JPH0740958B2 - Method for measuring dehydrogenase or substrate - Google Patents
Method for measuring dehydrogenase or substrateInfo
- Publication number
- JPH0740958B2 JPH0740958B2 JP62080514A JP8051487A JPH0740958B2 JP H0740958 B2 JPH0740958 B2 JP H0740958B2 JP 62080514 A JP62080514 A JP 62080514A JP 8051487 A JP8051487 A JP 8051487A JP H0740958 B2 JPH0740958 B2 JP H0740958B2
- Authority
- JP
- Japan
- Prior art keywords
- dehydrogenase
- substrate
- hydrogen peroxide
- measuring
- 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 title claims description 43
- 101710088194 Dehydrogenase Proteins 0.000 title claims description 26
- 239000000758 substrate Substances 0.000 title claims description 21
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 46
- 108091006149 Electron carriers Proteins 0.000 claims description 9
- 239000005515 coenzyme Substances 0.000 claims description 8
- 238000007357 dehydrogenase reaction Methods 0.000 claims description 7
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 3
- 239000001301 oxygen Substances 0.000 claims description 3
- 229910052760 oxygen Inorganic materials 0.000 claims description 3
- MASUWVVNWALEEM-UHFFFAOYSA-M 1-methoxy-5-methylphenazin-5-ium;methyl sulfate Chemical compound COS([O-])(=O)=O.C1=CC=C2N=C3C(OC)=CC=CC3=[N+](C)C2=C1 MASUWVVNWALEEM-UHFFFAOYSA-M 0.000 claims description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 claims description 2
- RXGJTUSBYWCRBK-UHFFFAOYSA-M 5-methylphenazinium methyl sulfate Chemical compound COS([O-])(=O)=O.C1=CC=C2[N+](C)=C(C=CC=C3)C3=NC2=C1 RXGJTUSBYWCRBK-UHFFFAOYSA-M 0.000 claims 1
- 238000006243 chemical reaction Methods 0.000 description 18
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 15
- RFSUNEUAIZKAJO-VRPWFDPXSA-N D-Fructose Natural products OC[C@H]1OC(O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-VRPWFDPXSA-N 0.000 description 14
- RLFWWDJHLFCNIJ-UHFFFAOYSA-N 4-aminoantipyrine Chemical compound CN1C(C)=C(N)C(=O)N1C1=CC=CC=C1 RLFWWDJHLFCNIJ-UHFFFAOYSA-N 0.000 description 12
- 239000000203 mixture Substances 0.000 description 8
- 102000003992 Peroxidases Human genes 0.000 description 6
- VMGAPWLDMVPYIA-HIDZBRGKSA-N n'-amino-n-iminomethanimidamide Chemical compound N\N=C\N=N VMGAPWLDMVPYIA-HIDZBRGKSA-N 0.000 description 6
- 108040007629 peroxidase activity proteins Proteins 0.000 description 6
- BOLDJAUMGUJJKM-LSDHHAIUSA-N renifolin D Natural products CC(=C)[C@@H]1Cc2c(O)c(O)ccc2[C@H]1CC(=O)c3ccc(O)cc3O BOLDJAUMGUJJKM-LSDHHAIUSA-N 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 5
- XJLXINKUBYWONI-DQQFMEOOSA-N [[(2r,3r,4r,5r)-5-(6-aminopurin-9-yl)-3-hydroxy-4-phosphonooxyoxolan-2-yl]methoxy-hydroxyphosphoryl] [(2s,3r,4s,5s)-5-(3-carbamoylpyridin-1-ium-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl phosphate Chemical compound NC(=O)C1=CC=C[N+]([C@@H]2[C@H]([C@@H](O)[C@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-DQQFMEOOSA-N 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- 238000004040 coloring Methods 0.000 description 5
- BAWFJGJZGIEFAR-NNYOXOHSSA-O NAD(+) Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 BAWFJGJZGIEFAR-NNYOXOHSSA-O 0.000 description 4
- BJEPYKJPYRNKOW-REOHCLBHSA-N (S)-malic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O BJEPYKJPYRNKOW-REOHCLBHSA-N 0.000 description 3
- RGHNJXZEOKUKBD-SQOUGZDYSA-N D-gluconic acid Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C(O)=O RGHNJXZEOKUKBD-SQOUGZDYSA-N 0.000 description 3
