CS268738B1 - Method of hydrogen peroxide's and oxidase substrates' quantitative fluorometric determination - Google Patents
Method of hydrogen peroxide's and oxidase substrates' quantitative fluorometric determination Download PDFInfo
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- CS268738B1 CS268738B1 CS884960A CS496088A CS268738B1 CS 268738 B1 CS268738 B1 CS 268738B1 CS 884960 A CS884960 A CS 884960A CS 496088 A CS496088 A CS 496088A CS 268738 B1 CS268738 B1 CS 268738B1
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- Prior art keywords
- hydrogen peroxide
- substrate
- peroxidase
- oxidase
- response
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- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 title claims abstract description 36
- 239000000758 substrate Substances 0.000 title claims abstract description 21
- 102000004316 Oxidoreductases Human genes 0.000 title claims abstract description 10
- 108090000854 Oxidoreductases Proteins 0.000 title claims abstract description 10
- 238000000034 method Methods 0.000 title claims description 10
- 239000000126 substance Substances 0.000 claims abstract description 11
- 102000003992 Peroxidases Human genes 0.000 claims abstract description 9
- 108040007629 peroxidase activity proteins Proteins 0.000 claims abstract description 9
- 108010093096 Immobilized Enzymes Proteins 0.000 claims abstract description 4
- 230000000694 effects Effects 0.000 claims description 2
- 230000002255 enzymatic effect Effects 0.000 abstract description 3
- 230000003647 oxidation Effects 0.000 abstract description 2
- 238000007254 oxidation reaction Methods 0.000 abstract description 2
- 238000011002 quantification Methods 0.000 abstract description 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 abstract 1
- 229910052739 hydrogen Inorganic materials 0.000 abstract 1
- 239000001257 hydrogen Substances 0.000 abstract 1
- 150000002978 peroxides Chemical class 0.000 abstract 1
- 238000004445 quantitative analysis Methods 0.000 abstract 1
- OIPILFWXSMYKGL-UHFFFAOYSA-N acetylcholine Chemical compound CC(=O)OCC[N+](C)(C)C OIPILFWXSMYKGL-UHFFFAOYSA-N 0.000 description 6
- 229960004373 acetylcholine Drugs 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 229940088598 enzyme Drugs 0.000 description 4
- 239000000284 extract Substances 0.000 description 4
- 238000005259 measurement Methods 0.000 description 3
- 239000002253 acid Substances 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- OEYIOHPDSNJKLS-UHFFFAOYSA-N choline Chemical compound C[N+](C)(C)CCO OEYIOHPDSNJKLS-UHFFFAOYSA-N 0.000 description 2
- 229960001231 choline Drugs 0.000 description 2
- XBDQKXXYIPTUBI-UHFFFAOYSA-N dimethylselenoniopropionate Natural products CCC(O)=O XBDQKXXYIPTUBI-UHFFFAOYSA-N 0.000 description 2
- NMHMNPHRMNGLLB-UHFFFAOYSA-N phloretic acid Chemical compound OC(=O)CCC1=CC=C(O)C=C1 NMHMNPHRMNGLLB-UHFFFAOYSA-N 0.000 description 2
- 102000012440 Acetylcholinesterase Human genes 0.000 description 1
- 108010022752 Acetylcholinesterase Proteins 0.000 description 1
- 108010000659 Choline oxidase 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
- YZCKVEUIGOORGS-NJFSPNSNSA-N Tritium Chemical compound [3H] YZCKVEUIGOORGS-NJFSPNSNSA-N 0.000 description 1
- 229940022698 acetylcholinesterase Drugs 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 230000029918 bioluminescence Effects 0.000 description 1
- 238000005415 bioluminescence Methods 0.000 description 1
- 210000005013 brain tissue Anatomy 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 235000019260 propionic acid Nutrition 0.000 description 1
- IUVKMZGDUIUOCP-BTNSXGMBSA-N quinbolone Chemical compound O([C@H]1CC[C@H]2[C@H]3[C@@H]([C@]4(C=CC(=O)C=C4CC3)C)CC[C@@]21C)C1=CCCC1 IUVKMZGDUIUOCP-BTNSXGMBSA-N 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
ŘeSení se týká způsobu kvantitativního fluorimetrického stanovení peroxidu vodíku a substrátů oxidáz. Tento způsob spočívá v injekci vzorku substrátu do kontinuálního proudu média. Stykem média s imobilizovaným enzymem odpovádajícím přislužnému substrátu vzniká při enzymatické oxidaci substrátu peroxid vodíku. Ten přichází dále do styku s lmohllizovanou peroxidázou. Médium obsahuje chemickou látku, která v přítomnosti peroxidu vodíku a peroxidázy je schopna vytvořit fluorescenční produkt. Vykazovaná fluorescence Je měřena fluorimetrem. Samotná kvantifikace se provádí na základě porovnáni íluorimetřické odezvy na stanovený vzorek substrátu a odezvy na známá standardní množství zkoumané látky.The solution concerns the quantitative method fluorimetric determination of peroxide hydrogen and oxidase substrates. This way consists in injecting a substrate sample into continuous media flow. Media contact with immobilized enzyme compatible the substrate is produced by enzymatic oxidation of the substrate hydrogen peroxide. It comes in contact with the lmohllized peroxidase. The medium contains a chemical that in the present hydrogen peroxide and peroxidase is capable to create a fluorescent product. Reported fluorescence is measured by a fluorimeter. Quantification itself is done on the basis of comparing the fluorimetric response to a determined substrate sample and response to known standard amounts investigated substances.
