JPH0343096A - Determination of substrate or enzyme - Google Patents
Determination of substrate or enzymeInfo
- Publication number
- JPH0343096A JPH0343096A JP15768390A JP15768390A JPH0343096A JP H0343096 A JPH0343096 A JP H0343096A JP 15768390 A JP15768390 A JP 15768390A JP 15768390 A JP15768390 A JP 15768390A JP H0343096 A JPH0343096 A JP H0343096A
- Authority
- JP
- Japan
- Prior art keywords
- chelating agent
- solution
- nicotinamide adenine
- reaction
- buffer
- 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
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 9
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 9
- 239000000758 substrate Substances 0.000 title claims description 7
- 239000002738 chelating agent Substances 0.000 claims abstract description 37
- 238000006243 chemical reaction Methods 0.000 claims abstract description 15
- 150000001875 compounds Chemical class 0.000 claims abstract description 11
- 239000012992 electron transfer agent Substances 0.000 claims abstract description 7
- 230000009467 reduction Effects 0.000 claims abstract description 7
- 239000007853 buffer solution Substances 0.000 claims abstract description 4
- PIICEJLVQHRZGT-UHFFFAOYSA-N Ethylenediamine Chemical compound NCCN PIICEJLVQHRZGT-UHFFFAOYSA-N 0.000 claims abstract 2
- 238000000034 method Methods 0.000 claims description 23
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 claims description 14
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 10
- 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 claims description 6
- ACFIXJIJDZMPPO-NNYOXOHSSA-N NADPH 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-N 0.000 claims description 3
- CWYNVVGOOAEACU-UHFFFAOYSA-N Fe2+ Chemical compound [Fe+2] CWYNVVGOOAEACU-UHFFFAOYSA-N 0.000 claims description 2
- VTLYFUHAOXGGBS-UHFFFAOYSA-N Fe3+ Chemical compound [Fe+3] VTLYFUHAOXGGBS-UHFFFAOYSA-N 0.000 claims description 2
- 230000009471 action Effects 0.000 claims description 2
- 229910001447 ferric ion Inorganic materials 0.000 claims description 2
- 229910001448 ferrous ion Inorganic materials 0.000 claims description 2
- BDDLHHRCDSJVKV-UHFFFAOYSA-N 7028-40-2 Chemical compound CC(O)=O.CC(O)=O.CC(O)=O.CC(O)=O BDDLHHRCDSJVKV-UHFFFAOYSA-N 0.000 claims 1
- XJLXINKUBYWONI-NNYOXOHSSA-N NADP zwitterion 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-NNYOXOHSSA-N 0.000 claims 1
- 239000000243 solution Substances 0.000 abstract description 29
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 20
- 229950006238 nadide Drugs 0.000 abstract description 11
- BAWFJGJZGIEFAR-NNYOXOHSSA-N NAD zwitterion 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-N 0.000 abstract description 10
- 238000005259 measurement Methods 0.000 abstract description 10
- 101710088194 Dehydrogenase Proteins 0.000 abstract description 8
- 229910019142 PO4 Inorganic materials 0.000 abstract description 4
- 230000008859 change Effects 0.000 abstract description 4
- 239000010452 phosphate Substances 0.000 abstract description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 abstract description 3
- 238000000862 absorption spectrum Methods 0.000 abstract description 2
- 238000007707 calorimetry Methods 0.000 abstract 2
- 230000003647 oxidation Effects 0.000 abstract 2
- 238000007254 oxidation reaction Methods 0.000 abstract 2
- 239000000872 buffer Substances 0.000 description 21
- 238000002835 absorbance Methods 0.000 description 18
- 230000000694 effects Effects 0.000 description 17
- 210000002966 serum Anatomy 0.000 description 17
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 description 15
- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 description 7
- GSEJCLTVZPLZKY-UHFFFAOYSA-N Triethanolamine Chemical compound OCCN(CCO)CCO GSEJCLTVZPLZKY-UHFFFAOYSA-N 0.000 description 6
- FPFSGDXIBUDDKZ-UHFFFAOYSA-N 3-decyl-2-hydroxycyclopent-2-en-1-one Chemical compound CCCCCCCCCCC1=C(O)C(=O)CC1 FPFSGDXIBUDDKZ-UHFFFAOYSA-N 0.000 description 5
- 102000004420 Creatine Kinase Human genes 0.000 description 5
- 108010042126 Creatine kinase Proteins 0.000 description 5
- XJLXINKUBYWONI-NNYOXOHSSA-O NADP(+) 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-NNYOXOHSSA-O 0.000 description 5
- 235000006408 oxalic acid Nutrition 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 4
- 238000004737 colorimetric analysis Methods 0.000 description 4
- 238000004040 coloring Methods 0.000 description 4
- 238000006911 enzymatic reaction Methods 0.000 description 4
- 230000004044 response Effects 0.000 description 4
- 229910052708 sodium Inorganic materials 0.000 description 4
- 239000011734 sodium Substances 0.000 description 4
- 239000008362 succinate buffer Substances 0.000 description 4
- DGEZNRSVGBDHLK-UHFFFAOYSA-N [1,10]phenanthroline Chemical compound C1=CN=C2C3=NC=CC=C3C=CC2=C1 DGEZNRSVGBDHLK-UHFFFAOYSA-N 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 239000013522 chelant Substances 0.000 description 3
- BDHFUVZGWQCTTF-UHFFFAOYSA-M sulfonate Chemical compound [O-]S(=O)=O BDHFUVZGWQCTTF-UHFFFAOYSA-M 0.000 description 3
- WYMDDFRYORANCC-UHFFFAOYSA-N 2-[[3-[bis(carboxymethyl)amino]-2-hydroxypropyl]-(carboxymethyl)amino]acetic acid Chemical compound OC(=O)CN(CC(O)=O)CC(O)CN(CC(O)=O)CC(O)=O WYMDDFRYORANCC-UHFFFAOYSA-N 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 2