- 108010079870 Sarcosine Dehydrogenase Proteins 0.000 description 3
- 102000013000 Sarcosine dehydrogenase Human genes 0.000 description 3
- 238000000691 measurement method Methods 0.000 description 3
- 229950006238 nadide Drugs 0.000 description 3
- IRQRBVOQGUPTLG-UHFFFAOYSA-M sodium;3-(n-ethyl-3-methylanilino)-2-hydroxypropane-1-sulfonate Chemical compound [Na+].[O-]S(=O)(=O)CC(O)CN(CC)C1=CC=CC(C)=C1 IRQRBVOQGUPTLG-UHFFFAOYSA-M 0.000 description 3
- KDYFGRWQOYBRFD-UHFFFAOYSA-N succinic acid Chemical compound OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 description 3
- XOAAWQZATWQOTB-UHFFFAOYSA-N taurine Chemical compound NCCS(O)(=O)=O XOAAWQZATWQOTB-UHFFFAOYSA-N 0.000 description 3
- XQXPVVBIMDBYFF-UHFFFAOYSA-N 4-hydroxyphenylacetic acid Chemical compound OC(=O)CC1=CC=C(O)C=C1 XQXPVVBIMDBYFF-UHFFFAOYSA-N 0.000 description 2
- 108020005199 Dehydrogenases Proteins 0.000 description 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 2
- -1 N-methyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine sodium Chemical compound 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 150000001448 anilines Chemical class 0.000 description 2
- OEYIOHPDSNJKLS-UHFFFAOYSA-N choline Chemical compound C[N+](C)(C)CCO OEYIOHPDSNJKLS-UHFFFAOYSA-N 0.000 description 2
- 238000006356 dehydrogenation reaction Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- QRMZSPFSDQBLIX-UHFFFAOYSA-N homovanillic acid Chemical compound COC1=CC(CC(O)=O)=CC=C1O QRMZSPFSDQBLIX-UHFFFAOYSA-N 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- FSYKKLYZXJSNPZ-UHFFFAOYSA-N sarcosine Chemical compound C[NH2+]CC([O-])=O FSYKKLYZXJSNPZ-UHFFFAOYSA-N 0.000 description 2
- ATHGHQPFGPMSJY-UHFFFAOYSA-N spermidine Chemical compound NCCCCNCCCN ATHGHQPFGPMSJY-UHFFFAOYSA-N 0.000 description 2
- JYSUYJCLUODSLN-UHFFFAOYSA-N 1,3-benzothiazol-2-ylhydrazine Chemical class C1=CC=C2SC(NN)=NC2=C1 JYSUYJCLUODSLN-UHFFFAOYSA-N 0.000 description 1
- 102000007698 Alcohol dehydrogenase Human genes 0.000 description 1
- 108010021809 Alcohol dehydrogenase Proteins 0.000 description 1
- 108010058733 Choline dehydrogenase Proteins 0.000 description 1
- 102100032363 Choline dehydrogenase, mitochondrial Human genes 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 108010050375 Glucose 1-Dehydrogenase Proteins 0.000 description 1
- 235000017784 Mespilus germanica Nutrition 0.000 description 1
- 240000002624 Mespilus germanica Species 0.000 description 1
- 235000000560 Mimusops elengi Nutrition 0.000 description 1
- ACFIXJIJDZMPPO-NNYOXOHSSA-J NADPH(4-) Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP([O-])(=O)OP([O-])(=O)OC[C@@H]2[C@H]([C@@H](OP([O-])([O-])=O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 ACFIXJIJDZMPPO-NNYOXOHSSA-J 0.000 description 1
- 108010077895 Sarcosine Proteins 0.000 description 1
- 108010055719 Spermidine dehydrogenase Proteins 0.000 description 1
- 102000019259 Succinate Dehydrogenase Human genes 0.000 description 1
- 108010012901 Succinate Dehydrogenase Proteins 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- 108010041155 Taurine dehydrogenase Proteins 0.000 description 1
- 235000007837 Vangueria infausta Nutrition 0.000 description 1
- 230000002378 acidificating effect Effects 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
