Spectrophotometry for testing activity of pyruvic acid dehydrogenase system

A pyruvate dehydrogenase, activity technology, applied in the determination/inspection of microorganisms, biochemical equipment and methods, color/spectral property measurement, etc., can solve the problems of no spectrophotometry, unstable radioactive substances, environmental pollution, etc. Achieve the effect of small standard error, high sensitivity and good repeatability

Inactive Publication Date: 2009-08-05
HUAZHONG NORMAL UNIV
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Problems solved by technology

Because the isotope labeling method uses dynamic measurement, it is not as convenient as the spectrophotometric method. At the same time, it also uses a relatively unstable radioactive substance, which is easy to cause environmental pollution.

Method used

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  • Spectrophotometry for testing activity of pyruvic acid dehydrogenase system
  • Spectrophotometry for testing activity of pyruvic acid dehydrogenase system
  • Spectrophotometry for testing activity of pyruvic acid dehydrogenase system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Embodiment 1: MTT method and DCPIP method measure the comparison of the enzyme activity of pyruvate dehydrogenation in the enzyme of lactic acid bacteria pyruvate dehydrogenation and pig heart pyruvate dehydrogenase complex (PDC)

[0019] Step 1: Enzyme pretreatment--lactobacillus pyruvate dehydrogenase (PDH) preparation, PDH powder is dissolved and diluted to 1 mg protein / ml. The porcine heart pyruvate dehydrogenase complex was diluted to 1 mg protein / ml with 50 mM potassium phosphate buffer pH 7.1 containing 20% ​​glycerol. Store at -20°C after aliquoting.

[0020] The second step: the preparation of the MTT method reaction mixture: the reaction mixture contains 50 mmol pH7.1 potassium phosphate buffer, 1 mmol / L MgCl 2 , 0.2 mmol / L thiamine pyrophosphate, 0.5 mmol / L MTT, 6.5 mmol / L phenazine dimethyl sulfate and 2.0 mmol / L sodium pyruvate were used as substrates for the reaction. The DCPIP method was modified with reference to the reaction system used by Nemeria e...

Embodiment 2

[0025] Embodiment 2: Determination of inhibition rate of inhibitor to pyruvate dehydrogenase activity

[0026] Step 1: Enzyme pretreatment--lactobacillus pyruvate dehydrogenase (PDH) preparation, PDH powder is dissolved with 50 millimolar pH7.1 potassium phosphate buffer solution containing 20% ​​glycerol and diluted to a concentration of 1 mg protein / ml.

[0027] The second step: with the second step of embodiment 1

[0028] The third step: the enzyme and the inhibitor IIM-1 (IIM-1: 1-(2,4-disubstituted phenoxyacetoxyalkylphosphonic acid monosodium salt compound) with a concentration of 100ppm) were incubated at 30°C for 30 -60 minutes

[0029] Step 4: At room temperature, add enzyme or enzyme and inhibitor to the reaction mixture to initiate the reaction, and measure the relationship between the absorbance of the reaction mixture and time, the results are shown in Figure 4.

[0030] In Fig. 4: pure enzyme is the complete reaction system adding enzyme solution to start the ...

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Abstract

A spectrophotometric method for the determination of pyruvate dehydrogenase activity. This method uses a spectrophotometer, in the presence of substrate sodium pyruvate and cofactors magnesium chloride, thiamine pyrophosphate, using phenazine dimethyl sulfate as electron transporter, thiazole blue as electron acceptor, thiazole blue Reducted by pyruvate dehydrogenase (E1) product hydroxyethyl-pyrophosphate thiamine, the maximum absorption peak of thiazole blue changes from wavelength 400-430 nanometers to 540-640 nanometers, by measuring the wavelength 540-640 nanometers light absorption The amount of increase determines the reduction of thiazolium blue and defines the activity of the enzyme. The reagents used in this method are more economical, more stable, and more sensitive than the 2,6-dichloroindoxyl method and the potassium ferricyanide method. The influence of reagents is not conducive to the determination of E1 activity in the pyruvate dehydrogenase complex. This method is not only suitable for the determination of purified pyruvate dehydrogenation activity, but also for pyruvate dehydrogenation in the pyruvate dehydrogenase complex after mitochondria extraction. Hydrogenase activity assay.

Description

Technical field: [0001] The invention relates to a method for measuring the activity of pyruvate dehydrogenase system. Specifically, based on the use of phenazine dimethyl sulfate as the electron carrier, thiazole blue as the electron acceptor, thiazolium blue is reduced by the catalytic product of pyruvate dehydrogenase (E1), and the reduction amount of thiazolium blue is determined to determine pyruvate. Spectrophotometric method of dehydrogenase system activity. Background technique [0002] The determination of pyruvate dehydrogenase activity is essential in the properties, gene cloning, separation, purification, identification and related research of pyruvate dehydrogenase system. Herbicides and fungicides targeting the ketoacid dehydrogenase system are also of great significance for the study of diseases related to abnormal metabolism of pyruvate. The invention studies the method for measuring activity of pyruvate dehydrogenase system, and establishes an economical a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/31C12Q1/32
Inventor 袁均林何亚辉贺红武彭浩杨旭丁书茂
Owner HUAZHONG NORMAL UNIV
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