Experiments on oxidation and phosphorylation in isolated mitochondria
Experiments on oxidation and phosphorylation in isolated mitochondria
When sufficient oxygen is supplied to plant tissues, plant cells can oxidize substrates completely. When glucose is fully oxidized as a respiratory substrate, CO2 is produced at the end, the absorbed O2 is reduced to water, and the oxidation of each gram molecule of glucose produces 38 grams of ATP.
Operation method
Experiments on oxidation and phosphorylation in isolated mitochondria
Principle
When supplied with sufficient oxygen to plant tissues, plant cells can oxidize substrates completely. When glucose is completely oxidized using glucose as the respiratory substrate, CO2 is finally produced, the absorbed O2 is reduced to water, and the oxidation of each gram of molecules of glucose produces 38 grams of molecules of ATP: most of this ATP is formed by a process known as oxidative phosphorylation, which is the main process for the formation of available energy in the cell, and it can now be affirmed that the tricarboxylic acid cycle, and its coupling to oxidative phosphorylation, takes place in the mitochondria. The rate at which these reactions take place in the mitochondria can be expressed in terms of oxygen consumption and also in terms of the amount of ATP formed. In this experiment, isolated mitochondria were prepared from mung bean cotyledons germinated in the dark or potato tubers sprouted in the dark, and O2 consumption and ATP production were qualitatively determined using succinic acid as a substrate, and malonic acid was applied to inhibit respiration to observe changes in oxygen consumption.
Materials and Instruments
Oxygen meters UV analyzers Frozen high-speed centrifuges Hematocrit pipettes Glass tubes Glass cylinders for chromatography Pipettes Reaction bottles Bowls DEAE Move I. Instrumental drugs II. Experimental Procedure The reaction solution was added to the main chamber of the reaction flask according to the table below: As shown, install the oxygen meter, oxygen electrode, and recorder. Check whether the instrument is normal. Add 0.5 ml of mitochondrial suspension into the side arm, insert the oxygen electrode, close the reaction bottle tightly, wait for the oximeter to stabilize, gently pour the mitochondrial suspension in the side arm into the reaction chamber, stir on a magnetic stirrer or gently shake the reaction bottle constantly. react for 10 min at 20°C. The change in oxygen partial pressure was recorded, and the change in O2 consumption of normal mitochondria and mitochondria with inhibitor was compared by the recording curve. Oxygen measurement device diagram (4) The air-dried thin-layer plate was observed under a UV analyzing lamp to observe the ATP formed. Experimental Records and Reports For more product details, please visit Aladdin Scientific website.
1. Extraction solution:0.1 M phosphate bufferpH 7.0 containing0.4 M sucrose.
0.005M EDTA 
2. Reaction solution:
(1) 0.1M phosphate buffer pH 7.0
(2) 1.5 M sucrose solution (25.67 g sucrose was fixed to 50 ml with distilled water ).
(3) ADP (adenosine diphosphate) 50 mg/ ml (50 mg ADP dissolved in 1 ml of water).
(4) CytC (Cytochrome C) 1mg/ ml (1mg CytC dissolved in 1 ml water).
(5) NAD (oxidized coenzyme I) 3.3 mg/ ml (3.3 mg NAD was dissolved in 0.5 ml of water, adjusted to pH 7.0 with NaOH, and reconstituted to 1 ml).
(6) CoA (Coenzyme A) (50 units each, 250 units equivalent to 1 mg).
(7) TPP (thiamine pyrophosphate) 50mg/ml (3.3mg NAD dissolved in 0.5 ml water, adjusted to pH7.0 with NaOH, and then reconstituted to 1 ml).
(8) 0.2 MMgCl2 (2.03 g MgCl2-6H2O was fixed in as 50 ml ).
(9) 1.1M glucose (10.9 glucose with water was fixed to 50 ml ).
(10) 0.1M succinic acid (0.118g succinic acid was dissolved in 5 ml of water, adjusted to pH 7.0 with NaOH, and reconstituted to 10 ml ).
3. 0.1M malonic acid (pH 7.0)
0.017g malonic acid was dissolved in 1 ml of water, adjusted to pH7.0 with NaOH and reconstituted to 2 ml.
4. 0.025 N HCl
1. Preparation of mitochondrial suspension
(1) Mung bean sprouts germinated in the dark at 25℃ for 3-4 days, cotyledons were removed, hypocotyls, roots and buds were removed.
(2) Take 30g of cotyledons, 40 ml of extract and a small amount of quartz sand, quickly grind them into a homogenate in a mortar (the mortar is placed in an ice-salt bath), and filter through a double layer of gauze.
(3) The filtrate was centrifuged at 3000 rpm for 10 min at 0°C and the precipitate was discarded.
(4) The supernatant was centrifuged at 12000 rpm for 20 min at 0°C, and the supernatant was discarded. The precipitate was suspended in 18 ml of extraction solution.
(5) The above suspension was centrifuged at 0℃ for 20 minutes at 12000rpm, the supernatant was discarded, and the precipitate was suspended in 2 ml of extraction solution, i.e. the mitochondrial suspension. Store in the refrigerator.
2. Instrument installation and determination of O2 consumption
(1) DEAE - - cellulose regeneration: after the use of cellulose soaked in 0.5N NaOH solution for 30 minutes - 1 hour, and then filtered with a Brinell funnel to remove the NaOH solution, and then soaked in 0.5NHCl solution for 30 minutes - 1 hour, rinsed with distilled water to neutral, standby.
Has been treated with standby DEAE ── cellulose (DE-22), pour too much water, the cellulose stirred evenly spread on a clean glass plate, and then gently vibrate the glass plate, so that the cellulose is evenly distributed, smooth surface. Put in 60 ℃ at the drying standby.
(2) Spotting: Pipette the ①standard APP 10mm3, ②reaction solution 20mm3, ③mitochondrial suspension 20mm3, ④reaction 10 minutes after the sample 20mm3 were spotted on the DEAE──cellulose thin layer, and blow-dry it with cold air.
(3) Spread the layer: Pour 0.025N HCl into the chromatography cylinder, put the cellulose thin layer flat into the inside, make the liquid level lower than the spotting line, cover the lid, take out immediately when the leading edge goes to 1 cm away from the upper end of the thin layer, and blow dry with hot air. 