To obtain the RNA molecules necessary for examination 0.3M KOH is added, which when incubated at 45 degrees Celsius for 30 minutes will break the RNA down into ribonucleotides, substances that will not precipitate during the next addition of HClO4. After incubation and cooling 1M perchloric acid is added, and the solutions are centrifuged at 2000 rpm for 5 minutes at room temperature. These supernatants will be decanted and known as N-S2 and C-S2. RNA is present in the nucleus because this is where mRNA is made, but it is also present in the cytoplasm since tRNA and mRNA are used by ribosomes in the synthesis of proteins. To allow for a test to compare which contains more, orcinal reagent will be added to each sample and also to test tubes containing RNA solution and water, and this will allow for comparison.
1.0M KOH is added to the precipitate and it will be incubated at 45 degrees Celsius for 30 minutes, making sure the precipitate is fully dissolved. After incubation the two solutions, NP and CP, will be tested for DNA. This will be done by adding diphenylamine reagent to some of NP and CP and incubating the solution at 90 degrees Celsius for 25 minutes, with the same process being performed on DNA solution and water to enable comparison. DNA is not free floating in the cell, but enclosed inside the nucleus coiled very tightly as chromosomes. The only trace of DNA outside the nucleus in this instance would be in the mitochondria, or if a plant cell were being used then it could also be found in chloroplasts. There should be a high expectation to see a vast amount of colouration change in the nuclear sample.
NP and CP will also be tested for the presence of protein. The first test involves adding biuret reagent to each solution, with it also being added to a protein solution and water to enable comparison. However this test may produce inconclusive results as both NP and CP will contain protein molecules, with proteins being the main constituent of DNA, yet proteins are produced by the ribosomes in the cytoplasm. A more quantitative set of results may be needed to distinguish which has most, and this can be achieved by doing a quantitative assay of protein. With the absorbance of the spectrophotometer set at 550 nm, solutions containing various amounts of standard protein 10mg/ml are made up with biuret solution in and the absorbance is measured against the reagent blank, giving a basis for comparison. Solutions containing NP and CP with biuret solution are prepared and the absorbance of each taken. This is measured against the others so that the mass of protein in NP and CP can be worked out and expressed in milligrams.
If the investigation were carried out with plant cells the results would be different, even though both are eukaryotic with similar basic structure. In most cases, upon looking at a plant cell through a microscope a large central vacuole is clearly visible. It takes up a large amount of space in the cytoplasm and performs a number of jobs, including the storage of waste material and nutrients. Both are secreted and used when necessary. This large organelle coupled with the previously mentioned cell wall provides the plant cell with a much more typical shape, whereas animal cells can differ greatly in their appearance, contain many small vacuoles and do not have a cell wall.
Results
Table 1. Colour change in solutions after adding iodine to test glycogen content
The results suggest that the cytoplasmic supernatant has a greater level of glycogen.
Table 2. Colour change in solutions after a test for RNA using Orcinal reagent
Results suggest that there is a much higher presence of RNA in the nuclear supernatant fraction 2.
Table 3. Colour change in solutions after a test for DNA using diphenylamine reagent
Results suggest no DNA in the cytoplasmic precipitate and a high amount in the nucleic precipitate.
Table 4. Colour change in solutions after a test for protein using biuret reagent.
Results are inconclusive, as the amount of protein in each sample is such that the resulting colours are too similar to show definite results
Table 5. Quantitative protein assay of various protein solutions, NP and CP, showing absorbance rates and protein mass per tube
To work out how much protein is in 10 ml of NP, the protein mass measurements of tubes 7 and 8 need to be averaged, and then multiplied by 20 since only 0.5 ml NP was tested. Similarly to work out the protein content of CP, the protein mass measurements of tubes 9 and 10 must be averaged and then multiplied by 100, since only 0.1 ml CP was tested. The calculations are on an attached sheet and a graph is also attached.
Discussion
The addition of the diphenylamine reagent turned the nucleic sample blue with very little colour change occurring in the cytoplasmic fraction, meaning that the amount of DNA in the nucleic sample is significantly larger than the amount in the non nucleic sample, supporting the theory. This is due to the simple fact that the nucleus is where all of the DNA is stored as chromosomes, and the only DNA outside of the cell in this instance is present in the mitochondria, which will not show since centrifugation was not strong enough to reduce them.
From previous knowledge it is known that RNA is present in both parts of the cell, but the addition of orcinal reagent produced a much darker green colour from the nucleic fraction than from the cytoplasmic fraction, showing that there is more RNA present in the nucleic sample. This means that there is a smaller amount of RNA being used in the cytoplasm for protein synthesis when compared to the amount of free ribonucleotides and newly formed RNA in the nucleus, again as expected.
Glycogen, in animals, is taken from food and used as a glucose store inside muscle and liver cells. The glucose resulting from its hydrolysis can be used in the nearby metabolic reactions of the cell, including re-synthesis of ATP. Results support the idea that no glycogen would be present in the cytoplasm, with the nucleic fraction turning a darker brown colour than the cytoplasmic fraction upon the addition of the iodine.
It was already obvious that there would be protein present in both samples due to DNA, RNA and enzymes having protein in them, and the Biuret test did not show enough difference in the colour of the samples to give a conclusive result. A quantitative assay proved that the amount of protein in the cytoplasm is much greater than the amount found in the nucleus, being due to the cytoplasm containing many more organelles and structures that are either formed from or create protein. The calculations and graph allowed for the approximate percentage of protein in the cell to be worked out.
Conclusion
It can be said that each of the results supports previous ideas on the subject. The iodine test for glycogen showed a high presence and colour change in the cytoplasm and none in the nucleus. The orcinal test showed a higher colour change, and therefore higher presence of RNA, in the nucleus than in the cytoplasm. The diphenylamine test showed that no DNA was present in the cytoplasmic fraction, whereas a high concentration of DNA in the nucleic fraction resulted in a high colour change. The biuret test for protein gave inconclusive results, but the quantitative assay allowed for the observation that there is nearly twice the amount of protein in the cytoplasm than in the nucleus, with the percentage of protein per 1000mg of liver being approximately 17.5%
References
1. Campbell and Reece, 2005, Biology 7th edition, Benjamin Cummins