- 150000001412 amines Chemical class 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 229960001231 choline Drugs 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 238000012921 fluorescence analysis Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- SUBFIBLJQMMKBK-UHFFFAOYSA-K iron(3+);trithiocyanate Chemical compound [Fe+3].[S-]C#N.[S-]C#N.[S-]C#N SUBFIBLJQMMKBK-UHFFFAOYSA-K 0.000 description 1
- 229940116298 l- malic acid Drugs 0.000 description 1
- VZCYOOQTPOCHFL-UPHRSURJSA-N maleic acid Chemical compound OC(=O)\C=C/C(O)=O VZCYOOQTPOCHFL-UPHRSURJSA-N 0.000 description 1
- 235000011090 malic acid Nutrition 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 1
- FSVCQIDHPKZJSO-UHFFFAOYSA-L nitro blue tetrazolium dichloride Chemical group [Cl-].[Cl-].COC1=CC(C=2C=C(OC)C(=CC=2)[N+]=2N(N=C(N=2)C=2C=CC=CC=2)C=2C=CC(=CC=2)[N+]([O-])=O)=CC=C1[N+]1=NC(C=2C=CC=CC=2)=NN1C1=CC=C([N+]([O-])=O)C=C1 FSVCQIDHPKZJSO-UHFFFAOYSA-L 0.000 description 1
- 238000006395 oxidase reaction Methods 0.000 description 1
- DCKVFVYPWDKYDN-UHFFFAOYSA-L oxygen(2-);titanium(4+);sulfate Chemical compound [O-2].[Ti+4].[O-]S([O-])(=O)=O DCKVFVYPWDKYDN-UHFFFAOYSA-L 0.000 description 1
- 150000002989 phenols Chemical class 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 230000027756 respiratory electron transport chain Effects 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 229940043230 sarcosine Drugs 0.000 description 1
- 238000004611 spectroscopical analysis Methods 0.000 description 1
- 229940063673 spermidine Drugs 0.000 description 1
- 239000001384 succinic acid Substances 0.000 description 1
- 229960003080 taurine Drugs 0.000 description 1
- 229910000348 titanium sulfate Inorganic materials 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Description
【発明の詳細な説明】 ［産業上の利用分野］ 本発明は脱水素酵素又は基質の測定法に関し、詳細には
ニコチンアミドアデニンジヌクレオチド（NAD+）やニコ
チンアミドアデニンジヌクレオチドフォスフェート（NA
DP+）等の補酵素を必要としない反応系の前記脱水素酵
素又は基質を正確且つ迅速に測定できると共に連続的な
自動分析においても支障なく実施できる様な方法に関す
るものである。TECHNICAL FIELD The present invention relates to a method for measuring a dehydrogenase or a substrate, and more particularly to nicotinamide adenine dinucleotide (NAD + ) and nicotinamide adenine dinucleotide phosphate (NA).
The present invention relates to a method capable of accurately and quickly measuring the dehydrogenase or substrate in a reaction system that does not require a coenzyme such as DP + ) and capable of carrying out continuous automatic analysis without any trouble.
［従来の技術］ 酵素や基質を測定する方法としては従来から各種の方法
が実施されているが、もっとも汎用されている発色測定
方法（分光学的測定方法）としては、一般的に下記の３
通りに大別される方法が知られている。[Prior Art] Various methods have been conventionally practiced as methods for measuring enzymes and substrates, but the most widely used color measurement method (spectroscopic measurement method) is generally the following 3
It is known how to divide into streets.
（Ｉ）反応系内で生じた過酸化水素を酵素的又は非酵素
的に発色させて定量する方法。(I) A method in which hydrogen peroxide generated in the reaction system is enzymatically or non-enzymatically developed and quantified.
（II）脱水素酵素を基質に作用させて生じたNADH（還元
型NAD+）やNADPH（還元型NADP+）の吸光度の変化から酵
素や基質を測定する方法。(II) A method of measuring an enzyme or a substrate from changes in the absorbance of NADH (reduced NAD + ) or NADPH (reduced NADP + ) produced by the action of a dehydrogenase on the substrate.
（III）反応系内で生じた水素を電子伝達体を介してホ
ルマザン発色に導く方法。(III) A method of leading hydrogen generated in the reaction system to formazan color development via an electron carrier.
