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Method and kit for investigating 5'-nucleotidase by coupling enzymatic reaction

A nucleotidase and inosinic acid technology, which is applied in biological testing, microbial measurement/inspection, biochemical equipment and methods, etc., can solve the problem of narrow selection of hydrogen donors

Inactive Publication Date: 2005-11-09
ZHEJIANG YAKE SCI & TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The disadvantage of this method is that the choice of hydrogen donor is too narrow, only DHBS is used

Method used

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  • Method and kit for investigating 5'-nucleotidase by coupling enzymatic reaction
  • Method and kit for investigating 5'-nucleotidase by coupling enzymatic reaction
  • Method and kit for investigating 5'-nucleotidase by coupling enzymatic reaction

Examples

Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0028] 1. Determine the IMP Michaelis constant Km

[0029] 1) Reagent composition

[0030] Reagent I Good's buffer pH 7.6 100mmol / L

[0031] 4-AA 2mmol / L

[0032] 5’-NT 150U / L (rattlesnake venom)

[0033] PNP 100U / L

[0034] XOD 200U / L

[0035] POD 600U / L

[0036] Reagent II Good's buffer pH 7.6 100mmol / L

[0037] IMP 0.001-10mmol / L

[0038] TOOS 2mmol / L

[0039] 2) Test parameters and operation steps

[0040] Temperature 37°C

[0041] Wavelength 550nm

[0042] Cuvette light path 1.0cm

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Abstract

Bertrand(1982) hydrolytic generates trophicardyl from trophicardyl acid by nucleotidase, and gains H2O2 through the reaction of the enzyme1 and enzyme2. Use of H2O2 from Trinder's reaction to combine hydrogen provider3,5-double CL-2-hydroxide benzene sulphur acid and 4- to create a amaranth in the situation of the catalase. By watching the velocity of increase of the degree of the amaranth absorbing light at 510 nms to gain 5' nucleotidase activity. The invention uses the aniline compound ADOS, ADPS, ALPS, TODB, TOOS and TOPS as hydrogen provider for Trinder's reaction and increases the reaction ability. The invention also involves a reagent box used for implementing the method mentioned above.

Description

technical field [0001] The invention relates to a technical scheme for measuring 5'-nucleotidase activity in a biological sample and a reagent produced by the scheme. In particular, it relates to a multi-enzyme-catalyzed reaction system coupled with 5'-nucleotidase to hydrolyze inosinic acid to generate inosine. Background technique [0002] Bertrand (1982) hydrolyzed inosine5'-monophosphate disodium salt (IMP) with 5'-nucleotidase (5'-NT) to produce inosine. Hydrogen peroxide (H 2 o 2 ). Using Trinder's Reaction (1969) H 2 o 2 Under the oxidation of peroxidase (Peroxidase, POD), 3,5-dichloro-2-hydroxybenzenesulfonic acid (3,5-dichloro-2-hydroxybenzenesulfonic acid, DHBS) and 4-aminopyrine (4- Aminophenazone) conjugates condense into purple colored quinone (Quinoneiminedye). The activity of 5'-NT was determined by continuous reaction and dynamic observation of the rate of increase of the absorbance at 510 nm of colored quinone. The disadvantage of this method is that...

Claims

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Application Information

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IPC IPC(8): G01N21/78
Inventor 蔡枫
Owner ZHEJIANG YAKE SCI & TECH
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