF, AdR, AMP, ADP,
ATP 0.2mM (total ATP-related compounds 1
.. The same measurements as in (d-1) were performed twice using 3mM). The determined concentrations of each component are as shown below, and were approximately quantitative.
NoI O,130,190,150,130,25
1,29No2 0.14 0.20 0.29 0.
14 0.28 1.41(単位mM)
(d−3)
)(! 、HX R,IMP、AMP、ADP、ATP
各0.2mM
(総A T P 155連化合物(ただしAd Rを含
まず) 1.2mM)
を含む被検液を用いて(d−1)における第5図(1)
に従って4回A11l定操作を行った。求められた各成
分の濃11は下記の通りであるが、略定品−的であった
。NoI O, 130, 190, 150, 130, 25
1,29No2 0.14 0.20 0.29 0.
14 0.28 1.41 (unit: mM) (d-3) ) (!, HX R, IMP, AMP, ADP, ATP
Figure 5 (1) in (d-1) using a test solution containing 0.2mM of each (1.2mM of total ATP 155 compound (excluding Ad R))
A11l constant operation was performed four times according to the procedure. The concentration 11 of each component determined is as shown below, and was approximately standard.
を含まず)
Not O,170,170,120,221,15
No2 0.14 0.18 0.27 0.22
1.27No3 0.13 0.18 0.28 Q
、22 1.28No4 0.13 0.17 0.
48 0.27 1.27(単位mM)
(d−4)
Hllo、11!IM
HXR,IMF 各0.1511IMAMP、ADP
、 ATP 各0.2 mM総ATP関連化合物(ただ
しAd Rを含まず)1.0mM
を含む被検液を用いて(d−3)と同様に4回Δ11j
2操作を行った。求められた各成分の濃度は下記の通り
であるが、略定hl的であった。(not including) Not O, 170, 170, 120, 221, 15
No2 0.14 0.18 0.27 0.22
1.27No3 0.13 0.18 0.28 Q
,22 1.28No4 0.13 0.17 0.
48 0.27 1.27 (unit: mM) (d-4) Hllo, 11! IM HXR, IMF 0.1511 each IMAMP, ADP
, ATP 4 times in the same manner as in (d-3) using a test solution containing 0.2 mM each of total ATP-related compounds (but not including AdR) and 1.0 mM.
Two operations were performed. The determined concentrations of each component are as follows, and were approximately constant HL.
総ATP関連化合
を含まず)
Not O,130,170,08−1,08No2
0.12 0.15 0.14 0.18 0.9
9No3 0.11 0.+4 0.18 0.22
0.98No4 0.12 0.17 0.0?
0.39 0.98(単位mM)
(d−5)
HX O,LmM
HE R,Ad R80,15a+M
AMP ’、ADP、ATP 各0.2a+M総AT
P関連化合物(ただしIMFを含まず)1.0mM
を含む被検液を用いて(d−1)における第5図(2)
に従って4回測定操作を行った。求められた各成分の濃
度は下記の通りであるが、略定埴的であった。Not including total ATP-related compounds) Not O, 130, 170, 08-1, 08 No2
0.12 0.15 0.14 0.18 0.9
9No3 0.11 0. +4 0.18 0.22
0.98No4 0.12 0.17 0.0?
0.39 0.98 (unit: mM) (d-5) HX O, LmM HE R, Ad R80, 15a+M AMP', ADP, ATP each 0.2a+M total AT
Figure 5 (2) in (d-1) using a test solution containing 1.0mM of P-related compound (but not IMF)
The measurement operation was performed four times according to the following. The determined concentrations of each component are as shown below, and were approximately constant.
■肝を含まず)
Nol O,130,160,1? 0.32
1.113No2 0.15 0.20 0.12 0
.30 1.18No3 0.14 0.19 0.
15 0.28 1.05No4 0.+3 0.1
9 0.1B 0.24 1.19(単位mM)
実施例(a)〜(d)の11111定において各酵素反
応は1分前後で終了するため、Do減少曲線を得るのに
(a)〜(c)の場合で僅かに5分程度であり、(d
)の場合でlO分程度である。なお1本発明においては
酵素の特異性を利用して多くの複合作用を連続させるた
めに、各酵素剤に他の酵素の混入がないことが重要であ
る。■Does not include liver) Nol O, 130, 160, 1? 0.32
1.113No2 0.15 0.20 0.12 0
.. 30 1.18No3 0.14 0.19 0.