- FCKYPQBAHLOOJQ-UHFFFAOYSA-N Cyclohexane-1,2-diaminetetraacetic acid Chemical compound OC(=O)CN(CC(O)=O)C1CCCCC1N(CC(O)=O)CC(O)=O FCKYPQBAHLOOJQ-UHFFFAOYSA-N 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- FSVCELGFZIQNCK-UHFFFAOYSA-N N,N-bis(2-hydroxyethyl)glycine Chemical compound OCCN(CCO)CC(O)=O FSVCELGFZIQNCK-UHFFFAOYSA-N 0.000 description 2
- QPCDCPDFJACHGM-UHFFFAOYSA-N N,N-bis{2-[bis(carboxymethyl)amino]ethyl}glycine Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(=O)O)CCN(CC(O)=O)CC(O)=O QPCDCPDFJACHGM-UHFFFAOYSA-N 0.000 description 2
- DRBBFCLWYRJSJZ-UHFFFAOYSA-N N-phosphocreatine Chemical compound OC(=O)CN(C)C(=N)NP(O)(O)=O DRBBFCLWYRJSJZ-UHFFFAOYSA-N 0.000 description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 229940098773 bovine serum albumin Drugs 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- GKQWYZBANWAFMQ-UHFFFAOYSA-M lithium;2-hydroxypropanoate Chemical compound [Li+].CC(O)C([O-])=O GKQWYZBANWAFMQ-UHFFFAOYSA-M 0.000 description 2
- VMGAPWLDMVPYIA-HIDZBRGKSA-N n'-amino-n-iminomethanimidamide Chemical compound N\N=C\N=N VMGAPWLDMVPYIA-HIDZBRGKSA-N 0.000 description 2
- 229960003330 pentetic acid Drugs 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 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 2
- 230000035945 sensitivity Effects 0.000 description 2
- ZYDGCYWJDWIJCS-UHFFFAOYSA-N 1-methoxyphenazine Chemical compound C1=CC=C2N=C3C(OC)=CC=CC3=NC2=C1 ZYDGCYWJDWIJCS-UHFFFAOYSA-N 0.000 description 1
- URDCARMUOSMFFI-UHFFFAOYSA-N 2-[2-[bis(carboxymethyl)amino]ethyl-(2-hydroxyethyl)amino]acetic acid Chemical compound OCCN(CC(O)=O)CCN(CC(O)=O)CC(O)=O URDCARMUOSMFFI-UHFFFAOYSA-N 0.000 description 1
- DHDHJYNTEFLIHY-UHFFFAOYSA-N 4,7-diphenyl-1,10-phenanthroline Chemical compound C1=CC=CC=C1C1=CC=NC2=C1C=CC1=C(C=3C=CC=CC=3)C=CN=C21 DHDHJYNTEFLIHY-UHFFFAOYSA-N 0.000 description 1
- 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 description 1
- 101000892220 Geobacillus thermodenitrificans (strain NG80-2) Long-chain-alcohol dehydrogenase 1 Proteins 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
- 102000005548 Hexokinase Human genes 0.000 description 1
- 108700040460 Hexokinases Proteins 0.000 description 1
- 229910021578 Iron(III) chloride Inorganic materials 0.000 description 1
- 102000003855 L-lactate dehydrogenase Human genes 0.000 description 1
- 108700023483 L-lactate dehydrogenases Proteins 0.000 description 1
- JVTAAEKCZFNVCJ-UHFFFAOYSA-M Lactate Chemical compound CC(O)C([O-])=O JVTAAEKCZFNVCJ-UHFFFAOYSA-M 0.000 description 1
- 108010013563 Lipoprotein Lipase Proteins 0.000 description 1
- 102100022119 Lipoprotein lipase Human genes 0.000 description 1
- 108091000080 Phosphotransferase Proteins 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000008033 biological extinction Effects 0.000 description 1
- MTFJSAGADRTKCI-VMPITWQZSA-N chembl77510 Chemical compound O\N=C\C1=CC=CC=N1 MTFJSAGADRTKCI-VMPITWQZSA-N 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000007357 dehydrogenase reaction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 230000000873 masking effect Effects 0.000 description 1
- 230000018984 mastication Effects 0.000 description 1
- 238000010077 mastication Methods 0.000 description 1
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 229940101270 nicotinamide adenine dinucleotide (nad) Drugs 0.000 description 1
- MGFYIUFZLHCRTH-UHFFFAOYSA-N nitrilotriacetic acid Chemical compound OC(=O)CN(CC(O)=O)CC(O)=O MGFYIUFZLHCRTH-UHFFFAOYSA-N 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 102000020233 phosphotransferase Human genes 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 239000001103 potassium chloride Substances 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 239000012064 sodium phosphate buffer Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000012546 transfer Methods 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)
Abstract
Description
【発明の詳細な説明】
〔産業上の利用分野〕
本発明は、基質又は酵素の改良された定量方法に関する
。DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to an improved method for quantifying substrates or enzymes.
近年、酵素法による生体成分の分析法が広く普及し、生
体中の酵素あるいは非酵素成分の測定系に、種々の共役
反応を利用する場合が非常に多くなった。中でも脱水素
酵素群は、検出が容易なＮＡＤ又はＮＡＤＰが関与する
ため適用範囲が広べ、クレアチンキナーゼ測定系におけ
るグルコース−６−リン酸脱水素酵素、トリグリセライ
ド測定系におけるグリセロール脱水素酵素などがそのよ
い例である。現在、ＮＡＤＨ又はＮＡＤＰＨの検出は、
一般にホルマザン発色系の共役によって行われているが
、この方法では色素が水に難溶性であり、又、反応条件
により発色度が異なるので正確な分子吸光係数を適用し
にくい。さらに測定器具に色素が吸着するなどの欠点が
ある。In recent years, enzymatic methods for analyzing biological components have become widespread, and various coupled reactions are increasingly used in measurement systems for enzymes or non-enzymatic components in living organisms. Among them, the dehydrogenase group has a wide range of applicability because it involves NAD or NADP, which is easy to detect. This is a good example. Currently, detection of NADH or NADPH is
Generally, this is carried out by conjugation of a formazan coloring system, but in this method, the dye is poorly soluble in water, and the degree of coloring varies depending on the reaction conditions, making it difficult to apply accurate molecular extinction coefficients. Furthermore, there are drawbacks such as dye adsorption to the measuring instrument.
