CS268737B1 - 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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- CS268737B1 CS268737B1 CS884959A CS495988A CS268737B1 CS 268737 B1 CS268737 B1 CS 268737B1 CS 884959 A CS884959 A CS 884959A CS 495988 A CS495988 A CS 495988A CS 268737 B1 CS268737 B1 CS 268737B1
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- Prior art keywords
- hydrogen peroxide
- substrate
- ach
- luminometer
- oxidase
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- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 title claims abstract description 40
- 239000000758 substrate Substances 0.000 title claims abstract description 21
- 102000004316 Oxidoreductases Human genes 0.000 title claims abstract description 9
- 108090000854 Oxidoreductases Proteins 0.000 title claims abstract description 9
- 238000000034 method Methods 0.000 title claims description 9
- 239000000126 substance Substances 0.000 claims abstract description 8
- 102000003992 Peroxidases Human genes 0.000 claims abstract description 7
- 238000004020 luminiscence type Methods 0.000 claims abstract description 7
- 108040007629 peroxidase activity proteins Proteins 0.000 claims abstract description 7
- 230000003647 oxidation Effects 0.000 claims abstract description 5
- 238000007254 oxidation reaction Methods 0.000 claims abstract description 5
- 108010093096 Immobilized Enzymes Proteins 0.000 claims abstract 2
- 150000002978 peroxides Chemical class 0.000 claims abstract 2
- 239000000284 extract Substances 0.000 claims description 4
- HWYHZTIRURJOHG-UHFFFAOYSA-N luminol Chemical compound O=C1NNC(=O)C2=C1C(N)=CC=C2 HWYHZTIRURJOHG-UHFFFAOYSA-N 0.000 claims description 3
- 238000002474 experimental method Methods 0.000 claims 1
- 230000003993 interaction Effects 0.000 claims 1
- 230000002255 enzymatic effect Effects 0.000 abstract description 3
- 238000011002 quantification Methods 0.000 abstract description 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 abstract 1
- 238000006243 chemical reaction Methods 0.000 abstract 1
- 229910052739 hydrogen Inorganic materials 0.000 abstract 1
- 239000001257 hydrogen Substances 0.000 abstract 1
- 238000011835 investigation Methods 0.000 abstract 1
- 230000035479 physiological effects, processes and functions Effects 0.000 abstract 1
- 238000004445 quantitative analysis Methods 0.000 abstract 1
- 102000004190 Enzymes Human genes 0.000 description 6
- 108090000790 Enzymes Proteins 0.000 description 6
- OIPILFWXSMYKGL-UHFFFAOYSA-N acetylcholine Chemical compound CC(=O)OCC[N+](C)(C)C OIPILFWXSMYKGL-UHFFFAOYSA-N 0.000 description 3
- 229960004373 acetylcholine Drugs 0.000 description 3
- 229960001231 choline Drugs 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 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
- 239000011521 glass Substances 0.000 description 2
- 239000004033 plastic Substances 0.000 description 2
- HUDPLKWXRLNSPC-UHFFFAOYSA-N 4-aminophthalhydrazide Chemical compound O=C1NNC(=O)C=2C1=CC(N)=CC=2 HUDPLKWXRLNSPC-UHFFFAOYSA-N 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
- 238000004458 analytical method Methods 0.000 description 1
- 239000011324 bead Substances 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
- 238000005415 bioluminescence Methods 0.000 description 1
- 230000029918 bioluminescence Effects 0.000 description 1
- 210000005013 brain tissue Anatomy 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- -1 choline peroxide Chemical class 0.000 description 1
- 238000011161 development 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
- 238000000691 measurement method Methods 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
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
ŘeSeni se týká způsobu kvantitativního luminometrického stanovení peroxidu vodíku a substrátů oxidáz. Tento způsob spočívá v injekci vzorku substrátu do kontinuelního proudu média. Stykem média s imobilizovaným enzymem odpovídajícím příslušnému substrátu vzniká při enzymatické oxidaci substrátu peroxid vodíku. Ten přichází dále do styku s imohilizovanou peroxidázou umístěnou v zorném poli luminometru. Médium obsahuje chemickou látku, která při reakci s peroxidem vodíku katalyzované peroxidázou vykazuje luminiscenci. Samotná kvantifikace se děje na základě srovnání mezi odezvami na neznámý stanovovaný vzorek substrátu a na známé množetví zkoumané látky. Tato stanovení je možno využít v oblasti biochemie, lékařské biochemie, fyziologie a farmag ceutického průmyslu.The solution relates to a quantitative method luminometric determination of peroxide hydrogen and oxidase substrates. This way consists in injecting a substrate sample into a continuous sample stream of media. Media contact with immobilized enzyme corresponding to the appropriate substrate is produced by enzymatic oxidation of the substrate hydrogen peroxide. The coming in contact with the imohilized peroxidase located in the field of view of the luminometer. The medium contains a chemical which is catalyzed by reaction with hydrogen peroxide peroxidase exhibits luminescence. Quantification itself happens on by comparing the responses to the unknown determined substrate sample and known the quantity of the substance under investigation. These determinations can be used in biochemistry medical biochemistry, physiology and pharmacy industry.