15 0.28 1.05No4 0. +3 0.1
9 0.1B 0.24 1.19 (unit: mM) In the 11111 constants of Examples (a) to (d), each enzyme reaction ends in around 1 minute, so in order to obtain the Do reduction curve, (a) to In case (c), it takes only about 5 minutes, and (d
), it is about 10 minutes. Note that in the present invention, it is important that each enzyme agent is free from contamination with other enzymes in order to utilize the specificity of enzymes to perform many complex actions in succession.
え乳立亘」
上記説明より明らかなように、本発明方法は従来のクロ
マトグラフ法や酵票法がATP関連化合物の組成分析に
数時間を要していたのに対し、la分程度で迅速に行う
ことができるばかりでなく、本発明者等が先に発明した
Do減少もしくはH2O2生成量を電気化学的信号によ
ってATP関連化合物の組成分析する方法を更に改良し
たものである。すなわち特定の酵素を利用することによ
ってAdRやAMPも定量することができるようになっ
た。従って本発明の方法によってATPI38連化合物
の組成を詳細に分析することができるので、魚介類や肉
類の鮮度測定に限らず、生化学、薬学、農学、医学、バ
イオテクノロジー等の広い領域に有用な分析技術である
0本発明に使用される酵素類の使用量も少なく極めて経
済的である。As is clear from the above explanation, the method of the present invention can quickly analyze the composition of ATP-related compounds in about 10 minutes, whereas conventional chromatography and fermentation methods require several hours. This method is a further improvement on the method previously invented by the present inventors for analyzing the composition of ATP-related compounds using electrochemical signals to determine the amount of Do reduction or H2O2 produced. That is, it has become possible to quantify AdR and AMP by using specific enzymes. Therefore, the method of the present invention allows detailed analysis of the composition of ATPI 38 compounds, which is useful not only for measuring the freshness of seafood and meat, but also for a wide range of fields such as biochemistry, pharmacy, agriculture, medicine, and biotechnology. The amount of enzymes used in the present invention, which is an analytical technique, is small and is extremely economical.
記録されたDo減少値やH2O2生成量を基にして、各
成分の濃度を手計算だけでなく、電算機連動による自動
演算も容易である。Based on the recorded Do reduction value and H2O2 production amount, it is easy to calculate the concentration of each component not only manually but also automatically using a computer.
従来技術に比べて、本発明の測定法は実験室ばかりでな
く、生産、流通の現場にも容易に実施され、食品産業の
振興、食品衛生の改善、消費者保護等の観点からも極め
て望ましいものである。Compared to conventional techniques, the measurement method of the present invention can be easily implemented not only in laboratories but also in production and distribution sites, and is extremely desirable from the viewpoints of promoting the food industry, improving food hygiene, and protecting consumers. It is something.
第1図は、本発明方法を実施するのに適した装置の反応
槽の縦断面図および装置系統図である。
第2〜5図はそれぞれIMFの定積; Ad Rの定:
、: 、 A M pの定;) ; A T P関連化
合物の混合液中のH! 、H! RlIMP、Ad R
およびA M Pおよび総ATP関連化合物の定量;に
おけるり。
減少曲線を示す。
第6〜8図はそれぞれIMF、Ad R,AMPの検+
、を線である。第6図(1)および第6図(2)は、そ
れぞれ予備反応にAc P、NTを用いた場合である。
特許出願人 オリエンタル酵母f業株式会社オリエン
タル電気株式会社
代 理 人 若 林 忠時間(
弁)
第2図
第4図
第5図
窩−←E参調
各艇−F!:砒−FIG. 1 is a longitudinal sectional view of a reaction tank and a system diagram of an apparatus suitable for carrying out the method of the present invention. Figures 2 to 5 are the constant product of IMF; constant product of Ad R:
, : , Determination of A M p ;) ; H in a mixture of A T P related compounds! ,H! RlIMP, Ad R
and quantification of AMP and total ATP-related compounds; Showing a decreasing curve. Figures 6 to 8 show the tests for IMF, Ad R, and AMP, respectively.