最近、これらの諸問題を解決する新しい発色系の利用が
報告されている。例えば、脱水素酵素−基質−ＮＡＤの
反応系にＦｅ’“、電子伝達剤及びＦｅｔ４″に特異的
に反応するキレート剤を添加した緩衝液中で次の反応を
行わせ、結果として生成した着色化合物を比色定量する
ことによる、トリグリセライドの測定方法が発表されて
いる（特開昭５４−８０１９２号公報）。Recently, the use of new coloring systems to solve these problems has been reported. For example, the following reaction is carried out in a buffer solution containing Fe'", an electron transfer agent, and a chelating agent that specifically reacts with Fet4" in the dehydrogenase-substrate-NAD reaction system, and the resulting colored A method for measuring triglyceride by colorimetrically quantifying a compound has been published (Japanese Patent Application Laid-open No. 80192/1983).
脱水素酵素
ＮＡＤ十基質 ＮＡＤＨ十生成物電子伝達剤
ＮＡＤ Ｈ＋ ２ Ｆｅ ’ ”−一−−→ＮへＤ＋２
Ｆｅ”Ｆｅ宜０＋ｎ（キレート剤）−一→錯化合物（着
色）この方法は、生成する着色物が水溶性であり、感度
もかなり高い。しかしながら、試薬ブランク植の経時変
化が著しいので、比色時には検体ごとに試薬ブランク値
による分光光度計のゼロ調整をしなければならず、測定
する試料件数が限られ、多検体処理には不適当であった
。Dehydrogenase NAD-substrate NADH-product Electron transfer agent NAD H+ 2 Fe' ”-1--→N to D+2
Fe"Fe 0 + n (chelating agent) -1 → complex compound (coloring) In this method, the colored product produced is water-soluble and the sensitivity is quite high. However, since the reagent blank plate changes significantly over time, colorimetry is not possible. Sometimes, the spectrophotometer must be zero-adjusted using a reagent blank value for each sample, which limits the number of samples to be measured, making it unsuitable for processing multiple samples.
本発明者は、この試薬ブランク値の経時変化を抑制し、
比色時の煩雑な操作を除くことにより多検体処理を可能
にするため、種々研究を行った結果、試薬ブランク値の
上昇は試薬中に含まれる種々の成分によるＦ、ｅ３４″
の非特異的還元に帰因し、とくに酵素蛋白やその夾雑物
量に依存して増大する傾向があることを見出し、更に、
それに効果的な隠ぺい剤、すなわちｐ ｅ ２ ＋と錯
化合物（キレート）を形成するキレート剤を使用するこ
とにより試薬ブランク値の経時変、化の抑制が可能であ
ることを見出した。本発明はかかる知見に基づくもので
ある。従って、本発明方法は、広く一般に脱水素酵素及
びＮＡＤ又はＮＡＤＰを含む測定系Ｃ：１１応用するこ
とが可能であり、ホルマザン法にかわる比色法として、
ＮＡＤＨ又はＮへＤＰＨの検出に適用することができる
。The present inventor suppressed the change in this reagent blank value over time,
In order to enable multi-sample processing by eliminating complicated operations during colorimetry, we conducted various studies and found that the increase in the reagent blank value is due to F, e34'' due to various components contained in the reagent.
We found that this is due to non-specific reduction of
It has been found that by using an effective masking agent, that is, a chelating agent that forms a complex compound (chelate) with p e 2 +, it is possible to suppress changes in the reagent blank value over time. The present invention is based on this knowledge. Therefore, the method of the present invention can be widely applied to measurement systems C:11 generally containing dehydrogenase and NAD or NADP, and as a colorimetric method in place of the formazan method.
It can be applied to the detection of DPH to NADH or N.
本発明は、脱水素酵素と酸化型ニコチンアミドアデニン
ジヌクレオチド（ＮＡＤ）又は酸化型ニコチンアミドア
デニンジヌクレオチドリン酸（ＮＡＤＰ）とを含む酵素
反応系により生成する還元型ニコチンアミドアデニンジ
ヌクレオチド（ＮＡＤＨ）又は還元型ニコチンアミドア
デニンジヌクレオチドリン酸（ＮＡ Ｄ Ｐ Ｈ）の量
に対応して、電子伝達剤の存在下で第二鉄イオン（Ｆｅ
″′″）から還元生成する第一鉄イオン（Ｆｅ”）の量
を、Ｆｅ”に特異的に反応するキレート剤の作用により
生成する着色化合物として比色定量する方法であって、
エチレンジアミン四酢酸（ＥＤＴＡ）をキレート剤とし
て含有するｐＨ５，０〜８．６の緩衝液を、一定時間反
応後に添加することを特徴とする、基質又は酵素の定量
方法に関する。The present invention provides reduced nicotinamide adenine dinucleotide (NADH) produced by an enzyme reaction system containing a dehydrogenase and oxidized nicotinamide adenine dinucleotide (NAD) or oxidized nicotinamide adenine dinucleotide phosphate (NADP). or corresponding to the amount of reduced nicotinamide adenine dinucleotide phosphate (NA D P H), ferric ion (Fe
A method for colorimetrically quantifying the amount of ferrous ions (Fe") produced by reduction from ethylenediaminetetraacetic acid) as a colored compound produced by the action of a chelating agent that specifically reacts with Fe". The present invention relates to a method for quantifying a substrate or an enzyme, which is characterized in that a buffer solution of pH 5.0 to 8.6 containing (EDTA) as a chelating agent is added after a certain period of reaction.
