Enzyme activity detection method for mitochondrial respiratory chain compound I and reagents

A technology for activity detection and mitochondria, which is used in material excitation analysis, fluorescence/phosphorescence, etc., and can solve the problems of large sample demand, poor accuracy, and low sensitivity.

Inactive Publication Date: 2013-12-04
北京中科非凡生物技术有限公司
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] However, the currently used detection methods for the enzyme activity of the mitochondrial respiratory chain complex I are colorimetric methods, that is, the enzyme activity of the mitochondrial respiratory chain complex is detected by a spectrophotometer. The disadvantages of this method are low sensitivity and poor accuracy. The demand for samples is high

Method used

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  • Enzyme activity detection method for mitochondrial respiratory chain compound I and reagents
  • Enzyme activity detection method for mitochondrial respiratory chain compound I and reagents
  • Enzyme activity detection method for mitochondrial respiratory chain compound I and reagents

Examples

Experimental program
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Effect test

Embodiment 1

[0076] Embodiment one Mitochondria Extraction from Human Blood Leukocytes

[0077] Take 2ml of human whole blood, add 8ml of lysate (0.1mM EDTA) to it, treat it for 15min to break up the red blood cells, centrifuge at 3000rpm for 10min, discard the supernatant, collect the precipitate that is the white blood cells, wash the white blood cells twice with normal saline, and centrifuge at 3500rpm for 5min. Discard the supernatant and collect the white blood cell pellet. Add 5ml of cell suspension (0.25M Sucrose, 5mM HEPES, 0.5mM EGTA, pH7.4) to the pellet to suspend the cells, homogenize 20 times with a glass homogenizer, centrifuge the homogenate at 1000g for 10 minutes, discard the pellet, and collect the supernatant Centrifuge the supernatant at 10,000 g for 10 minutes, discard the supernatant, and collect the precipitate as mitochondria, which can be stored at -80°C.

[0078]

Embodiment 2

[0079] Embodiment two Extraction of Porcine Myocardial Mitochondria

[0080] Weigh 750g of porcine myocardium with the fat tissue cut off, add 2.25L of 0.25M (containing 0.01M, pH8 phosphate buffer) sucrose solution, mash for 75 seconds in three times, add appropriate amount of 6N KOH before mashing to maintain pH7.2 -7.4, KD-70 centrifuge at 2600-2800rpm for 10 minutes, filter with four layers of gauze, and discard the precipitate. The supernatant was centrifuged in Beckman J2-21 at 1200rpm for 25 minutes, and the resulting pellet was mitochondria, which were suspended in 0.25M (including 0.01M, pH8 phosphate buffer) sucrose solution. Mitochondria could be stored at -80°C.

[0081]

Embodiment 3

[0082] Embodiment three Extraction and Purification of Complex I of the Mitochondrial Respiratory Chain in Porcine Myocardium

[0083] The pig heart mitochondria prepared in Example 2 were diluted to a protein concentration of 30 mg / ml with 0.25 M sucrose, centrifuged at 2000 rpm for 25 minutes in a Beckman J2-21, and the supernatant was discarded. The precipitate was suspended in TSH buffer solution (50mM Tris pH8, 0.67M sucrose, 1mM Histidine), adjusted the protein concentration to 25mg / ml with TSH buffer solution, and then added 10% pH9 potassium deoxycholate to 0.3mg / mg protein, in 72g Add solid KCl at an amount of / L. After KCl is completely dissolved, centrifuge at 30,000 rpm for 30 minutes on a Beckman L8-80 ultracentrifuge. The precipitate is green (containing cytochrome oxidase, which can be used for further extraction of this enzyme). The red supernatant was diluted with 0.25 times the volume of pre-cooled deionized water, and then centrifuged at 30,000 rpm for...

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Abstract

The invention provides an enzyme activity analysis and detection method for mitochondrial respiratory chain compound I (NADH-Q reductase, EC1.6.5.3). The invention also provides reagents for detecting the enzyme activity of the mitochondrial respiratory chain compound I. The above method and the reagents can be used for analyzing and detecting the enzyme activity of the mitochondrial respiratory chain compound I in samples to be detected, and therefore the method and the reagents can be used for analysis, examination and diagnosis of clinic diseases. Based on the method, the reagents have advantages of convenience, rapidness and high sensitivity, and are convenient for popularization and application.

Description

technical field [0001] The invention belongs to the technical field of biomedicine and at the same time belongs to the field of clinical diagnosis and detection of genetic metabolic diseases. Specifically, the present invention provides a method for measuring the enzymatic activity of mitochondrial respiratory chain complex I (NADH-Q reductase, NADH:ubiquinone reductase, EC1.6.5.3). A reagent for the analysis and detection of the enzyme activity of substance I (NADH-Q reductase, NADH:ubiquinone reductase, EC1.6.5.3). Background technique [0002] Mitochondria is a kind of organelle in eukaryotic cells, which is covered by two membranes, the outer membrane is smooth, the inner membrane folds inward to form cristae, there is a cavity between the two membranes, called the inmembrane space, and the inside of the mitochondria is the matrix. There are respiratory chain enzymes and ATPase complexes on the inner membrane of mitochondria. As the "power plant" (power plant) of the c...

Claims

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Application Information

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IPC IPC(8): G01N21/64
Inventor 安祺孙宏博
Owner 北京中科非凡生物技术有限公司
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