Description
CS 268 737 B1 1EN 268 737 B1 1
Vynález se týká způsobu kvantitativního luminometrického stanovení peroxiduvodíku a substrátů oxidáz.BACKGROUND OF THE INVENTION The present invention relates to a method for the quantitative luminometric determination of hydrogen peroxide and oxidase substrates.
Vytvoření analytických metod umožňujících kvantitativní stanovení biologickyvýznamných látek s vysokou citlivostí je velkým problémem v biochemických labora-tořích. Pro měření některých látek, které je možno působením oxidáz přeměnit na pe-roxid vodíku, je používáno luminiscence, při které je do kyvety luminometru napipe-tován přisluěný stanovovaný substrát, jemu odpovídající oxidéza, peroxidáza a lumi-nol příp. jiný akceptor elektronů. Oxidáza převede substrát na peroxid vodíku, kte-rý v přítomnosti peroxidázy reaguje s luminolem za vzniku luminiscence. Velikostluminiscence, registrované luminometrem, odpovídá původnímu množství stanovovanéhosubstrátu. Metoda i její aplikace jsou podrobně popsány v knize M.A.Detuca /ed./,Methods in Bnzymology, vol. 57, Bioluminiscence and Chemilumiscence (Academie PressNew York, 1978/.The development of analytical methods allowing the quantitative determination of biologically significant substances with high sensitivity is a major problem in biochemical laboratories. For the measurement of certain substances which can be converted to hydrogen peroxide by oxidase treatment, luminescence is used in which the respective substrate to be determined, the corresponding oxidation, peroxidase and luminal is pipetted into the luminometer cuvette. another electron acceptor. The oxidase converts the substrate to hydrogen peroxide, which reacts with luminol in the presence of peroxidase to form luminescence. The luminescence, registered by the luminometer, corresponds to the original amount of the determined substrate. The method and its application are described in detail in M. A. Detuca / ed./, Methods in Bnzymology, vol. 57, Bioluminescence and Chemilumiscence (Academic PressNew York, 1978).
Tato metoda, přestože je vysoce citlivá, má své nevýhody. Nutnost přidání no-vého množství enzymů pro každé jednotlivé měření znamená vysokou spotřebu specific-kých enzymů. Měření samotné je pomalé a pracné. Tyto nevýhody odstraňuje způsob mě-ření podle vynálezu. Jsou v něm uplatněny dva základní prvky novosti. Injekce mě-řených vzorků substrátů je prováděna přímo do kontinuálního proudu média, čímž od-padá přidávání jednotlivých enzymů ke každému vzorku a použité enzymy jsou navíczmobilizovány na vhodném nosiči, se kterým proudící médium přichází do styku.This method, although highly sensitive, has its drawbacks. The need to add a new amount of enzymes for each individual measurement means high consumption of specific enzymes. The measurement itself is slow and laborious. These disadvantages are eliminated by the measurement method according to the invention. It incorporates two basic elements of novelty. Injection of the measured substrate samples is done directly into a continuous stream of media, thereby eliminating the addition of individual enzymes to each sample and the enzymes used are additionally immobilized on a suitable carrier with which the flowing medium contacts.