即ち、簡単に言えば、本発明方法は、電子伝達剤の存在
下に、脱水素酵素反応により生成したＮへＤｒ（又はＮ
ＡＤＰＨ量に対応してＦｅ”から還元生成するＦｅ”を
錯化合物として比色するにあたり、余剰のＦ Ｃ３＋と
測定波長に吸収スペクトルを示さないキレート化合物を
生成するキレート剤を添加して反応を停止させるととも
に試薬ブランク値の経時変化を抑制することを特徴とす
る基質又は酵素の改良された定量方法である。That is, to put it simply, the method of the present invention involves adding Dr (or N) to N produced by a dehydrogenase reaction in the presence of an electron transfer agent.
In colorimetrically comparing Fe", which is produced by reduction from Fe" in response to the amount of ADPH, as a complex compound, the reaction is stopped by adding excess F C3+ and a chelating agent that produces a chelate compound that does not show an absorption spectrum at the measurement wavelength. This is an improved method for quantifying substrates or enzymes, which is characterized by suppressing changes in reagent blank values over time.
本発明方法におけるキレート剤、即ち、エチレンジアミ
ン四酢酸（ＥＤＴＡ）の効果は、Ｆｅ”と安定な錯化合
物（キレート）を形成し、Ｆｅ”が還元されるのを抑制
する作用で説明することができる。従って、反応時にキ
レート剤を共存させると、反応に由来するＦｅｈの還元
を阻害する一可能性がある。そこで、実際に本発明方法
を実施する際には、基質の定量の場合には反応が終結し
た時点でキレート剤を加え、酵素の定量の場合には、規
定時間反応させた直後にキレート剤を（必要により酵素
反応停止剤と共に）加える。キレート剤が同時に酵素反
応を停止する作用を有する場合もある。The effect of the chelating agent, i.e., ethylenediaminetetraacetic acid (EDTA) in the method of the present invention, can be explained by the effect of forming a stable complex compound (chelate) with Fe'' and suppressing the reduction of Fe''. . Therefore, if a chelating agent is present during the reaction, there is a possibility that reduction of Feh derived from the reaction may be inhibited. Therefore, when actually carrying out the method of the present invention, in the case of quantifying a substrate, the chelating agent is added when the reaction is completed, and in the case of quantifying the enzyme, the chelating agent is added immediately after the reaction has been carried out for a specified time. Add (along with an enzyme reaction stopper if necessary). In some cases, the chelating agent also has the effect of stopping the enzymatic reaction.
本発明で使用するキレート剤はエチレンジアミン四酢酸
（ＥＤＴＡ）である。The chelating agent used in the present invention is ethylenediaminetetraacetic acid (EDTA).
キレート剤は、その濃度とｐＨにより、効果がかなり左
右されるため、使用する際には条件設定が重要となる。Since the effectiveness of chelating agents is significantly affected by their concentration and pH, it is important to set the conditions when using them.
次に、本発明のキレート剤であるエチレンジアミン四酢
酸（ＥＤＴＡ） 、及びこれと同様の効果を有する以下
のキレート剤等について、各種の条件を具体的に示す。Next, various conditions will be specifically shown regarding the chelating agent of the present invention, ethylenediaminetetraacetic acid (EDTA), and the following chelating agents having similar effects.
ヒドロキシエチルエチレンジアミン三酢酸（ＥＤＴＡ−
ＯＨ）
ジエチレントリアミン五酢酸（ＤＴＰＡ）ジアミノプロ
パノール四酢酸（ＤＰＴＡ−ＯＨ）
ニトリロ三酢酸（ＮＴＡ）
トランスシクロヘキサンジアミン四酢酸（ＣｙＤＴＡ）
ジヒドロキシエチルグリシン（ＤＨＥＧ）リン酸塩
表１及び表２は、後述の参考例１の乳酸脱水素酵素（Ｌ
ＤＨ）測定試薬を用いて試験を実施した結果を示すもの
である。表１には、ｐＨが試薬ブランク値の経時変化に
及ぼす影響を示した。この結果から、ｐＨの選択によっ
ては、かえって逆効果となってしまうことがわかる。表
２には、キレート剤の濃度が試薬ブランク値の経時変化
と呈色に及ぼす影響を示した。この結果から、キレート
剤が高濃度になるに従って経時的に試薬ブランク値は減
少するが、反応による呈色をも減少させる傾向にあるこ
とがわかる。従ってＬＤＨを測定する場合のキレート剤
としては、１〜８ｍＭ−ＥＤＴＡ−ＯＨ（ｐＨ８，６）
や１〜８ｍＭ−ＤＰＴＡ−ＯＨ（ｐＨ８，６） 、４〜
８ｍＭ−ＣｙＤＴＡ （ｐＨ８，６）が有効と考えられ
る。Hydroxyethylethylenediaminetriacetic acid (EDTA-
OH) Diethylenetriaminepentaacetic acid (DTPA) Diaminopropanoltetraacetic acid (DPTA-OH) Nitrilotriacetic acid (NTA) Transcyclohexanediaminetetraacetic acid (CyDTA) Dihydroxyethylglycine (DHEG) phosphate Tables 1 and 2 are for reference below. Lactate dehydrogenase (L
This figure shows the results of a test using the DH) measurement reagent. Table 1 shows the influence of pH on the change in reagent blank values over time. This result shows that depending on the pH selection, the opposite effect may occur. Table 2 shows the influence of the concentration of the chelating agent on the change in reagent blank value over time and color development. From this result, it can be seen that as the concentration of the chelating agent increases, the reagent blank value decreases over time, but there is also a tendency to reduce coloration due to reaction. Therefore, when measuring LDH, the chelating agent is 1-8mM-EDTA-OH (pH 8.6).
or 1-8mM-DPTA-OH (pH 8,6), 4-
8mM-CyDTA (pH 8.6) is considered effective.