上記（Ｉ）の方法は特に酸化酵素反応の様に、反応系内
において直接過酸化水素を生じる場合に一般的に用いら
れている方法であり、従って脱水素酵素又は基質を測定
する方法としては適用されていない。そこで脱水素酵素
が関与する系では（II）又は（III）の方法によるのが
一般的であり、殊に（III）の方法はNAD+やNADP+等の補
酵素を必要としない脱水素酵素を測定する場合には常識
的な方法として汎用されている。The above method (I) is a method generally used when hydrogen peroxide is directly produced in the reaction system, such as an oxidase reaction. Therefore, the method for measuring dehydrogenase or substrate is Not applicable Therefore, in the system in which dehydrogenase is involved, the method (II) or (III) is generally used. In particular, the method (III) is a dehydrogenase that does not require coenzymes such as NAD + and NADP +. Is commonly used as a common sense method for measuring.
上記（III）の方法（ホルマザン発色法）は、例えばサ
ルコシン脱水素酵素（SDH）を分光学的に測定する場合
において、下記の反応式で示す様に脱水素反応により生
じた水素を、フェナジンメトサルフェート（PMS）や１
−メトキシ−５−メチルフェナジニウムメチルサルフェ
ート（１−mPMS）或は９−ジメチルアミノベンゾ−α−
フェナゾキソニウムクロライド（メルドラーブルー）等
の電子伝達体を介して紫色のホルマザン発色に導いて行
なわれるものである。In the method (III) (formazan color development method), for example, when spectroscopically measuring sarcosine dehydrogenase (SDH), hydrogen generated by the dehydrogenation reaction is converted into phenazinemethoxide as shown in the following reaction formula. Sulfate (PMS) and 1
-Methoxy-5-methylphenazinium methylsulfate (1-mPMS) or 9-dimethylaminobenzo-α-
It is carried out by forming a purple formazan color through an electron carrier such as phenazoxonium chloride (Medlar blue).
（但し、式中NTBはニトロテトラゾリウムブルー,NTBH2
はNTBの還元型,1−mPMSH2は１−mPMSの還元型であ
る。） ［発明が解決しようとする問題点］ しかしながら上記ホルマザン発色法は、発色の際に生じ
る色素物質が沈着するという特性がある為チューブやセ
ルを汚染し易く、従って連続的な自動分析には適用し難
く、この方法は用手法によらざるを得ないという欠点を
有していた。 (However, in the formula, NTB is nitrotetrazolium blue, NTBH 2
Is the reduced form of NTB and 1-mPMSH 2 is the reduced form of 1-mPMS. ) [Problems to be Solved by the Invention] However, the above-mentioned formazan coloring method is apt to contaminate tubes and cells due to the characteristic that a pigment substance generated during coloring is deposited, and is therefore applicable to continuous automatic analysis. It is difficult to do, and this method has a drawback that it cannot help but depend on the method used.
本発明はこうした従来技術のもつ技術的課題を解決する
為になされたものであって、その目的とするところは、
補酵素を必要としない脱水素酵素反応における脱水素酵
素又は基質を正確迅速に測定でき、チューブやセルを汚
染する様物質を発生させることなく、自動分析に最適な
測定方法を提供することにある。The present invention was made in order to solve the technical problems of these conventional techniques, and its purpose is to:
An object of the present invention is to provide an optimal measurement method for automatic analysis, which can accurately and quickly measure dehydrogenase or substrate in a dehydrogenase reaction that does not require coenzyme, and does not generate substances that contaminate tubes and cells. .
［問題点を解決する為の手段］ 上記目的を達成し得た本発明とは、補酵素を必要としな
い脱水素酵素反応における脱水素酵素又は基質を測定す
るに当たり、脱脂素酵素反応により基質から生じる電子
を電子伝達体を経由して酸素に受容させることにより過
酸化水素を生成せしめ、生じた過酸化水素を酵素的又は
非酵素的に定量する点に要旨を有する脱水素酵素又は基
質の測定法である。[Means for Solving Problems] According to the present invention which has achieved the above-mentioned object, in measuring a dehydrogenase or a substrate in a dehydrogenase reaction that does not require a coenzyme, a substrate is removed from the substrate by a defatty enzyme reaction. Measurement of dehydrogenase or substrate, which is characterized in that hydrogen peroxide is generated by accepting generated electrons into oxygen via an electron carrier to generate hydrogen peroxide, and quantifying the generated hydrogen peroxide enzymatically or nonenzymatically Is the law.