, is a line. FIG. 6(1) and FIG. 6(2) show the cases where Ac P and NT were used in the preliminary reaction, respectively. Patent applicant: Oriental Yeast f-Gyo Co., Ltd. Oriental Electric Co., Ltd. Agent: Tadashi Wakabayashi (
(valve) Figure 2 Figure 4 Figure 5 Fosse - ← E participating boats - F! :砒-
Claims (1)
オシドホスホリラーゼ(NP)とキサンチンオキシダー
ゼ(XO)を複合的に作用させて、溶存酸素の減少量又
は過酸化水素生成量の電気化学的信号により測定する方
法において、被検液にNPとXOを複合的に作用をさせ
る前に、(i)酸ホスファターゼ(AcP)もしくはヌ
クレオチダーゼ(NT)を作用させるか、(ii)アデ
ノシンデアミナーゼ(ADA)を作用させるか、(ii
i)ヌクレオチダーゼ(NT)を作用させたのちに、A
DAを作用させることによつて、それぞれイノシン酸(
IMP)、アデノシン(AdR)、アデノシン1リン酸
(AMP)を測定し、かくしてこれらの化合物の少くと
も1つを測定することよりなるアデノシン3リン酸関連
化合物の測定方法。 2、特許請求の範囲第1項の記載方法において、被検液
を(i)、(ii)、(iii)のすべての操作に付す
る前に、粗アルカリホスファターゼ(粗AP)を作用さ
せることによって、アデノシン3リン酸(ATP)+ア
デノシン2リン酸(ADP)+アデノシン1リン酸(A
MP)+アデノシン(AdR)+イノシン酸(IMP)
+イノシン(HxR)+ヒポキサンチン(Hx)(ただ
し、各成分のいずれかが欠けていても差支えな い)、AMP、AdR、IMP、HxRおよびHxのそ
れぞれを測定することよりなるアデノシン3リン酸関連
化合物の測定方法。[Claims] 1. Nucleoside phosphorylase (NP) and xanthine oxidase (XO) are combined to act on adenosine triphosphate (ATP)-related compounds to reduce the amount of dissolved oxygen or the amount of hydrogen peroxide produced. In the method of measurement using chemical signals, before the test liquid is subjected to the combined action of NP and XO, (i) acid phosphatase (AcP) or nucleotidase (NT) is applied, or (ii) adenosine deaminase is (ADA) or (ii
i) After acting with nucleotidase (NT), A
By acting with DA, inosinic acid (
1. A method for measuring adenosine triphosphate-related compounds, which comprises measuring adenosine monophosphate (IMP), adenosine (AdR), and adenosine monophosphate (AMP), and thus measuring at least one of these compounds. 2. In the method described in claim 1, crude alkaline phosphatase (crude AP) is allowed to act on the test liquid before subjecting it to all operations (i), (ii), and (iii). adenosine triphosphate (ATP) + adenosine diphosphate (ADP) + adenosine monophosphate (A
MP) + adenosine (AdR) + inosinic acid (IMP)
+ inosine (HxR) + hypoxanthine (Hx) (however, there is no problem even if one of each component is missing), AMP, AdR, IMP, adenosine triphosphate-related by measuring each of HxR and Hx How to measure compounds.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP61153969A JPS6311848A (en) | 1986-07-02 | 1986-07-02 | Handy measurement of compound related to adenosine-3-phosphoric acid |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP61153969A JPS6311848A (en) | 1986-07-02 | 1986-07-02 | Handy measurement of compound related to adenosine-3-phosphoric acid |
Publications (1)
Publication Number | Publication Date |
---|---|
JPS6311848A true JPS6311848A (en) | 1988-01-19 |
Family
ID=15574030
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP61153969A Pending JPS6311848A (en) | 1986-07-02 | 1986-07-02 | Handy measurement of compound related to adenosine-3-phosphoric acid |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPS6311848A (en) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1999007877A1 (en) * | 1997-08-05 | 1999-02-18 | The University Court Of The University Of St. Andrews | Biosensor for detecting adenosine |
US7122333B2 (en) | 2001-11-21 | 2006-10-17 | Unitika Ltd. | Method and reagent for visually measuring ATP |
WO2021241446A1 (en) | 2020-05-25 | 2021-12-02 | 横河電機株式会社 | Method for detecting target molecule in specimen, and target molecule detection kit |
-
1986
- 1986-07-02 JP JP61153969A patent/JPS6311848A/en active Pending
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1999007877A1 (en) * | 1997-08-05 | 1999-02-18 | The University Court Of The University Of St. Andrews | Biosensor for detecting adenosine |
US7122333B2 (en) | 2001-11-21 | 2006-10-17 | Unitika Ltd. | Method and reagent for visually measuring ATP |
WO2021241446A1 (en) | 2020-05-25 | 2021-12-02 | 横河電機株式会社 | Method for detecting target molecule in specimen, and target molecule detection kit |
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