1. Homogenisation is the grinding up of tissue and lysing (breaking open) cell to release organelles. In this experiment, cauliflower floret was homogenised in mannitol solution using a kitchen blender on liquefy setting and the organelles was released inside the mannitol solution. This will then produce the homogenate. The homogenate consists of the organelles freely broken apart in the mannitol solution.
2. Differential centrifugation is a method used to separate cell organelles on the basis of differences of size. Bigger particles known as pellet settle at the bottom of the centrifuge tube after centrifugation and the smaller particles will be floating in liquid above the larger particles. This liquid of smaller particles is known as supernatant.
Differential centrifugation is done at specific speeds to pellet organelles at the bottom based on their differences of size . Bigger particles sediment easier at a lower speed followed by smaller particles at a higher speed. So, the bigger the organelles, the less speed required to pellet the organelle and the smaller the organelle, the high speed needed to pellet the organelle. For example, cauliflower homogenate which is composed of organelles such as nuclei, mitochondria etc. will pellet mainly nuclei at the bottom when it is centrifuged at low speed for a short time. Further centrifugation at a higher speed will pellet smaller particles (e.g., mitochondria).
Density gradient centrifugation separates organelles based on their densities and not by size. It produces a purified separation of particles compared to differential centrifugation because a density matrix is provided for the particles to move through in density gradient centrifugation. This help to reduce contamination.
RESULTS AND DATA INTERPRETATION
3. When the homogenate and each fraction were viewed under a microscope, several organelles were identified and its briefly described below;
a) Homogenate: many different organelles were identified under a microscope. It showed large green dots which is identified as nuclei, small black dots which is also identified as mitochondria and other blurry dots which could not be identified but presumably it is one of the cell organelles.
b) Fraction 1: two organelles were identified under a microscope. It showed loads of large green dots which is identified as nuclei and other blurry dots.
c) Fraction 2: Loads of small black dots were seen under a microscope and this is identified as mitochondria.
d) Fraction 3: Hardly could you see anything under a microscope. This could be that the organelles in that fraction are very tiny that the microscope could not pick up at that magnification.
Based on the results described above, majority of the mitochondria was found in fraction 2 because you could see more black dots in fraction 2 than in the other fractions.
4. Results of succinate dehydrogenase assay
Sample / Fraction Succinate Malonate
H ( homogenate) +++ +++
1 (pellet 1) – –
2 (pellet 2) +++ +
3 (supernatant) ++ ++
B (boiled homogenate) +
C (ascorbic acid) ++++ ++++
a. Based on the succinate column alone ,I can conclude that fraction 2 contains mitochondria because it showed a deep red colour compared with the homogenate and the other fractions.
b. The purpose of the malonate control was to inhibit the activity of succinate dehydrogenase and used as a negative control. This control help refine interpretation of my results because in the malonate section, fraction 2 was faint pink suggesting the malonate has inhibit the enzyme activity.
c. The purpose of the boiled control was to confirm that reduction of INT is due to the activity of succinate dehydrogenase. Because when the homogenate is boiled the enzymes are denatured and hence cannot function as they should and hence no INT will be reduced to give the red-purple colour. This control helped in my interpretation of results.
DISCUSSION AND CONCLUSIONS
a. Succinate dehydrogenase oxidises succinate to fumarate in step 6 of the citric acid cycle. It does this with the help of Succinate-coenzyme Q reductase (SQR), which catalyses this oxidation reaction, whilst the reduction of ubiquinone to ubiquinol takes place. FAD (the enzyme’s cofactor} is reduced to FADH2 at the same time the oxidation reaction is taking place. The enzyme is bound to the inner membrane of the mitochondria, making up part of complex II of the electron transport chain, which is where this take place (Nicholls and Ferguson, 1992).
Succinate dehydrogenase is a good mitochondrial marker because of its stability and high abundance in the mitochondria of eukaryote. This enzyme is found only in mitochondria and it is useful in oxidising succinate to release electron which can be transferred to INT, producing the reduced ,coloured INT.2H.
b. Detergents can also be used to detect the presence of a specific protein.