表３及び表４は、同様に、後述の参考例３のトリグリセ
ライド測定に用いるキレート剤を検討した結果を示すも
のである。これらの表から、１〜８ｍＭ（特に１〜４
ｍＭ） −ＥＤＴＡ （ｐＨ８，６）、１〜８ｍＭ−Ｄ
ＴＰＡ−ＯＨ（ｐＨ８，６） 、４〜８ｍＭ−Ｃｙ Ｄ
ＴＡ （ｐＨ１０，０）及び５０ｍＭリン酸緩衝液（ｐ
Ｈ６，０）が使用可能なものと判断することができる。Tables 3 and 4 similarly show the results of examining chelating agents used for triglyceride measurement in Reference Example 3, which will be described later. From these tables, 1-8mM (especially 1-4mM)
mM) -EDTA (pH 8,6), 1-8mM-D
TPA-OH (pH 8,6), 4-8mM-CyD
TA (pH 10,0) and 50mM phosphate buffer (p
H6,0) can be determined to be usable.
後述の参考例２に記載の方法に従ってクレアチンキナー
ゼ（ＣＫ）を測定する場合には、ここで生じる色素が中
性以上のＰＨで不安定であるため、キレート剤を弱酸性
の条件で用いることが望ましい。When measuring creatine kinase (CK) according to the method described in Reference Example 2 below, it is recommended to use a chelating agent under weakly acidic conditions because the pigment produced here is unstable at pH above neutral. desirable.
又、表５に示す様に、使用する緩衝液の種類によって効
果の異なるものもある。濃度を変化させた場合の実験結
果を表６に示した。この結果から、０．５〜２ｍＭのＥ
ＤＴＡＳＥＤＴＡ−ＯＨＳＤＴＰＡ及びＣｙＤＴＡが有
効であることがわかる。Furthermore, as shown in Table 5, the effects may vary depending on the type of buffer used. Table 6 shows the experimental results when the concentration was varied. From this result, 0.5-2mM E
It can be seen that DTASEDTA-OHSDTPA and CyDTA are effective.
本発明方法に用いられるＦｅ”＋は、硫酸第二鉄アンモ
ニウム、塩化第二鉄などであり、Ｆｅ”に特異的に反応
するキレート剤としては、例えば、バソフエナンスロリ
ンスルホン酸ナトリウム、２−ピリジルアルドキシム、
３−（２−ピリジル）−５，６−ビス（４−スルホニル
） −１，２，４−トリアジンスルホン酸ナトリウム、
オルトフェナンスロリンが適当である。また、電子伝達
剤としては、たとえばフェナジンメトサルフェー） （
ＰＭＳ）、メルトラブル−、メトキシフェナジンメ！・
サルフェート（Ｍ−ＰＭＳ） 、ディアフォラーゼが適
している。緩衝液としては、トリエタノールアミン緩衝
液の他、反応に関わる酵素等の反応至適条件を満足する
ような成分と９Ｈを選択することができる。The Fe''+ used in the method of the present invention is ferric ammonium sulfate, ferric chloride, etc., and the chelating agent that specifically reacts with Fe'' is, for example, sodium bathophenanthrolinsulfonate, 2- pyridylaldoxime,
Sodium 3-(2-pyridyl)-5,6-bis(4-sulfonyl)-1,2,4-triazine sulfonate,
Orthophenanthroline is suitable. In addition, as an electron transfer agent, for example, phenazine methosulfate) (
PMS), Mel Trouble, Methoxyphenazine Me!・
Sulfate (M-PMS) and diaphorase are suitable. As the buffer, in addition to the triethanolamine buffer, 9H and components that satisfy the optimum reaction conditions for enzymes involved in the reaction can be selected.
表１ （１） ０度は全て４ｍＭとした。 Table 1 (1) All 0 degrees were 4mM.
（２）キレート剤を５０ｍ１Ｊシュウ酸含有０．１Ｍコ
ハク酸緩衝液（ｐＨ６，０）に溶解。(2) Dissolve the chelating agent in 50ml of 0.1M succinate buffer containing 1J oxalic acid (pH 6.0).
（３）キレート剤を５０ｍＭシュウ酸含有０．１Ｍ ト
リエタノールアミン緩衝液（ｐＨ８，６）に溶解。(3) Dissolve the chelating agent in 0.1M triethanolamine buffer (pH 8.6) containing 50mM oxalic acid.
（４）キレート剤を５０ｍＭシュウ酸含有０．１Ｍ炭酸
緩衝液（ｐＨ１０，ｏ）に溶解。(4) Dissolve the chelating agent in 0.1M carbonate buffer (pH 10, o) containing 50mM oxalic acid.
表２
（１）キレート剤添加直後に測定した吸光度に対する、
６０分後に測定した吸光度の百分率で示した。Table 2 (1) Regarding the absorbance measured immediately after adding the chelating agent,
It is expressed as a percentage of the absorbance measured after 60 minutes.
表３ （１）濃度は、全て４ｍＭとした。Table 3 (1) All concentrations were 4mM.
