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A method for rapid detection of acetaldehyde dehydrogenase activity using a fluorescence spectrophotometer

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

Inactive Publication Date: 2018-04-06
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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  • A method for rapid detection of acetaldehyde dehydrogenase activity using a fluorescence spectrophotometer
  • A method for rapid detection of acetaldehyde dehydrogenase activity using a fluorescence spectrophotometer
  • A method for rapid detection of acetaldehyde dehydrogenase activity using a fluorescence spectrophotometer

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Embodiment 1

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

[0033] (1), prepare 0.1mol / L Tris-HCl buffer solution, pH8.0, the steps are as follows:

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

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

[0036] (3), the Tris-HCl buffer solution preparation concentration that is 0.01mol / L with pH8.0, concentration is 5 * 10 -4 -0.39×10 -5 mol / L coenzyme I solution, and measure the fluorescence value of coenzyme I solution, then take the fluorescence value of coenzyme I solution as the abscissa and the concentration of coenzyme I as the ordinate to obtain the standard curve between the fluorescence value of coenzyme I solu...

Embodiment 1

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

[0054] (1), prepare 0.1mol / L Tris-HCl buffer solution, pH8.0, the steps are as follows:

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

[0056] (2) Take the cell culture solution and centrifuge at 4°C and 5000r / min for 15 minutes, add phosphate buffer solution 3 times the volume of the cells to the collected cells, and then perform the operation at 150W ultrasonic power, 6 seconds at an interval of 8 seconds, and a total ultrasonic time of 28 minutes. Cells were disrupted, then centrifuged at 4°C and 10,000r / min for 20min, and the supernatant was collected as crude acetaldehyde dehydrogenase enzyme solution;

[0057] The phosphate buffer is calcula...

Embodiment 2

[0065] Adopt HPLC to detect the method for acetaldehyde dehydrogenase activity, specifically comprise the following steps:

[0066] (1), prepare 0.1mol / L Tris-HCl buffer solution, pH8.0, the steps are as follows:

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

[0068] (2) Take the cell culture solution and centrifuge at 4°C and 5000r / min for 15 minutes, add phosphate buffer solution 3 times the volume of the cells to the collected cells, and then perform the operation at 150W ultrasonic power, 6 seconds at an interval of 8 seconds, and a total ultrasonic time of 28 minutes. The cells were disrupted, and then centrifuged at 4°C and 10,000r / min for 20min to collect the supernatant as the crude acetaldehyde dehydrogenase enzyme solution;

[0069] The phosphate buffer is calculated per liter, containing 0.24g KH ...

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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 by using a fluorescence spectrophotometer, which belongs to the technical field of enzyme activity detection in enzyme engineering. Background technique [0002] The ethanol absorbed by the human body is mainly metabolized in the liver. After ethanol enters the body, it is dehydrogenated into acetaldehyde under the action of alcohol dehydrogenase (ADH), and then oxidized to acetic acid under the catalysis of acetaldehyde dehydrogenase (ALDH). It is decomposed into CO2 and water through the tricarboxylic acid cycle (TCA), and releases energy to generate ATP. [0003] [0004] Acetaldehyde is a highly toxic substance and a potential carcinogen in the body, which can interfere with the synthesis and repair of DNA, and has highly toxic, mutagenic, and carcinogenic effects on human tissues and organs. The metabolism of acetaldehyde dehydrogenase The activ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/64
Inventor 龚钢明魏晓聪吴范宏
Owner SHANGHAI INST OF TECH