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A novel method for measuring hydrogen pumping capability of mitochondria

A mitochondrial and new method technology, applied in the field of scientific research and experiments, can solve the problems of reduced repeatability and reliability, many interference factors, and large error in results, and achieve excellent accuracy and repeatability, good quality, and low environmental interference. Effect

Active Publication Date: 2018-05-15
LINYI UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The technical problem solved by the present invention is: the method for studying the hydrogen pumping capacity of mitochondria in the prior art is complicated to operate and has many interference factors, which leads to large error in the results and reduces the repeatability and reliability; A New Approach to Capabilities

Method used

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  • A novel method for measuring hydrogen pumping capability of mitochondria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] A novel method for measuring the hydrogen pumping capacity of mitochondria comprising the following steps:

[0048] S1: Place the miniature magnetic stirrer (purchased from Shanghai Sile S21-1) under the cuvette holder of the spectrophotometer (purchased from Hitachi UV-3000, Japan);

[0049] S2: Set the spectrophotometer to the time scanning state, and the scanning wavelength is 475nm;

[0050]S3: Put the cuvette (1×1×3cm) into the spectrophotometer, and add 1.6ml 150mM KCl as the reaction solution; put the magnetic stirrer rotor in the cuvette, and start the magnetic stirrer; then adjust zero, start scanning;

[0051] S4: Add 5.7 μl of 14 mM 8-hydroxy-1,3,6-pyrene trisulfonate to the cuvette reaction solution to make the final concentration 50 μM;

[0052] S5: After the absorption line becomes flat, add 2μl 2mM ferrocytochrome c, 2μl 0.2mg / ml valinomycin, 5μl 5mM 2,3-dimethoxy-5- Methyl-6-(10-bromo)-1,4-benzoquinone and 50 μl of 0.5g / ml mitochondrial solution;

[...

Embodiment 2

[0072] Embodiment 2: The difference from Example 1 is that the separation and purification method of the mitochondrial solution is:

[0073] 1) Preparation of crude mitochondrial product:

[0074] Put the organism to be tested into the grinding buffer, homogenate and grind for 10 minutes, then crush it with an ultrasonic cell disruptor in an ice bath at 4°C, filter it at 250 mesh, centrifuge the filtrate at 10°C, 3000r / min for 10 minutes, and collect the precipitate , which is the crude product of mitochondria; the composition of the grinding buffer is: 47mmol L -1 4-Hydroxyethylpiperazineethanesulfonic acid-tromethamine, 1.8mmol·L -1 NaCl, 120mmol·L -1 Sucrose, 8mmol·L -1 beta-mercaptoethanol, 20mg·L -1 Polyvinylpyrrolidone, 9mg·L -1 Bovine serum albumin, 26 μg L -1 EDTA, the grinding buffer pH is 7.5;

[0075] 2) Enzymatic treatment:

[0076] In the crude mitochondrial product obtained in step 1), add the suspension buffer at a volume ratio of 1:10, mix well, then a...

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Abstract

The invention discloses a novel method for measuring hydrogen pumping capability of mitochondria, and belongs to the field of scientific research and experiments. The method includes S1) putting a magnetic stirrer below an absorption cell rack of a spectrophotometer; S2) setting the spectrophotometer into a time scanning state; S3) adding an absorption cell into the spectrophotometer, adding KCl into the absorption cell, starting the magnetic stirrer and starting scanning; S4) adding 8-hydroxy-1,3,6-pyrenetrisulfonic acid; S5) adding ferrocytochrome c, valinomycin, 2,3-dimethoxy-5-methyl-6-(10-bromo)-1,4-benzoquinone and a mitochondrion solution; S6) recording an absorption value A1 after an absorption line is flat; S7) adding 1 [mu]L of sodium ferricyanide having a concentration of 5 mM,and recording an absorption value A2; S8) adding a proton carrier that is [(3-chlorophenyl)hydrazono]malononitrile, and recording an absorption value A3; and S9) adding sodium ferricyanide, and recording an absorption value A4; and S10) calculating the hydrogen pumping capability according to an equation that is (H<+> / e)=x / y=(A1-A2) / (A3-A4). The method is reasonable in design, simple to operate and low in interference by the environment, and a measurement result is good in precision and repeatability. The method is suitable for use in common laboratories.

Description

technical field [0001] The invention relates to the technical field of scientific research experiments, in particular to a new method for measuring the hydrogen pumping ability of mitochondria. Background technique [0002] Organisms oxidize and decompose nutrients such as carbohydrates, proteins, and fats through respiration, and at the same time, through oxidative phosphorylation, convert the energy stored in these organic substances into chemical energy to continuously supply the needs of physiological activities. [0003] These processes are mainly carried out and completed in the mitochondria. When the mitochondria carry out electron transfer, the H in the mitochondria will be simultaneously + pumped outside the mitochondrial membrane, thus forming H inside and outside the membrane + The concentration difference is the proton kinetic potential. Driven by the proton kinetic potential, the ATPase on the inner membrane of mitochondria can synthesize ATP, which is the di...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/31
CPCG01N21/31
Inventor 曲元刚
Owner LINYI UNIVERSITY
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