（２）キレート剤を０．１Ｍコハク酸緩衝液に溶解（ｐ
Ｈ６，０）（３）キレート剤をＯ，１Ｍ）リエタノール
アミン緩衝液に溶解（ｐＨ８，６）
（４）キレート剤をＯ，１Ｍ炭酸緩衝液に溶解（ｐＨ１
Ｏ，Ｏ）表４
表５
キレート剤の溶解緩衝液
０．１Ｍコハク酸緩衝液（ｐＨ５，０）０．１Ｍコハク
酸緩衝液（ｐＨ６，０）０、１Ｍ３．３−ジメチルグル
タル酸緩衝液（ｐＨ５，０）０、１Ｍ３．３−ジメチル
グルタル酸緩衝液（ｐＨ６，０）濃度は全てＩｍＭ
表６
（１）０．１Ｍ
３．３
一ジメチルグルタル酸（ｐＨ６，０）
に溶解
〔以下余白〕
〔実施例〕
次に実施例により本発明を更に詳しく説明するが、これ
は本発明を限定するものではない。(2) Dissolve the chelating agent in 0.1M succinate buffer (p
H6,0) (3) Dissolve the chelating agent in O, 1M) reethanolamine buffer (pH 8,6) (4) Dissolve the chelating agent in O, 1M carbonate buffer (pH 1
O, O) Table 4 Table 5 Chelating agent dissolution buffer 0.1M succinate buffer (pH 5,0) 0.1M succinate buffer (pH 6,0) 0,1M 3.3-dimethylglutarate buffer ( pH 5,0) 0,1M 3.3-dimethylglutarate buffer (pH 6,0) All concentrations are ImM Table 6 (1) Dissolved in 0.1M 3.3-dimethylglutarate (pH 6,0) [Margin below] [Example] Next, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited thereto.
施：血 の酸脱 ＬＤＨ活の
定
ｌ）試薬
溶液Ａ ： １１．６ｍＭオルトフェナンスロリンと４
００ｍＭ乳酸リチウムとを含む２００ｍＭトリエタノー
ルアミン緩衝液（ｐＨ８，０）。Treatment: Blood acid removal and determination of LDH activity l) Reagent solution A: 11.6mM orthophenanthroline and 4
200mM triethanolamine buffer (pH 8,0) containing 00mM lithium lactate.
溶液Ｂ ：２０ｍＭ−ＮＡＤと、３Ｕ／ｄデイアフオラ
ーゼと、１２５ｍＭ硫酸第二鉄アンモニウムとを含む５
０ｍＭ トリエタノールアミン緩衝液（ｐＨ７，２）。Solution B: 5 containing 20mM NAD, 3U/d diafluorase, and 125mM ferric ammonium sulfate.
0mM triethanolamine buffer (pH 7.2).
溶液Ｃ（キレート剤）：５０ｍＭシュウ酸と、４ｍＭ−
ＥＤＴＡとを含む０．１Ｍ ）リエタノールアミン緩衝
液（ｐＨ８，６）。Solution C (chelating agent): 50mM oxalic acid, 4mM-
0.1M) reethanolamine buffer (pH 8.6) containing EDTA.
２）操作方法
血清３０μｌと溶液Ｂ０．５ｒＩＬｌとを試験管にとり
、溶液へ〇、５−を加えて３７”Ｃで正確に１０分間反
応させた後、ただちに溶液Ｃ２７１Ｌｌを添加して室温
に戻す。別に、血清を加えずに、同様に操作したものを
試薬ブランクとして、５１０ｎｍの吸光度を測定する。2) Procedure: Take 30μl of serum and 0.5rILl of solution B into a test tube, add 〇 and 5- to the solution and react at 37''C for exactly 10 minutes, then immediately add 271Ll of solution C and return to room temperature. Separately, absorbance at 510 nm is measured using a reagent blank prepared in the same manner without adding serum.
次に、血清試料と同一方法で処理した既知ＬＤＨ活性の
溶液によって示される吸光度と測定吸光度との比較によ
って、血清試料のＬ Ｄ Ｈ活性を算出する。The L DH activity of the serum sample is then calculated by comparing the measured absorbance with the absorbance exhibited by a solution of known LDH activity treated in the same manner as the serum sample.
１：血 の乳酸脱 ＬＤＨ活 の団完
ｌ）試薬
溶液Ａ ： １１．６ｍＭオルトフェナンスロリンと４
００ｍＭ乳酸リチウムとを含む２００ｍＭトリエタノー
ルアミン緩衝液（ｐＨ８，０）。1: Blood lactate removal and LDH activation group 1) Reagent solution A: 11.6mM orthophenanthroline and 4
200mM triethanolamine buffer (pH 8,0) containing 00mM lithium lactate.
溶液Ｂ ：２０ｍＭ−ＮＡＤと、３Ｕ／−ディアフォラ
ーゼと、１２５ｍＭ硫酸第二鉄アンモニウムとを含む５
０ｍＭ ）リエタノールアミン緩衝液（ｐＨ７，２）。Solution B: 5 containing 20mM NAD, 3U/-diaphorase and 125mM ferric ammonium sulfate.
0mM) reethanolamine buffer (pH 7,2).
溶液Ｃ（キレート剤）：５０ｍＭシュウ酸と、４［ＩＩ
Ｍ −ＥＤＴＡ−ＯＨとを含む０．１Ｍ）リエタノール
アミン緩衝液（ｐＨ８，６）。Solution C (chelating agent): 50mM oxalic acid and 4[II
0.1 M) reethanolamine buffer (pH 8,6) containing M-EDTA-OH.
２）操作方法
血清３０μｌと溶液Ｂ０．５Ｔｌｄ！とを試験管にとり
、溶液Ａ０．５−を加えて３７℃で正確に１０分間反応
させた後、ただちに溶液Ｃ２ｎ１１を添加して室温に戻
す。別に、血清を加えずに、同様に操作したものを試薬
ブランクとして、５１０ｎｍの吸光度を測定する。次に
、血清試料と同一方法で処理した既知ＬＤＨ活性の溶液
によって示される吸光度と測定吸光度との比較によって
、血清試料のＬＤＨ活性を算出する。第１図に示すとお
り、上記の諸条件下で、直線的な吸光度の応答が約１２
００ウロブレスキー（Ｗｒｏｂｌｅｗｓｋｉ）単位のＬ
ＤＨ活性のレベルまで認められた。2) Procedure: 30 μl of serum and 0.5 Tld of solution B! and into a test tube, add solution A0.5- and react at 37°C for exactly 10 minutes, then immediately add solution C2n11 and return to room temperature. Separately, absorbance at 510 nm is measured using a reagent blank prepared in the same manner without adding serum. The LDH activity of the serum sample is then calculated by comparing the measured absorbance with the absorbance exhibited by a solution of known LDH activity treated in the same manner as the serum sample. As shown in Figure 1, under the above conditions, the linear absorbance response was approximately 12
L in 00 Wroblewski
Even the level of DH activity was observed.