［作用］ 本発明方法は上述の如く構成されるが、要は補酵素を必
要としない脱水素酵素反応系において電子伝達系を介し
て酸素に電子を受容させることにより過酸化水素を生成
せしめ、この過酸化水素の量を酵素的又は非酵素的に定
量することによって、ホルマザン発色法が持つ欠点を解
消し（即ちチューブやセルを汚染することなく）、迅速
且つ正確に脱水素酵素又は基質を測定し得る様になった
のである。[Operation] The method of the present invention is configured as described above, but the point is that hydrogen peroxide is produced by allowing oxygen to accept an electron through an electron transfer system in a dehydrogenase reaction system that does not require a coenzyme, By quantifying the amount of this hydrogen peroxide enzymatically or non-enzymatically, the disadvantages of the formazan coloring method are eliminated (that is, without contaminating the tube or cell), and the dehydrogenase or substrate can be rapidly and accurately detected. It became possible to measure.
本発明方法における代表的な反応系を例示すると下記の
如くである。A typical reaction system in the method of the present invention is as follows.
但し H2O2:過酸化水素 ４−AA:4−アミノアンチピリン TOOS:N−メチル−Ｎ−（２−ヒドロキシ−３−スルホプ
ロピル）−ｍ−トルイジンナトリウム POD:ペルオキシダーゼ 本発明方法は前述した趣旨より明らかな様に、NAD+やNA
DP+等の補酵素を必要としない脱水素酵素を対象とした
ものであるが、この様な脱水素酵素については何ら限定
されず上記の反応系において電子伝達体を介して過酸化
水素を生成できるすべての酵素を意味する。この様な酵
素として下記の様なものが挙げられる。 However, H 2 O 2 : hydrogen peroxide 4-AA: 4-aminoantipyrine TOOS: N-methyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine sodium POD: peroxidase The method of the present invention has the above-mentioned purpose. More clearly, NAD + and NA
This is intended for dehydrogenases that do not require coenzymes such as DP + , but such dehydrogenases are not limited at all and hydrogen peroxide is generated via an electron carrier in the above reaction system. It means all possible enzymes. Examples of such enzymes include the following.
（ａ）サルコシン脱水素酵素（EC1.5.99.1） （ｂ）アミノ脱水素酵素（EC1.4.99.3） （ｃ）スクシネイト脱水素酵素（EC1.3.99.1） （ｄ）コリン脱水素酵素（EC1.1.99.1） （ｅ）アルコール脱水素酵素（EC1.1.99.8） （ｆ）Ｄ−フラクトース脱水素酵素（EC1.1.99.11） （ｇ）タウリン脱水素酵素（EC1.4.99.2） （ｈ）ケトグルコネイト脱水素酵素（EC1.3.99.4） （ｉ）グルコース脱水素酵素（EC1.1.99.10） （ｊ）マレイト脱水素酵素（EC1.1.99.16） （ｋ）スペルミジン脱水素酵素（EC1.5.99.6） 又本発明方法における基質とは各脱水素酵素が特異的に
作用する物質を意味し、上述した脱水素酵素の場合は下
記の如くである。(A) Sarcosine dehydrogenase (EC1.5.99.1) (b) Amino dehydrogenase (EC1.4.99.3) (c) Succinate dehydrogenase (EC1.3.99.1) (d) Choline dehydrogenase ( EC1.1.99.1) (e) Alcohol dehydrogenase (EC1.1.99.8) (f) D-fructose dehydrogenase (EC1.1.99.11) (g) Taurine dehydrogenase (EC1.4.99.2) (H) Ketogluconate dehydrogenase (EC1.3.99.4) (i) Glucose dehydrogenase (EC1.1.99.10) (j) Maleate dehydrogenase (EC1.1.99.16) (k) Spermidine dehydrogenase ( EC1.5.99.6) Further, the substrate in the method of the present invention means a substance on which each dehydrogenase specifically acts, and in the case of the above-mentioned dehydrogenase, it is as follows.