Způsob kvantitativního stanovení peroxidu vodíku a substrátů oxidáz spočívátedy v injekci vzorku substrátu do kontinuálního proudu média. Stykem média s zmo-bilizovaným enzymem odpovídajícím příslušnému substrátu vzniká při enzymové oxida-ci substrátu peroxid vodíku. Vzniklý peroxid vodíku prochází pak s médiem kolonkous zmobilizovanou peroxidázou na nosiči, který je propustný pro světelné záření,např. drobné skleněné nebo plastové kuličky, stěny skleněné nebo plastové kapiláryapod.The method of quantitatively determining hydrogen peroxide and oxidase substrates consists in injecting a substrate sample into a continuous stream of medium. By contacting the medium with the polymorphized enzyme corresponding to the respective substrate, hydrogen peroxide is produced by the enzymatic oxidation of the substrate. The resulting hydrogen peroxide is then passed through a light-permeable carrier, which is permeated by a peroxidase-immobilized medium, e.g. small glass or plastic beads, glass or plastic capillaries, and a base.
Medium obsahuje chemickou látku např. luminol, isoluminol apod., která přireakci s peroxidem vodíku katalyzované peroxidázou slouží jako akceptor elektronůa při oxidaci vykazuje luminiscenci. Proto je zmobilizovaná peroxidáza umístěnapřímo v zorném poli luminometru.The medium contains a chemical eg luminol, isoluminol and the like, which reacts with hydrogen peroxide catalyzed hydrogen peroxide to serve as an electron acceptor and exhibits luminescence in oxidation. Therefore, the mobilized peroxidase is placed directly in the field of view of the luminometer.
Světelný signál, vznikající při enzymatické redukci peroxidu vodíku, je přímoúměrný koncentraci peroxidu vodíku a tedy i původního vzorku substrátu pro použitouzmobilizovanou oxidazu. Samotná kvantifikace se děje na základě srovnání mezi odez-vami na neznámý stanovovaný vzorek substrátu a na známé standardní množství zkouma-né látky.The light signal produced by the enzymatic reduction of hydrogen peroxide is directly proportional to the concentration of hydrogen peroxide and hence the original substrate sample used for the mobilized oxidase. The quantification itself is based on a comparison between responses to an unknown determined substrate sample and to a known standard amount of test substance.
Princip tohoto způsobu stanovení je možno využít v oblasti biochemie, lékařskébiochemie, fyziologického a farmaceutického výzkumu. Charakteristickým příklademlátek, které mohou být tímto způsobem stanoveny, jsou glukosa, cholin a cholesterol. PříkladThe principle of this method of determination can be used in the fields of biochemistry, medical biochemistry, physiological and pharmaceutical 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 extrak-tech z mozkové tkáně. K tomuto účelu byla použita kombinace tří zmobilizovaných en-zymů na třech za sebou následujících kolonách: acetylcholinestaráza /vytváří z ace-tylcholinu cholin/, cholinoxidáza /vytváří z cholinu peroxid vodíku/ a peroxidázaThe method described was used to determine the amount of acetylcholine (ACh) in brain tissue extracts. For this purpose, a combination of three mobilized enzymes was used on three consecutive columns: acetylcholinestarase / forms choline /, choline oxidase / produces hydrogen peroxide / choline peroxide / and peroxidase from acetylcholine
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Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CS884959A CS268737B1 (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 |
|---|---|---|---|
| CS884959A CS268737B1 (en) | 1988-07-08 | 1988-07-08 | Method of hydrogen peroxide's and oxidase substrates' quantitative fluorometric determination |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CS495988A1 CS495988A1 (en) | 1989-07-12 |
| CS268737B1 true CS268737B1 (en) | 1990-04-11 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CS884959A CS268737B1 (en) | 1988-07-08 | 1988-07-08 | Method of hydrogen peroxide's and oxidase substrates' quantitative fluorometric determination |
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| Country | Link |
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| CS (1) | CS268737B1 (en) |
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1988
- 1988-07-08 CS CS884959A patent/CS268737B1/en unknown
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| Publication number | Publication date |
|---|---|
| CS495988A1 (en) | 1989-07-12 |
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