According to numerous research that have been carried out by scientists across the world, it has been established that in higher organisms, cellular respiration occurs in the mitochondria. Mitochondria are small cell organelles where chemical and cellular reaction occurs in the living organisms. During cellular respiration, carbohydrates, amino acids and fatty acids are broken down to release carbon dioxide and water. Energy released is transferred to ATP or used to reduce molecules such as NAD+ and FAD and some given out as heat. Part of the reaction occurs in the Krebs cycle or may occur in the biochemical pathways that feed intermediates in the Krebs cycle and yet more occur as a result of the electron transport chain. Succinate dehydrogenase is located in the inner mitochondrial membrane and is one of the enzymes that takes part in the process. SDH stimulates a series of the redox reaction that that transfers two electrons from succinate to FAD to form FADH2 while at the same time forming fumerate from the succinate.
FADH2 and NADH + H are able to transfer electrons into a couple of redox reactions in the inner membrane through mediation of by the electron transport chain. At the end of the reaction, the electrons are fed to molecular oxygen which is reduced and combines with the protons to form water. This experiment will focus on the activity of SDH which is an important part of the citric acid cycle and is found in the inner membrane of the mitochondria of cauliflower.
Materials and Apparatus
Pestle, mortar, spatula, sharp sand, fresh cauliflower, scalpel or razor blade, spectrophotometer, cooled ultracentrifuge, ignition tubes, cuvettes, 7* graduated 1.0ml pipettes, gloves, paraffin’s scissors, ice, 3* cuvette stands and ignition tube racks.
Reagents
Grinding buffer (0.3 M mannitol; 0.006M KH2PO4; K2HPO4-PH 7.2)
Assay buffer (0.3M mannitol; 0.006M KHPO4; 0.014M K2HPO4– 0.01M KCL; 0.005M MgCl2– PH 7.2)
0.04M Azide.
5 * 10 4 M 2, 6- dicholophenolindophenol.
0.002 Sodium Succinate.
0.2 Disodium Malonate.
0.002M Disodium Malonate.
0.0002 Disodium Malonate.
Procedure
A cauliflower was placed on the bench and 25g of the outer 2 to 3 mm of the surface of florets removed. The tissue was then placed in a chilled mortar and 50ml of ice cold water grinding buffer measured and about its half and about 5g of cooled sand added to the mortar. The mixture was then ground vigorously for 2 minutes using a chilled pestle. The other half of the remaining buffer was added to the mortar and ground vigorously for a further 2 minutes.
The material was then filtered through four layers of cheesecloth into a chilled centrifuge tube. The tube was then wired out into the tube and labelled ‘nuclear fraction’.
The tube was then centrifuged at 2160 rpm while ensuring that the opposing tubes were balanced.
In the next step, a clean chilled centrifuge tube was labelled as ‘mitochondrial fraction’ and the post nuclear supernatant decanted into it.
The supernatant was then centrifuged at 8820 rpm for 30 minutes at 0-40C.
In the meantime 10.0 ml of clean grinding buffer was added to the tube containing the nuclear pellet and then the tube re-suspended using a stirring rod and then agitated using a Pasteur pipette.
After the second tube was centrifuged for 30 minutes, the mitochondrial fraction settled at the bottom of the tube as a pellet, the post mitochondrial supernatant was then poured out into the sink and 10.0 ml of grinding buffer added to the tube.
The mitochondrial pellet was then re-suspended in the grinding buffer, and scrapped off the sides of the tube using a spatula then dispersed using a Pasteur pipette. The pellet was dispersed until no lump was seen floating in the tube.
The mitochondrial suspension was then stored in ice.
Preparation of the Assay tubes
We divided ourselves into two groups and separately prepared the nuclear mitochondrial and assay tubes suspensions.
The tubes were then labelled 1-13 and the corresponding cuvettes also labelled 1-13 on their ground surfaces. A separate graduated pipette for each reagent were used to measure the required reagent for each tube in the following order.
Assay buffer.
Azide.
DCIP.
Malonate and/ or Succinate.