（ａ）サルコシン （ｂ）アミン （ｃ）コハク酸 （ｄ）コリン （ｅ）アルコール （ｆ）Ｄ−フラクトース （ｇ）タウリン （ｈ）ケトグルコン酸 （ｉ）グルコース （ｊ）Ｌ−リンゴ酸 （ｋ）スペルミジン 但し、本発明方法で用いる基質は上記の様に各脱水素酵
素に対応したものに限定されるものではなく、他の基質
であっても特異的に作用する場合もあり得る。(A) sarcosine (b) amine (c) succinic acid (d) choline (e) alcohol (f) D-fructose (g) taurine (h) ketogluconic acid (i) glucose (j) L-malic acid (k) Spermidine However, the substrate used in the method of the present invention is not limited to those corresponding to each dehydrogenase as described above, and other substrates may act specifically.
尚本発明で様いる電子伝達体としては上記反応系で示し
た１−mPMSの他、前述したPMSやメルドラーブルー等を
用いることが好ましい。これらの電子伝達体を使用する
場合は、１種又は２種以上を混合して使用することもで
きる。又これらの電子伝達体を使用する場合の濃度につ
いては何ら限定されるものではないが、好ましくは0.00
01〜1.0mg/ml程度である。In addition to the 1-mPMS shown in the above reaction system, it is preferable to use the above-mentioned PMS, Meldler blue or the like as the electron carrier in the present invention. When using these electron carriers, one kind or a mixture of two or more kinds may be used. The concentration when these electron mediators are used is not particularly limited, but is preferably 0.00
It is about 01 to 1.0 mg / ml.
本発明方法は上述した様に、一連の反応によって過酸化
水素を生成せしめ、この過酸化水素を酵素的又は非酵素
的に定量するものであるが、過酸化水素の定量法につい
ては何ら限定されるものではない。As described above, the method of the present invention produces hydrogen peroxide by a series of reactions and quantifies this hydrogen peroxide enzymatically or non-enzymatically, but the method for quantifying hydrogen peroxide is not limited at all. Not something.
過酸化水素を酵素的に定量する方法の１つとしては、過
酸化水素にペルオキシダーゼと色原体を作用させる方法
がある。色原体としては次のような組合せがある。As one of the methods for enzymatically quantifying hydrogen peroxide, there is a method in which hydrogen peroxide is reacted with peroxidase and a chromogen. The following combinations are available as chromogens.
４−アミノアンチピリン／アニリン誘導体系 ４−アミノアンチピリン／フェノール誘導体系 ベンゾチアゾリノンヒドラゾン誘導体／アニリン誘導体
系 上記した反応系においては、生じた過酸化水素に４−ア
ミノアンチピリン（４−AA）及びＮ−メチル−Ｎ−（２
−ヒドロキシ−３−スルホプロピル）−ｍ−トルイジン
ナトリウム（TOOS）等の発色剤を添加し、ペルオキシダ
ーゼ（POD）によって酵素的に発色せしめ、その後分光
学的に過酸化水素量を定量する方法について例示した。
酵素的な過酸化水素測定法の他の例としては、例えばホ
モバニリン酸（又はＰ−ヒドロキシフェニル酢酸）とPO
Dを添加することによって蛍光物質を生成させ、この蛍
光物質を測定する様な蛍光分析法を挙げることができ
る。この蛍光分析法によれば、上記の分光学的方法より
も更に高精度に過酸化水素を定量することができる。4-Aminoantipyrine / aniline derivative system 4-Aminoantipyrine / phenol derivative system Benzothiazolinone hydrazone derivative / aniline derivative system In the above-mentioned reaction system, 4-aminoantipyrine (4-AA) and N -Methyl-N- (2
-Hydroxy-3-sulfopropyl) -m-toluidine sodium (TOOS) is added as a color-developing agent to enzymatically develop color with peroxidase (POD), and then spectroscopically quantifies the amount of hydrogen peroxide. did.
Other examples of the enzymatic hydrogen peroxide measuring method include, for example, homovanillic acid (or P-hydroxyphenylacetic acid) and PO.
