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Method for rapidly detecting activity of acetaldehyde dehydrogenase by using fluorescence spectrophotometer

A fluorescence spectrophotometry, acetaldehyde dehydrogenase technology, applied in fluorescence/phosphorescence, material excitation analysis, etc., can solve the problems of inability to monitor enzyme activity in real time, long time, complicated steps, etc.

Inactive Publication Date: 2015-07-22
SHANGHAI INST OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] One of the purposes of the present invention is to solve the problem of low detection sensitivity when using ultraviolet spectrophotometry to detect acetaldehyde dehydrogenase enzyme activity, and when using HPLC method, the steps are more complicated, the time is longer, and it is impossible to monitor the enzyme activity in real time. In order to solve technical problems such as activity, a method for quickly detecting the activity of acetaldehyde dehydrogenase by using a fluorescence spectrophotometer is provided. The detection method has the advantages of fast detection speed and high sensitivity.

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  • Method for rapidly detecting activity of acetaldehyde dehydrogenase by using fluorescence spectrophotometer
  • Method for rapidly detecting activity of acetaldehyde dehydrogenase by using fluorescence spectrophotometer
  • Method for rapidly detecting activity of acetaldehyde dehydrogenase by using fluorescence spectrophotometer

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Embodiment 1

[0032] A method for rapidly detecting the activity of acetaldehyde dehydrogenase using a fluorescence spectrophotometer, which specifically includes the following steps:

[0033] (1) Prepare 0.1 mol / L Tris-HCl buffer, pH 8.0, the steps are as follows:

[0034] 0.1mol / L Tris-HCl (1000mL): Tris base 12.11g; ddH 2 O 800mL; HCl 49mL, after the three are mixed and fully dissolved, add concentrated hydrochloric acid dropwise to adjust the pH to 8.0, and the solution is set to 1000mL;

[0035] (2) The excitation wavelength is 320nm, and the emission wavelength is 440nm;

[0036] (3) Prepare a concentration of 5×10 with Tris-HCl buffer with a pH of 8.0 and a concentration of 0.01mol / L -4 -0.39×10 -5 mol / L Coenzyme I solution, and measure the fluorescence value of the Coenzyme I solution, and then use the fluorescence value of the Coenzyme I solution as the abscissa and the concentration of Coenzyme I as the ordinate to obtain a standard curve between the fluorescence value of the Coenzyme I so...

Embodiment 1

[0053] The method for detecting acetaldehyde dehydrogenase activity by ultraviolet spectrophotometer method includes the following steps:

[0054] (1) Prepare 0.1 mol / L Tris-HCl buffer, pH 8.0, the steps are as follows:

[0055] 0.1mol / L Tris-HCl (1000mL): Tris base 12.11g; ddH 2 O 800mL; HCl 49mL After the three are mixed and fully dissolved, add concentrated hydrochloric acid dropwise to adjust the pH to 8.0, and dissolve to 1000mL

[0056] (2) Take the cell culture solution and centrifuge at 5000r / min for 15min at 4℃, add phosphate buffer solution 3 times the cell volume to the collected cells, and then perform ultrasound power 150W, ultrasound 6s interval 8s, total ultrasound time 28min The cells were disrupted, and then centrifuged for 20 minutes at a temperature of 4°C and a rotation speed of 10000r / min, and the supernatant was collected as the crude acetaldehyde dehydrogenase enzyme solution;

[0057] The phosphate buffer solution is calculated per liter, containing 0.24g KH 2 ...

Embodiment 2

[0065] The method for detecting acetaldehyde dehydrogenase activity by HPLC includes the following steps:

[0066] (1) Prepare 0.1 mol / L Tris-HCl buffer, pH 8.0, the steps are as follows:

[0067] 0.1mol / L Tris-HCl (1000mL): Tris base 12.11g; ddH 2 O 800mL; HCl 49mL, after the three are mixed and fully dissolved, add concentrated hydrochloric acid dropwise to adjust the pH to 8.0, and the solution is set to 1000mL;

[0068] (2) Take the cell culture solution and centrifuge at 5000r / min for 15min at 4℃, add phosphate buffer solution 3 times the cell volume to the collected cells, and then perform ultrasound power 150W, ultrasound 6s interval 8s, total ultrasound time 28min The cells were broken, and the supernatant was collected as crude acetaldehyde dehydrogenase enzyme solution by centrifugation at a temperature of 4°C and a speed of 10000r / min for 20 minutes;

[0069] The phosphate buffer solution is calculated per liter, containing 0.24g KH 2 PO 4 , 1.44g Na 2 HPO 4 , 8.0g NaCl, 0....

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Abstract

The invention discloses a method for rapidly detecting activity of acetaldehyde dehydrogenase by using a fluorescence spectrophotometer. The method comprises the steps of firstly preparing coenzyme I solutions of different concentration gradients from a Tris-HCl buffer solution and a coenzyme I, detecting fluorescence values of the coenzyme I solutions of different concentrations, establishing a coenzyme I concentration-fluorescence value corresponding standard curve, controlling the temperature of a reaction system, prepared from crude aldehyde dehydrogenase enzyme liquid, a 0.1mol / L Tris-HCl buffer solution with the pH value of 7.0-9.0 and the like, to be 25-40 DEG C, maintaining for 10-30 minutes, then, determining the minutely fluorescence change value of the crude aldehyde dehydrogenase enzyme liquid in the reaction system under the conditions that the excitation wavelength is 320-380nm and the emission wavelength is 440-480nm, and finally, calculating the activity unit of acetaldehyde dehydrogenase according to an equation, namely the enzyme activity unit=delta fluorescence value / 3. The determination method is simple and convenient in operation and high in sensitivity.

Description

Technical field [0001] The invention relates to a method for rapidly detecting the activity of acetaldehyde dehydrogenase using a fluorescence spectrophotometer, and belongs to the technical field of enzyme activity detection in enzyme engineering. Background technique [0002] The human body absorbs and ingests ethanol from the intestine, and is mainly metabolized in the liver. After ethanol enters the body, it is firstly converted into acetaldehyde by dehydrogenation of alcohol under the action of alcohol dehydrogenase (ADH), and then under the catalysis of acetaldehyde dehydrogenase (ALDH), acetaldehyde is oxidized to produce acetic acid. It is decomposed into CO2 and water through the tricarboxylic acid cycle (TCA), and releases energy to generate ATP. [0003] [0004] Acetaldehyde is a very toxic substance and is a potential carcinogen in the body. It can interfere with DNA synthesis and repair. It is highly toxic, mutagenic and carcinogenic to human tissues and organs. It i...

Claims

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Application Information

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IPC IPC(8): G01N21/64
Inventor 龚钢明魏晓聪吴范宏
Owner SHANGHAI INST OF TECH
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