ｌ）試薬
溶液へ： ６ｍ）Ｊ ３−（２−ピリジル）−５，６−
ビス（４−スルホニル）−１，２，４−トリアジンスル
ホン酸ナトリウムと、４０ｍＭクレアチンリン酸をを含
む２００ｍＭ トリエタノールアミン緩衝液（ｐＨ７
，６）。l) To the reagent solution: 6m) J 3-(2-pyridyl)-5,6-
200 mM triethanolamine buffer (pH 7) containing sodium bis(4-sulfonyl)-1,2,4-triazine sulfonate and 40 mM creatine phosphate.
, 6).
溶液Ｂ：２ｍＭＮＡＤＰと、４０ｕＭ−ＰＭＳと、４ｍ
Ｍ了デノシンー２−リン酸と、６ｍＭ硫酸第二鉄アンモ
ニウムと、−１２ｍＭ塩化マグネシウムと、８０ｍＭグ
ルコースと、０．１ｗ／ｖ％ウシ血清アルブミンと、０
．８Ｕ／−グルコース６リン酸脱水素酵素と、１．３Ｕ
／ｍｌへキソキナーゼとを含む５０ｍＭ ）リエタノー
ルアミン緩衝液（ｐＨ７，２）。Solution B: 2mM NADP, 40uM-PMS, 4m
Myodenosine-2-phosphate, 6mM ferric ammonium sulfate, -12mM magnesium chloride, 80mM glucose, 0.1% w/v bovine serum albumin, 0
．． 8U/-glucose 6-phosphate dehydrogenase and 1.3U
50mM) reethanolamine buffer (pH 7.2) containing /ml hexokinase.
溶液Ｃ（キレート剤）： １ｍＭ−ＤＴＰＡを含む０．
１Ｍ ３．３−ジメチルグルタル酸緩衝液−（ｐＨ６
，０２”）。Solution C (chelating agent): 0.0% containing 1mM-DTPA.
1M 3.3-dimethylglutarate buffer (pH 6
, 02”).
２）操作方法
血清ｌＯμｌと溶液Ａ０．２５ｍ１とを試験管にとり、
溶液８０．２５ｍ１を加えて３７℃で正確に１０分間反
応させた後、ただちに溶液Ｃ４ｒＩｄ！を添加し、室温
に戻す。別に、血清を加えずに同様に操作したものを試
薬ブランクとして、５６４ｎｍの吸光度を測定する。2) Procedure: Take 10 μl of serum and 0.25 ml of solution A into a test tube.
After adding 80.25 ml of solution and reacting at 37°C for exactly 10 minutes, immediately add solution C4rId! Add and return to room temperature. Separately, absorbance at 564 nm is measured using a reagent blank prepared in the same manner without adding serum.
次に、血清試料と同一方法で処理した既知ＣＫ活性溶液
によって示される吸光度と測定吸光度との比較によって
血清試料のＣＫ活性を算出する。第２図に示すとおり、
上記の諸−条件下で直線的な吸光度の応答が２００１．
Ｕ、／ｊ７のレベルまで認められた。Next, the CK activity of the serum sample is calculated by comparing the measured absorbance with the absorbance exhibited by a known CK activity solution treated in the same manner as the serum sample. As shown in Figure 2,
Under the above conditions, the linear absorbance response was 2001.
It was recognized up to the level of U, /j7.
例３：血 のトリグセライドの 定
ｌ）試薬
溶液Ａ：５．８ｍＭバソフェナンスロリンスルホン酸ナ
トリウムと、２００ｍＭ塩化カリウムとを含む２００ｍ
Ｍ’トリエタメトリアミン緩衝液（ｐＨ８，０）。Example 3: Determination of triglyceride in blood l) Reagent solution A: 200mM containing 5.8mM sodium bathophenanthrolin sulfonate and 200mM potassium chloride.
M'trietamethramine buffer (pH 8,0).
溶液Ｂ ： ４０ｍＭ−ＮＡＤと、４０μＭメトキシＰ
ＭＳと、２５ｍＭ硫酸第二鉄アンモニウムと、０．１ｗ
／Ｖ％ウシ血清アルブミンと、４０００／−リポプロテ
ィンリパーゼと、３２Ｕ／−グリセロール脱水素酵素と
を含む５０ｍＭ ）リエタノールアミン緩衝液（ｐＨ７
，２）。Solution B: 40mM-NAD and 40μM methoxy P
MS, 25mM ferric ammonium sulfate, 0.1w
/V% bovine serum albumin, 4000/- lipoprotein lipase, and 32 U/- glycerol dehydrogenase in 50 mM) reethanolamine buffer (pH 7).
,2).
溶液Ｃ： ５０ｍＭリン酸ナトリウム緩衝液。Solution C: 50mM sodium phosphate buffer.
２）操作方法
血清１０μｌと、溶液へ〇、 ２５ｍ１とを試験管にと
り、溶液Ｂ Ｏ，２５ｔｄを加えて、３７℃で２０分間
インキュベートした後、ただちに溶液Ｃ４−を添加して
室温に戻す。別に、血清を加えずに同様に操作したもの
を試薬ブランクとして５３５ｎｍの吸光度を測定する。2) Procedure: Transfer 10 μl of serum and 25 ml of solution into a test tube, add solution BO, 25 td, and incubate at 37°C for 20 minutes. Immediately add solution C4- and return to room temperature. Separately, absorbance at 535 nm is measured using a reagent blank that was operated in the same manner without adding serum.