A fluorescent analysis method may be mentioned in which a fluorescent substance is generated by adding D and the fluorescent substance is measured. According to this fluorescence analysis method, hydrogen peroxide can be quantified with higher accuracy than the above spectroscopic method.
一方過酸化水素を非酵素的に定量する方法についても何
ら限定されるものではなく、従来から知られている各種
の方法を任意に選定して実施すればよい。例えば、硫酸
チタンやチオシアン酸鉄（III）等の呈色試薬を用いる
比色法を挙げることができ、その他硫酸酸性試料溶液の
過マンガン酸塩滴定等の容量法、或はポーラログラフィ
ーによる方法等を挙げることができる。On the other hand, the method for non-enzymatically quantifying hydrogen peroxide is not limited at all, and various conventionally known methods may be arbitrarily selected and implemented. For example, a colorimetric method using a coloring reagent such as titanium sulfate or iron (III) thiocyanate can be mentioned. Other methods such as a volumetric method such as permanganate titration of a sulfuric acid acidic sample solution, or a polarographic method Etc. can be mentioned.
［実施例］ 以下本発明の実施例を示すが、下記実施例は本発明を限
定する性質のものではない。[Examples] Examples of the present invention will be shown below, but the following examples are not intended to limit the present invention.
実施例１ 下記の手順でＤ−フラクトースの測定を行なった。Example 1 D-fructose was measured by the following procedure.
まずＤ−フラクトースにＤ−フラクトース脱水素酵素を
作用させて脱水素反応を進行させた。このときの反応組
成及び条件は下記の如くである。First, D-fructose dehydrogenase was allowed to act on D-fructose to advance the dehydrogenation reaction. The reaction composition and conditions at this time are as follows.
反応組成１ マッキルバイン緩衝液（pH4.5） ……1.4ml Ｄ−フラクトース溶液（0,16,32,48mg/） ……0.3ml Ｄ−フラクトース脱水素酵素（EC1.1.99.11,200U/ml）
……0.3ml １−mPMS（0.5mg/ml） ……0.1ml 反応条件 温度:30℃ pH:4.5 時間:15分 次に下記反応組成物を添加して、過酸化水素を酵素的に
発色させた。Reaction composition 1 McIlvain buffer (pH4.5) ...... 1.4ml D-fructose solution (0,16,32,48mg /) ・ ・ ・ 0.3ml D-fructose dehydrogenase (EC1.1.99.11,200U / ml)
…… 0.3 ml 1-mPMS (0.5 mg / ml) …… 0.1 ml Reaction conditions Temperature: 30 ℃ pH: 4.5 hours: 15 minutes Next, add the following reaction composition to make hydrogen peroxide enzymatically develop color. It was
反応組成２ ４−AA（10mg/ml） ……0.3ml TOOS（10mg/ml） ……0.3ml POD（100U/ml） ……0.3ml 発色した各試料について、水をブランクとして波長550m
mにおける吸光度を測定した。Reaction composition 2-4-AA (10mg / ml) ...... 0.3ml TOOS (10mg / ml) ...... 0.3ml POD (100U / ml) ...... 0.3ml For each colored sample, water was used as a blank and wavelength was 550m.
The absorbance at m was measured.
その結果を第１図に示すが、Ｄ−フラクトースが正確且
つ迅速にしかもチューブやセルを汚染することなく測定
された。The results are shown in FIG. 1, and D-fructose was measured accurately and rapidly without contaminating tubes or cells.
実施例２ 実施例１と同じ基質及び酵素並びに電子伝達体を使用
し、37℃、pH4.5の条件で下記の反応組成にてＤ−フラ
クトース脱水素酵素反応を進行させた。Example 2 Using the same substrate, enzyme and electron carrier as in Example 1, a D-fructose dehydrogenase reaction was allowed to proceed with the following reaction composition at 37 ° C and pH 4.5.