Table 1. Localisation of succinic dehydrogenase activity (All amount in ml)
|
Assay Tube |
Buffer |
Azide |
DCIP |
Succinate (0.2M) |
Nuclear Suspension |
Mitochondrial Suspension |
|
1 (Blank) |
3.1 |
0.5 |
– |
0.5 |
0.9 |
– |
|
2 |
3.1 |
0.5 |
0.5 |
– |
0.9 |
– |
|
3 |
2.6 |
0.5 |
0.5 |
0.5 |
0.9 |
– |
|
4 (Blank) |
3.1 |
0.5 |
– |
0.5 |
– |
0.9 |
|
5 |
3.1 |
0.5 |
0.5 |
– |
– |
0.9 |
|
6 |
2.6 |
0.5 |
0.5 |
0.5 |
– |
0.9 |
Table 2. Effect of malonate on succinic dehydrogenase activity (all amount in ml)
|
Assay Tube |
Buffer |
Azide |
DCIP |
Succinate (0.2M) |
Malonate (0.2M) |
Mitochondrial Suspension |
|
7 |
2.6 |
0.5 |
0.5 |
– |
0.5 |
0.9 |
|
8 |
2.1 |
0.5 |
0.5 |
0.5 |
0.5 |
0.9 |
Table 3. Competitive ratio of malonate to succinate (ml)
|
Assay Tube |
Buffer |
Azide |
DCIP |
Succinate (0.20M) |
Malonate (0.02) |
Malonate (0.002) |
Malonate (0.0002) |
Mitochondrial Suspension |
|
9 (Blank) |
3.4 |
0.5 |
– |
0.5 |
– |
– |
– |
0.6 |
|
10 |
2.9 |
0.5 |
0.5 |
0.5 |
– |
– |
– |
0.6 |
|
11 |
2.1 |
0.5 |
0.5 |
0.5 |
0.5 |
– |
– |
0.6 |
|
12 |
2.1 |
0.5 |
0.5 |
0.5 |
– |
0.5 |
– |
0.6 |
|
13 |
2.1 |
0.5 |
0.5 |
0.5 |
– |
– |
0.5 |
0.6 |
The tubes were then covered with a small amount of parafilm and carefully inverted twice to mix the contents and then returned to the rack and maintained at room temperature. The spectrophotometer was then set to give a reading of 600nm.
The tubes containing the nuclear and mitochondrial suspensions were carefully re-suspended and the correct volume was added to tubes labelled 1, 4 and 9. The tubes were then covered with a small amount of parafilm and inverted twice to thoroughly mix the contents.
Some of the contents of the tubes were transferred to the corresponding labelled cuvettes and then the cuvettes placed near the spectrophotometer. The results were then recorded on the proforma.
Zeroing the spectrophotometer using cuvette number 1 (blank 1)
In the next step, 0.9 ml of nuclear suspension was added to tube 2, then the tube inverted to mix the contents thoroughly and then transferred to the corresponding cuvette and the absorbance measured at 600nm. Time and absorbance value were recorded at the room temperature and the cuvette returned to the rack.
Steps 1, 2 and 3 were repeated for tube 3.
Zeroing the spectrophotometer with cuvette number 4 (blank 2)
0.9 ml of mitochondrial suspension was added to tube 2. It was inverted twice to mix the contents. It was then transferred to the corresponding cuvette and the absorbance measured at 600nm. The time and the absorbance value were recorded on the proforma and the cuvette returned to the rack and maintained at room temperature.
The step was repeated for tube 6, 7 and 8.
Zeroing the spectrophotometer using cuvette number 9
0.6ml of mitochondrial suspension was added to tube number 10 and the tube inverted twice to mix its contents. The mixture was then transferred to the corresponding cuvette and its absorbance measured at 600nm. Time and the absorbance value were recorded and the cuvette returned to the rack and maintained at room temperature.
This step was repeated for tubes 11, 12 and 13. The readings were then taken at 5 minutes interval for all the treatments.
Conclusions
Because there was a reduction in the concentration of DCIP concentration in the solutions that we prepared, we found it necessary to isolate some tissues of mitochondria. In contrast to what we thought, both the pellet of mitochondria and supernatant reduced DCIP at almost the same rate. Because mitochondrial pellet and supernatant reduced the concentration of DCIP, succinate dehydrogenase is a must and also mitochondrial tissue in the two solutions. We realized that there occurred more enzyme activity in the supernatant as compared to the mitochondrial pellet. Working out the rate of enzyme activity in the first isolated fraction, we found out that its kinetic was very low as a result of dilution in 35ml as compared to the mitochondrial pellet in which it was re-suspended at 5ml.
There are numerous reasons why our results did not coincide with the expected values. It is possible that centrifugation was not rightly performed since the samples was not consistently maintained at 0-40C. The re-suspension of the pellet with the assay buffer may not have been done properly. The clumps of pellets are likely to not have been dispersed effectively and therefore not being able to react fully with the other part of the solution. There are chances that excess DCPIP may have been added leading to a higher reading of absorbance because of a reduced rate of loss of colour. Differential isolation when performed in the right manner is an effective method of mitochondrial isolation. Separation when this method is used is based in the difference in the size of the components of the cell. However, where very small organelles are involved sucrose gradient centrifugation would be suitable since it allows for separation according to the size and the shape.
References
Nelson, D.L., Cox, M.M. (2007) Lehninger: Principles of Biochemistry, 5th edition, Freeman, New York.
Sadava et.al, (2014) Lite: The Science of Biology (6th edition).Sincuer Association Inc.
Campbell et al, (2011) Biology. Addison- Wesley.
Gilbert H. F (2000) Basic Concepts in Biochemistry, Second Edition, McGraw Hill, New York, NY.
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