Experimental inhibition of succinate dehydrogenase and malonate
Experimental inhibition of succinate dehydrogenase and malonate
Succinate dehydrogenase is an important enzyme in the tricarboxylic acid cycle, and the determination of the presence or absence of succinate dehydrogenase activity in cells can be used to preliminarily characterize the existence of the tricarboxylic acid cycle pathway.
(Source: Biochemistry and Molecular Biology Laboratory Guide, Shao Xueling et al, Wuhan University Press, 2003)
Operation method
Methylene blue decolorization method
Principle
Succinate dehydrogenase dehydrogenates succinate to form fenugreek and gives the hydrogen removed to a hydrogen acceptor. When methylene blue is used as the hydrogen acceptor, methylene blue is reduced by hydrogen to form colorless methylene white. The reaction is as follows: succinic acid + methylene blue → fumaric acid + methylene white + blue acid malonic acid is a competitive inhibitor of succinate dehydrogenase the more the amount of bacteria or the higher the activity of dehydrogenase methylene blue decolorization time required is shorter, therefore, the inverse of the time required for the decolorization of methylene blue can be used to indicate the activity of the enzyme or the bacterial growth of the situation.
Materials and Instruments
E. coli Move (i) Succinate dehydrogenase activity test Caveat ①The bacteria solution added to the third tube was boiled in boiling water for 5 min beforehand as a control tube;② Do not vibrate the test tube when observing the color change, so as not to affect the color change by oxygen leakage into the tube;③ Since methylene blue is easily oxidized by oxygen in the air, the experiment needs to be carried out under anaerobic conditions. Liquid paraffin can be used to seal the reaction solution. For more product details, please visit Aladdin Scientific website.
Sodium succinate, methylene blue, phosphoric acid, malonic acid solution, liquid paraffin.
Test tubes Pipettes Electrothermal water baths
1. Add about 5 ml of 0.15 mol/L, pH7.3 buffer to 1 beveled strain tube, scrape off the bacterial body on the beveled surface with a glass rod, oscillate, pour it into a centrifuge tube, centrifuge at 2500 r/min for 5 min, decant out the washing solution, and then add the bacterial body to 6 ml of 0.15 mol/L, pH7.3 phosphate buffer, oscillate, and then make the bacterial suspension to be used.
2. Take two test tubes and add 1 ml of 0.02 mol/L sodium succinate solution, 1 ml of 0.15 mol/L phosphate buffer, pH 7.3, and 0.3 ml of 0.001 mol/L methylene blue solution to each tube. Add 1 ml of bacterial suspension to 1 test tube and 1 ml of bacterial suspension that has been boiled for 5 min (added after cooling) to the other test tube as a control. Mix well and immediately add 0.5 to 1 ml of liquid paraffin.
3. Place the test tube in a 37 ℃ water bath at a constant temperature, observe and record the time required for the color change of methylene blue.
(ii) Inhibition of succinate dehydrogenase by malonic acid
Take 3 test tubes numbered and add reagents as per the table below: 
First, add sodium succinate, sodium malonate, water, phosphate buffer, methylene blue and other reagents in the test tube, mix well, immediately add a layer of liquid paraffin, the reaction at a constant temperature of 37 ℃, from the start of the liquid paraffin addition to record the time required for methylene blue whitening.