反応組成１ マッキルバイン緩衝液（pH4.5） ……1.7ml Ｄ−フラクトース溶液（1M） ……0.1ml Ｄ−フラクトース脱水素酵素 ……0.1ml １−mPMS ……0.1ml 上記の組成及び条件で各時間（１〜７分間）反応させた
後、SDS溶液（300mg/ml）を0.1ml加えその反応を停止さ
せた。その後実施例１と同様にして、過酸化水素を生成
せしめると共に各試料における過酸化水素の量を定量し
た。Reaction composition 1 McIlvain buffer (pH4.5): 1.7 ml D-fructose solution (1M): 0.1 ml D-fructose dehydrogenase: 0.1 ml 1-mPMS: 0.1 ml Each of the above composition and conditions After reacting for a time (1 to 7 minutes), 0.1 ml of SDS solution (300 mg / ml) was added to stop the reaction. Thereafter, in the same manner as in Example 1, hydrogen peroxide was produced and the amount of hydrogen peroxide in each sample was quantified.
その結果を第２図に示すが、Ｄ−フラクトース脱水素酵
素の活性が正確に（直線的に）測定されているのが良く
分かる。The results are shown in FIG. 2, and it can be seen that the activity of D-fructose dehydrogenase is accurately (linearly) measured.
［発明の効果〕 以上述べた如く本発明によれば、既述の手順に従うこと
によって、補酵素を必要としない脱水素酵素反応におけ
る脱水素酵素又は基質を正確且つ迅速に測定できる様に
なり、しかもこの方法はホルマザン発色法の様な沈着物
質を生じることなく、連続的な自動分析にも最適な方法
である。[Effects of the Invention] According to the present invention as described above, by following the procedure described above, it becomes possible to accurately and rapidly measure a dehydrogenase or a substrate in a dehydrogenase reaction that does not require a coenzyme, Moreover, this method is the most suitable method for continuous automatic analysis without producing deposited substances as in the formazan coloring method.
第１図は実施例１においてＤ−フラクトースを測定した
結果を示すグラフ、第２図は実施例２においてＤ−フラ
クトース脱水素酵素を測定した結果を示すグラフであ
る。FIG. 1 is a graph showing the results of measuring D-fructose in Example 1, and FIG. 2 is a graph showing the results of measuring D-fructose dehydrogenase in Example 2.
Claims (2)
ける脱水素酵素又は基質を測定するに当たり、前記脱水
素酵素反応により基質から生じる電子を電子伝達体を経
由して酸素に受容させることにより、過酸化水素を生成
せしめ、生じた過酸化水素を酵素的又は非酵素的に定量
することを特徴とする脱水素酵素又は基質の測定法。1. When measuring a dehydrogenase or a substrate in a dehydrogenase reaction that does not require a coenzyme, by accepting an electron generated from the substrate by the dehydrogenase reaction to oxygen via an electron carrier. A method for measuring a dehydrogenase or a substrate, which comprises producing hydrogen peroxide and quantifying the produced hydrogen peroxide enzymatically or nonenzymatically.
ート、１−メトキシ−５−メチルフェナジニウムメチル
サルフェート及び９−ジメチルアミノベンゾ−α−フェ
ナゾキソニウムクロライドから選ばれる１種又は２種以
上である特許請求の範囲第１項に記載の測定法。2. The electron carrier is one or more selected from phenazine methosulfate, 1-methoxy-5-methylphenazinium methylsulfate and 9-dimethylaminobenzo-α-phenazoxonium chloride. The measuring method according to claim 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP62080514A JPH0740958B2 (en) | 1987-03-31 | 1987-03-31 | Method for measuring dehydrogenase or substrate |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP62080514A JPH0740958B2 (en) | 1987-03-31 | 1987-03-31 | Method for measuring dehydrogenase or substrate |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS63245697A JPS63245697A (en) | 1988-10-12 |
| JPH0740958B2 true JPH0740958B2 (en) | 1995-05-10 |
Family
ID=13720425
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP62080514A Expired - Lifetime JPH0740958B2 (en) | 1987-03-31 | 1987-03-31 | Method for measuring dehydrogenase or substrate |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0740958B2 (en) |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS59210899A (en) * | 1983-05-13 | 1984-11-29 | Wako Pure Chem Ind Ltd | Determination of reduced-type coenzyme and reagent therefor |
| JPS6219100A (en) * | 1985-07-16 | 1987-01-27 | Wako Pure Chem Ind Ltd | Measurement of bile acid |
-
1987
- 1987-03-31 JP JP62080514A patent/JPH0740958B2/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| JPS63245697A (en) | 1988-10-12 |
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