Detection method and reagent of enzyme activity of pyruvate dehydrogenase complex

A technology for pyruvate dehydrogenase and detection reagents, which is applied in the field of determination of enzyme activity of pyruvate dehydrogenase complex, which can solve the problems of low sensitivity, high interference of enzyme activity detection, and difficulty in enzyme activity determination.

Active Publication Date: 2014-07-09
北京中科非凡生物技术有限公司
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  • Abstract
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  • Application Information

AI Technical Summary

Problems solved by technology

However, skin or skeletal muscle biopsy is an invasive examination, which is difficult for children and parents to accept, and it is difficult to collect normal values. It is very difficult to measure the enzyme activity of skin or skeletal muscle tissue in clinical practice.
Relatively speaking, peripheral blood is easy to obtain and has minimal trauma. However, due to the small content of mitochondria in peripheral blood lymphocytes, it is very difficult to extract mitochondria and isolate mitochondrial proteins.
In the existing reports, most of the specimens used to detect PDHC enzyme activity use muscle and other tissues to purify PDHC, and then detect the enzyme activity of the PDHC pure enzyme complex. The enzyme activity detection of PDHC in liquid is very disturbing, so the accuracy of the results is the primary consideration when establishing the method

Method used

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  • Detection method and reagent of enzyme activity of pyruvate dehydrogenase complex
  • Detection method and reagent of enzyme activity of pyruvate dehydrogenase complex
  • Detection method and reagent of enzyme activity of pyruvate dehydrogenase complex

Examples

Experimental program
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Effect test

Embodiment 1

[0060] Embodiment one Mitochondria Extraction from Human Blood Leukocytes

[0061] Take 5ml of human peripheral whole blood, add 20ml of lysate (0.1mM EDTA), process for 15min to break the red blood cells, centrifuge at 3000rpm for 10min, discard the supernatant, collect the precipitate that is white blood cells, wash the white blood cells twice with normal saline, centrifuge at 3500rpm for 5min, discard Supernatant, leukocyte pellet was collected. 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 10,000g for 10 minutes, discard the supernatant, and collect the precipitate as mitochondria, which can be stored at -80°C.

Embodiment 2

[0062] Embodiment two Extraction of Porcine Skeletal Muscle Mitochondria

[0063] Weigh 750g of porcine skeletal muscle from which adipose tissue has been 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 1200 rpm for 25 minutes, and the resulting pellet was mitochondria, which could be stored at -80°C.

Embodiment 3

[0064] Embodiment three Preparation of Crude Enzyme Solution of Mitochondrial Pyruvate Dehydrogenase Complex

[0065] Suspend the mitochondria obtained in Examples 1 and 2 with a suspension medium (containing 0.25M Sucrose, 5mM HEPES, 0.5mM EGTA, pH7.4), and then sonicate for 10s, with an interval of 10s, and sonicate 10 times in total. Then the BCA legal protein concentration is 1ug / ul, and the suspension is the crude enzyme solution containing the pyruvate dehydrogenase complex. After the concentration is determined, it is placed in an environment at 4°C for use.

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Abstract

The invention provides a method for enzyme activity analysis and detection of pyruvate dehydrogenase complex (PDH complex, PDHC), and the invention also provides a reagent for determining the enzyme activity of PDHC. The method and the agent can be used for analysis and determination of the enzyme activity of PDHC in samples, and thus can be used for analysis, examination and diagnosis of clinical diseases. The principle of the invention is that: PDHC can orderly perform catalytic decarboxylation of pyruvic acid to generate products of CO2, acetyl coenzyme A, and NADH. Therefore, detection of the generation of the final product NADH means the detection of the enzyme activity of PDHC. NADH can emit fluorescence at a specific excitation wavelength, and the reaction rate is in direct proportion to the product fluorescence intensity, so the enzyme activity of PDHC in a sample can be calculated by detecting the fluorescence value change. The detection method of the enzyme activity of PDHC provided in the invention can realize rapid, specific, and sensitive determination of the enzyme activity of PDHC in a sample. Based on the method, the reagent for determining the enzyme activity of PDHC provided by the invention has the characteristics of convenience, rapidness, and high sensitivity, and facilitates 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 enzyme activity of pyruvate dehydrogenase complex (Pyruvate dehydrogenase complex, PDH complex, PDHC), and the present invention also provides the enzyme activity analysis as pyruvate dehydrogenase complex and detection reagents. Background technique [0002] Pyruvate, formerly known as pyrogluconic acid, is one of the intermediate products involved in the basic metabolism of the whole organism. Pyruvate can realize the interconversion between sugar, fat and amino acid in the body through acetyl CoA and tricarboxylic acid cycle. Therefore, pyruvate plays an important pivotal role in the metabolic connection of the three major nutrients. The oxidative decarboxylation reaction of pyruvate is a key step in the comple...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64
Inventor 郝淑静刘卫德齐晓丽刘晓敏
Owner 北京中科非凡生物技术有限公司
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