CN101659986A

CN101659986A - Method for testing activity of H2O2-producing oxidase

Info

Publication number: CN101659986A
Application number: CN200810107036A
Authority: CN
Inventors: 周静; 汪天
Original assignee: Individual
Current assignee: Individual
Priority date: 2008-08-27
Filing date: 2008-08-27
Publication date: 2010-03-03

Abstract

The invention provides a method for testing the activity of H2O2-producing oxidase. In the method, oxidase is used to oxidize a substrate to form H2O2, the H2O2 content is tested to realize the quantitative test of the activity of the oxidase, a reaction color development reagent is N,N-dimethylaniline and 4-aminophenazone and reacts with the H2O2 to form a violet product, the optical density of the product is determined at a position where the wavelength is 550 nanometers, 0.001deltaOD550.min<-1> is taken as an enzyme activity unit (U), and the activity rate of the oxidase is indirectly tested by detecting the concentration of the product of the oxidase. Compared with the prior art, the method improves the minimum test amount of the concentration of H2O2 to 0.65mumol.L<-1> from 1.25mumol.L<-1>, thereby improving the test rate of the activity of the oxidase and being capable of improving test effect by prolonging reaction time due to the excellent stability and reproducibility of a colored product of the oxidase.

Description

Resultant is H 2O 2The oxidases activity test method

Technical field:

The present invention relates to the bioenzyme activity detection range, being specifically related to the resultant is H ₂O ₂The active detection of oxidases, comprise H in the plant materials ₂O ₂The detection of content.

Background technology:

Oxidases is the important substance in the organism oxidative metabolism process, has determined the generation and the elimination of biological intravital oxidative metabolism process and secondary metabolites.Make plant under different growing environments, have different adaptive states.(Polyamines PAs) is a class secondary metabolites that produces in the organism nitrogen metabolism process, and plant materials is being subjected to biologically during with abiotic coercing, and intravital polyamines concentration changes sharp as polyamines.(Polyamine oxidase PAO) is the key enzyme of PAs oxidation in the catalysis biological body to polyamine oxidase, by the PAs level of adjusting cell and the concentration of resultant, participates in reaction and the growth and development process of various plant materialss to environment stress.Thereby PAO is an important step of PAs metabolism research; And polyphenoloxidase is the key enzyme of oxidation phenols, is the key enzyme of research phenols oxidative metabolism.Oxydase like that also has a lot in plant materials.The oxidasic common feature of this class has product H when being oxidation substrates ₂O ₂Generation.Thereby can detect H ₂O ₂Content is to reach the detection by quantitative to oxidase activity.Traditional detection method is the active detection method of PAO of Smith proposition in 1972 and application.But, in a lot of plants, all detect activity less than PAO because its susceptibility is lower and to detect product stability relatively poor etc.Influenced the popularity that traditional detection method is used.

Summary of the invention:

It is H that purpose of the present invention just provides a kind of resultant ₂O ₂The oxidases activity test method, this detection method can improve the verification and measurement ratio of oxidase activity, H ₂O ₂Concentration lowest detection amount can be from 1.25 μ molL ^-1Bring up to 0.65 μ molL ^-1

Its concrete operations technology is as follows:

(1) makes the oxydase extracting solution: take by weighing 0.5g examination material and add 1.6mL phosphoric acid buffer (0.1molL ^-1, pH 6.5) and in ice bath, grind the back with the centrifugal 20min of 10000 * g (4 ℃), supernatant liquor is the oxydase extracting solution;

(2) contain 25 μ L N in the composition of making colour developing liquid: 100mL colour developing liquid (use 0.1mol, the phosphoric acid buffer of pH 6.5 is joined), accelerine, the amino ammonia of 10mg 4-is for the pyrrole quinoline;

(3) make reaction mixture: this reaction mixture contains 2.5mL phosphoric acid buffer (0.1molL ^-1, pH 6.5), the 0.2mL liquid that develops the color, 0.2mL oxydase extracting solution, 0.1mL superoxide enzyme solution (250UmL ^-1);

(4) reaction mixture is added 20mmo lL ^-1After the substrate 0.1mL starting reaction, mixed reaction solution places 25 ℃ to react 30min.Described substrate is decided according to oxidasic kind, and as detecting polyamine oxidase, its substrate is a polyamines; Detect polyphenoloxidase, its substrate is various phenols;

(5) with spectrophotometric determination wavelength 550nm place optical density value, with 0.001 Δ OD ₅₅₀Min ^-1It is enzyme unit (U) alive.

(6) be contrast with the extracting solution that adds 5% perchloric acid.

Method of the present invention, H ₂O ₂The lowest detection amount of concentration can be from 1.25 μ molL ^-1Bring up to 0.65 μ molL ^-1, improved the verification and measurement ratio of oxidase activity.And its colour developing product has satisfactory stability and circulation ratio, and can prolong the reaction times improves the detection effect.

Claims

1, a kind of resultant is H ₂O ₂The oxidases activity test method, the concrete operations technology is as follows:

(4) reaction mixture is added 20mmolL ^-1After the substrate 0.1mL starting reaction, mixed reaction solution places 25 ℃ to react 30min, and described substrate is decided according to oxidasic kind, and as detecting polyamine oxidase, its substrate is a polyamines; Detect polyphenoloxidase, its substrate is various phenols;

(5) with spectrophotometric determination wavelength 550nm place optical density value, with 0.001 Δ OD ₅₅₀Min ^-1It is enzyme unit (U) alive;

(6) be contrast with the extracting solution that adds 5% perchloric acid.