Biological marker-earthworm internal organ p450 content measuring method

A biomarker and measurement method technology, applied in the field of determination of biomarker-earthworm viscera P450 content, can solve problems such as experimental failure, and achieve the effect of simplifying the measurement process

Inactive Publication Date: 2008-07-02
SHENYANG INST OF APPL ECOLOGY CHINESE ACAD OF SCI
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Problems solved by technology

For example, Achazi et al. 16 After the earthworm microsomes were prepared by differential centrifugation, the heme was further separated by electrophoresis through molecular sieves, trying to separate interfering substances from cytochrome P450, but the experiment failed in the end.

Method used

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  • Biological marker-earthworm internal organ p450 content measuring method

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Embodiment

[0021] Biomarker-a method for measuring the content of P450 in the viscera of earthworms, the operation steps are as follows:

[0022] 1) Soak the live earthworms after the substances in the body have been drained in ice-cold glycerin solution, and dissect them quickly to get their viscera. The internal organs are washed with a large amount of KCl solution until the eluate is colorless;

[0023] 2) Transfer the washed viscera to the homogenization buffer, break the cells with a glass tissue grinder, centrifuge at 10,000g for 20min in an ultracentrifuge at a low temperature of 3°C, and centrifuge the supernatant again at 150,000g for 90min, discard The supernatant retains the microsomal pellet;

[0024] 3) Mix the microsomes in the preservation buffer, measure the total amount of P450 by ultraviolet spectrophotometry, and measure the protein content of the microsome suspension by the Coomassie brilliant blue method.

[0025] Before the determination of the P450 content, the l...

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Abstract

The invention relates to cytochrome P450, specifically to a method for measuring the content of a biomarker-cytochrome P450 in earthworm viscera, which comprises the following steps of: (1) soaking a live earthworm in glycerol solution, and immediately dissecting and taking out the viscera thereof; (2) cleaning the viscera with cold saline water, transferring to a homogenization buffer, breaking cells with a glass tissue grinder, centrifuging with a ultra-speed centrifuge at low temperature (3DEG C) and 10,000g for 20min, centrifuging the supernatant at 150,000g for 90min, removing the supernatant, and collecting microsome sediment; and (3) dissolving the microsome sediment in a storage buffer, mixing, and measuring the total amount of cytochrome P450 and the protein content in the microsome suspension. The total amount of cytochrome P450 is quantified with sodium hydrosulfite-reduced CO difference spectra method. The invention has the advantages of simplicity, rapidness, low consumption, and accurate qualification and quantitation.

Description

technical field [0001] The invention relates to cytochrome P450, in particular to a method for measuring the content of biomarker P450 in the internal organs of earthworms. Background technique [0002] Cytochrome P450 (CYP) is a kind of metabolic enzyme system with important physiological functions that widely exists in different organisms such as animals, plants and microorganisms. It is composed of a variety of isozymes with similar structures and properties. As the first metabolic stage enzyme system in organisms, the P450 enzyme system can catalyze a large amount of lipophilic foreign substances and undertake the metabolic detoxification of a large number of liver drugs (Document 1. Shen Jun, Xu Peipei, Jin Xipeng. 1997. Determination of cytochrome P450 in the liver Method improvement. Industrial Hygiene and Occupational Diseases, 23(4): 236-238; Literature 2. Leng Xinfu, Qiu Xinghui. 2001. The structure, function and application prospect of cytochrome P450 enzyme syste...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/48G01N1/28G01N21/25
Inventor 张薇宋玉芳孙铁珩刘淼
Owner SHENYANG INST OF APPL ECOLOGY CHINESE ACAD OF SCI
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