Description
1 CS 268378 B11 CS 268378 B1
Vynález se týká způsobu kvantitativního fluorimetrického stanovení peroxidu vodí-ku a substrátů oxidáz.The present invention relates to a method for the quantitative fluorimetric determination of hydrogen peroxide and oxidase substrates.
Vytvoření analytických metod umožňujíc&h kvantitativní stanoveni biologicky význam-ných látek s vysokou citlivostí je velkým problémem v biochemických laboratořích. Proměření některých látek, které je možno působením oxidáz přeměnit na peroxid vodíku, Jepoužíváno íluorimetrie, při které je do kyvety fluorimetru napipetován příslušný stano-vený substrát, Jemu odpovídající oxidáza, peroxidáza a chemická látka, která po enzyma-tické reakci katalyzované peroxidázou vykazuje fluorescenci. Oxidáza převede substrátna peroxid vodíku, který v přítomnosti peroxidázy oxiduje látku schopnou fluorescence.Velikost fluorescenčního efektu odpovídá množství stanoveného substrátu. Metody tohototypu Jsou popsány v knize M.A.Detuca (ed.), Methods in Enzymology, vol. 57, Bioluminis-cence and Chemiluminiscence (Academie Press, New York, 1978). Tato metoda, přestože jecitlivá, má své nevýhody. Nutnost přidání nového množství enzymů pro každé jednotlivéměření znamená vysokou spotřebu specifických enzymů. Měření samotné Je pomalé a pracné.The creation of analytical methods allowing quantitative determination of biologically important substances with high sensitivity is a major problem in biochemical laboratories. Measurements of some of the substances that can be converted to hydrogen peroxide by oxidase treatment are used by fluorimetry in which the respective determined substrate is pipetted into the fluorimeter cuvette, the corresponding oxidase, the peroxidase, and the chemical that has fluorescence upon peroxidase catalyzed enzymatic reaction. The oxidase converts the substrate hydrogen peroxide, which in the presence of peroxidase oxidizes the fluorescent substance. The size of the fluorescent effect corresponds to the amount of substrate determined. Methods of this Method are described in M. A. Detuca (ed.), Methods in Enzymology, vol. 57, Bioluminescence and Chemiluminescence (Academic Press, New York, 1978). This method, though sensitive, has its drawbacks. The need to add new amounts of enzymes for each individual measurement means high consumption of specific enzymes. The measurement itself is slow and laborious.
Tyto nevýhody odstraňuje způsob měření podle vynálezu. Jsou v něm uplatněny dva zá-kladní prvky novosti. Injekce měřených vzorků se provádí přímo do kontinuálního proudumédia, ěímž odpadá přidávání jednotlivých enzymů ke každému vzorku a použité enzymyjsou navíc imobilizovány na vhodném nosiěl, se kterým proudící medium přichází do styku.These disadvantages are eliminated by the measurement method according to the invention. There are two basic elements of novelty applied. Injection of the measured samples is done directly into the continuous stream, eliminating the addition of the individual enzymes to each sample, and the enzymes used are additionally immobilized on a suitable carrier with which the flowing medium comes into contact.