次に、血清試料のトリグリセライド含量を血清試料と同
一方法で処理した既知トリグリセライド含量の溶液によ
って示される吸光度と測定吸光度との比較によって算出
する。第３図に示すとおり、上記の諸条件下で直線的な
吸光度の応答がほぼ１０００ｍｇ／ｄｌのトリグリセラ
イドのレベルまで認められた。The triglyceride content of the serum sample is then calculated by comparing the measured absorbance with the absorbance exhibited by a solution of known triglyceride content treated in the same manner as the serum sample. As shown in FIG. 3, under the above conditions, a linear absorbance response was observed up to a triglyceride level of approximately 1000 mg/dl.
以上述べたことから、本発明方法は広く一般に脱水素酵
素及びＮＡＤ又はＮＡＤＰを含む測定系への応用が充分
可能であ、す、比色時の煩雑な操作を必要とせず、−信
頼度の高い測定を多検体にわたり可能とする。又、本発
咀方法は高い感度を期待できるため、反応時間の短縮や
試料の微量化も可能であることが明らかである。From the above, the method of the present invention can be widely applied to measurement systems containing dehydrogenase and NAD or NADP, does not require complicated operations during colorimetry, and has improved reliability. Enables high-quality measurements over multiple samples. Furthermore, since the present mastication method can be expected to have high sensitivity, it is clear that it is possible to shorten the reaction time and reduce the amount of sample.
第１図は血清中のＬＤＨ活性の測定におけるウロプレウ
スキー単位と吸光度との関係を示すグラフであり、第２
図はクアレアチンキナーゼ（ＣＫ）活性測定における測
定可能領域を示すグラフであり、そして第３図はトリグ
リセライドの測定における測定可能領域を示すグラフで
ある。Figure 1 is a graph showing the relationship between Uroplewski units and absorbance in measuring LDH activity in serum;
The figure is a graph showing the measurable area in the measurement of quareatine kinase (CK) activity, and FIG. 3 is a graph showing the measurable area in the measurement of triglyceride.
Claims (1)
クレオチド（ＮＡＤ）又は酸化型ニコチンアミドアデニ
ンジヌクレオチドリン酸（ＮＡＤＰ）とを含む酵素反応
系により生成する還元型ニコチンアミドアデニンジヌク
レオチド（ＮＡＤＨ）又は還元型ニコチンアミドアデニ
ンジヌクレオチドリン酸（ＮＡＤＰＨ）の量に対応して
、電子伝達剤の存在下で第二鉄イオン（Ｆｅ＾３＾＋）
から還元生成する第一鉄イオン（Ｆｅ＾２＾＋）の量を
、Ｆｅ＾２＾＋に特異的に反応するキレート剤の作用に
より生成する着色化合物として比色定量する方法であっ
て、 エチレンジアミン四酢酸（ＥＤＴＡ）をキレート剤とし
て含有するｐＨ５．０〜８．６の緩衝液を、一定時間反
応後に添加することを特徴とする、基質又は酵素の定量
方法。(1) Reduced nicotinamide adenine dinucleotide (NADH) or Corresponding to the amount of reduced nicotinamide adenine dinucleotide phosphate (NADPH), ferric ion (Fe^3^+) in the presence of an electron transfer agent
A method for colorimetrically quantifying the amount of ferrous ions (Fe^2^+) produced by reduction from ethylenediamine as a colored compound produced by the action of a chelating agent that specifically reacts with Fe^2^+, the method comprising: A method for quantifying a substrate or an enzyme, which comprises adding a buffer solution containing tetraacetic acid (EDTA) as a chelating agent and having a pH of 5.0 to 8.6 after a certain period of reaction.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP15768390A JPH0343096A (en) | 1990-06-18 | 1990-06-18 | Determination of substrate or enzyme |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP15768390A JPH0343096A (en) | 1990-06-18 | 1990-06-18 | Determination of substrate or enzyme |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP15396181A Division JPS5856698A (en) | 1981-09-30 | 1981-09-30 | Improved method for quantitative determination of substrate or enzyme |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH0343096A true JPH0343096A (en) | 1991-02-25 |
| JPH0442000B2 JPH0442000B2 (en) | 1992-07-10 |
Family
ID=15655108
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP15768390A Granted JPH0343096A (en) | 1990-06-18 | 1990-06-18 | Determination of substrate or enzyme |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0343096A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0681611A4 (en) * | 1993-01-28 | 1998-07-29 | Boehringer Mannheim Corp | Compositions useful in anaerobic determination of analytes. |
| EP1466987A1 (en) * | 2001-12-28 | 2004-10-13 | ARKRAY, Inc. | Method of colorimetry and reagent for use therein |
| US7550273B2 (en) | 2001-12-28 | 2009-06-23 | Arkray, Inc. | Colorimetric method and reagent used for the same |
-
1990
- 1990-06-18 JP JP15768390A patent/JPH0343096A/en active Granted
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0681611A4 (en) * | 1993-01-28 | 1998-07-29 | Boehringer Mannheim Corp | Compositions useful in anaerobic determination of analytes. |
| EP1466987A1 (en) * | 2001-12-28 | 2004-10-13 | ARKRAY, Inc. | Method of colorimetry and reagent for use therein |
| EP1466987A4 (en) * | 2001-12-28 | 2006-04-19 | Arkray Inc | Method of colorimetry and reagent for use therein |
| US7550273B2 (en) | 2001-12-28 | 2009-06-23 | Arkray, Inc. | Colorimetric method and reagent used for the same |
| US8574896B2 (en) | 2001-12-28 | 2013-11-05 | Arkray, Inc. | Colorimetric method and reagent used for the same |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0442000B2 (en) | 1992-07-10 |
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