Způsob kvantitativního stanovení peroxidu vodíku a substrátů oxidáz spočívá tedyv injekci vzorku substrátu do kontinuálního proudu média. Stykem média s imobilizovanýmenzymem odpovídajícím příslušnému substrátu vzniká při enzymatické oxidaci substrátuperoxid vodíku. Vzniklý peroxid vodíku prochází pak s médiem kolonkou s imobillzovanouperoxidázou. Médium obsahuje chemickou látku, která v přítomnosti peroxidu vodíku a pe-roxidázy je schopna vytvořit fluorescenční produkt, nejlépe 3-/p.hydroxyfenyl/ propio-novou kyselinu, komovanillnovou kyselinu případně hydrofenyloctovou kyselinu. Tato látkaje akceptorem elektronů při enzymatické redukci peroxidu vodíku katalyzované peroxidá-zou. Fluorescenci je při vhodné kombinaci vlnových délek pro excitaci a emisi možno kvan-titativně měřit na připojeném fluorimetru. Jeho signál Je přímo úměrný koncentraci pero-xidu vodíku a tedy i původnímu vzorku substrátu pro danou imobillzovanou oxidázu. Samot-ná kvantifikace se provádí na základě porovnání fluorimetrické odezvy na stanovený vzoreksubstrátu a odezvy na známé standardní množství zkoumané látky.Thus, a method for quantitatively determining hydrogen peroxide and oxidase substrates is to inject a substrate sample into a continuous media stream. By contacting the medium with the immobilized enzyme corresponding to the respective substrate, hydrogen peroxide substrate is produced during enzymatic oxidation of the substrate. The resulting hydrogen peroxide then passes through the medium with an imobillinated peroxidase column. The medium contains a chemical which, in the presence of hydrogen peroxide and peroxidase, is capable of forming a fluorescent product, preferably 3- (p-hydroxyphenyl) propionic acid, como acid or hydrophenylacetic acid. This substance is an electron acceptor in the enzymatic reduction of peroxide-catalyzed hydrogen peroxide. Fluorescence can be quantitatively measured on the attached fluorimeter at a suitable wavelength combination for excitation and emission. Its signal is directly proportional to the concentration of hydrogen peroxide and hence to the original substrate sample for the given imbulated oxidase. The quantification itself is carried out by comparing the fluorimetric response to the determined substrate sample and the response to the known standard amount of test substance.
Princlptohoto způsobu stanovení je možno využít v oblasti biochemie, lékařské bio-chemie, fyziologického a farmakologického výzkumu. Charakteristickým příkladem látek,které mohou být tímto způsobem stanoveny, jsou glukosa, cholin a cholesterol. PříkladThis method can be used in the fields of biochemistry, medical bio-chemistry, physiological and pharmacological research. Typical examples of substances that can be determined in this manner are glucose, choline, and cholesterol. Example
Popsaný způsob byl použit ke stanovení množství acetylcholinu /ACh/ v extraktechz mozkové tkáně. K tomuto účelu byla použita kombinace tří imobilizovaných enzymů natřech za sebou následujících kolonách: acetylcholinesteráza /vytváří z acetylcholinucholin/, cholinoxidáza /vytváří z cholinu peroxid vodíku / a peroxidáza /vytváří fluo-rescentní produkt interekci mezi peroxidem vodíku a 3-/p-rhydroxyfenyl/-propionovou ky-selinou, Jež byla přítomna v proudícím médiu/. Do kontinuálního proudu média byla ve dvouminutových intervalech vstřikovány jednak vzorky známých množství Ach /20 pmol, 40 pmol,100 pmol atd/, jednak 20 ul množství tkáňových extraktů. OdpověS fluorimetru na standart-ní množství ACh v průběhu pokusu neměnila. Porovnáním mezi odpovědí na známá množstvíACh a na tkáňové extrakty bylo možno vypočítat koncentraci ACh v jednotlivých extraktech.The method described was used to determine the amount of acetylcholine (ACh) in the brain tissue extract. For this purpose, a combination of three immobilized enzymes was used, including three consecutive columns: acetylcholinesterase (generates acetylcholinucholine), choline oxidase / produces hydrogen peroxide from choline (and peroxidase) forms a fluoroscent product by interaction between hydrogen peroxide and 3- (β-hydroxyphenyl) propionic acid, which was present in the flowing medium. Samples of known amounts of Ach / 20 pmol, 40 pmol, 100 pmol, etc., and 20 µl of tissue extracts were injected into the continuous media stream at two-minute intervals. The response of the fluorimeter to the standard amount of ACh did not change during the experiment. By comparing the response to the known amounts of ACh and the tissue extracts, the concentration of ACh in the individual extracts could be calculated.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CS884960A CS268738B1 (en) | 1988-07-08 | 1988-07-08 | Method of hydrogen peroxide's and oxidase substrates' quantitative fluorometric determination |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CS884960A CS268738B1 (en) | 1988-07-08 | 1988-07-08 | Method of hydrogen peroxide's and oxidase substrates' quantitative fluorometric determination |
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| Publication Number | Publication Date |
|---|---|
| CS496088A1 CS496088A1 (en) | 1989-07-12 |
| CS268738B1 true CS268738B1 (en) | 1990-04-11 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CS884960A CS268738B1 (en) | 1988-07-08 | 1988-07-08 | Method of hydrogen peroxide's and oxidase substrates' quantitative fluorometric determination |
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| CS (1) | CS268738B1 (en) |
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1988
- 1988-07-08 CS CS884960A patent/CS268738B1/en unknown
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| Publication number | Publication date |
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| CS496088A1 (en) | 1989-07-12 |
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