TITLE The Effect of Temperature on Membranes
OBJECTIVE The objectives of this experiment are:
To practise experimental and investigative skills
To investigate the effect of different temperatures on the plasma membrane structure
PROBLEM STATEMENT Betacyanin, a type of betalains, is a pigment commonly found in the vacuoles of beetroot cells. When the beetroot is cooked without its outer skin intact, a large amount of red dye will leak out from it. It is thought that the process of heating the beetroot itself causes the dye to leak out. This experiment aims to confirm the relation between high temperatures and the leakage of betalains from the beetroot cells.
HYPOTHESIS It is proposed that as the temperature increases, the permeability of the cell membrane also increases, which can be seen by the increasing optical density of the solution dipped with the beetroot sample. The null hypothesis for this experiment is that even as the temperature increases, the permeability of the plasma membrane remains the same and so the optical density of light of all solutions is the same.
INTRODUCTION In biology, the term membrane refers to the layer of a certain material which functions as a selective barrier between two states of matter. The layer only allows specific particles, molecules or substances to pass through it. The plasma membrane, a type of membrane, is a structure that encloses each and every plant and animal cells. It is actually an incredibly thin film with a thickness of 8 nanometres. It holds an important role in regulating cellular function by maintaining the composition of the cell. This is achieved by regulating traffic of substances in and out of the cell. The plasma membrane mainly consists of lipi ds, which form the bulk of the t he membrane. Besides lipids, plasma membrane also consists of carbohydrates, cholesterol (another form of l ipids) 1 and protein .
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The plasma membrane layer is composed of a double layer of phospholipids, molecules that 2 have a phosphate head that is hydrophilic and a lipid tail that is hydrophobic . The structure of a phospholipid molecule can be seen in the diagram below:
Diagram 1: The structure of a phospholipid molecule (Image source: http://bioweb.wku.edu/courses/biol115/wyatt/biochem/lipid/P-lipid.gif) The phospholipids are arranged so that the hydrophilic heads are directed outwards and the 3 lipid tails meet at the inside of the bilayer. They are arranged in a fluid mosaic model , proposed by S.J. Singer and Garth Nicholson in 1972. The molecules are said to be constantly undergoing displacement as opposed to only being held in one place. This model also states that the pattern of proteins on the layer is constantly changing.
Diagram 2: The fluid mosaic model (Image source: http://www.ncbi.nlm.nih.gov/books/NBK9898/bin/ch12f3.jpg)
Plasma membranes are said to be selectively permeable, meaning that it allows only small 4 water soluble molecules to pass through it by simple diffusion and water molecules by osmosis . However, the presence of transmembrane proteins in the form of carrier proteins and pore protein 5 allows large molecules to enter or exit the cell by facilitated diffusion and active transport . 2
Plasma membranes also contain proteins, which are embedded in the plasma membrane. These proteins carry out functions such as providing transport for certain molecules into and out from the 6 cell, enzymatic activity, intercellular junction and the identification of other cells . Beetroot ( Beta vulgaris sp.) is a type of plant which has a root usually eaten as food. The root has a purplish red colour due to the presence of betalains, a type of pigment stored in the cell 7 vacuoles . The specific type of betalain found in beetroots is known as betacyanins, which exhibit a range of colour from red to violet. Normally, when a beetroot is cooked in a pot of water with its outer skin intact, the leakage of the red dye is minimal. However, if the skin is peeled, a substantial amount of dye will leak out of the beetroot. Temperature is defined as the average kinetic energy of the molecules or particles in an object or system measureable with a thermometer. Protein molecules are known to be denatured when they are exposed to high temperatures. It occurs because heat energy increases the kinetic energy of the molecules in protein structures, making 8 them vibrate faster and more violently . This eventually causes the hydrogen bonds in proteins to be disrupted, denaturing them in the process. Spectrophotometry refers to the quantitative measurement of the reflection or transmission properties of a substance or material as a function of wavelength 9. A spectrophotometer is usually used to measure the transmittance or the reflectance of solutions. In this experiment, the device is used to measure the optical density of the solution. The amount of pigment present in the solution influences the reading of optical density of the solution so that a solution with more pigments in it will bring about a higher reading for optical density.
VARIABLES o
o
o
o
o
o
Manipulated: Different temperatures of water bath (0 C, 25 C, 35 C, 45 C, 55 C, and 65 C) Responding: Permeability of plasma membrane to pigments (shown by the optical density) Controlled: Length, diameter and total surface area of beetroot sections, beetroot sample, time taken for all beetroot samples left in water bath, type of solution used in water bath
MATERIALS Raw beetroot, cork borer, ruler, distilled water, dropper, spectrophotometer, beakers, water bath of o o o o temperatures 35 C, 45 C, 55 C and 65 C, knife, forceps, white tile, 2 boiling tube racks, 8 boiling tubes, thermometer, cuvettes, stopwatch, small measuring cylinders, lint -free tissue paper
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METHODOLOGY 1. Several sections are cut from a single beetroot using a size 4 cork borer. Eight 1 cm long slices are cut from these sections. Precaution is taken so that the beetroot juice is not spilled on the skin or clothing. 2. The sections are placed in a beaker of distilled water. They are left for 5 minutes to wash away excess dye. 3 3. Eight labelled boiling tubes each containing 5cm distilled water are placed into water baths at O O O O O O 0 C, 25 C, 35 C, 45 C, 55 C and 65 C. They are left for 5 minutes until the water reaches the required temperature. One of the beetroot sections is placed into each of the boiling tubes. They are left for 30 minutes in the water baths. 4. The beetroot sections are removed and the tubes are shaken to disperse the dye in the water. 5. The spectrophotometer is switched on and it is set to read % absorbance. 6. The filter is set to the wavelength of 500nm. 3 7. Using a pipette, 2cm of distilled water is accurately measured into a cuvette. The cuvette is placed into the spectrophotometer, making sure that the light is shining through the smooth sides. 8. The spectrophotometer is adjusted to read zero absorbance for clear distilled water. The setting is not altered again during the experiment. 3 9. 2cm of the dye solution is placed into a cuvette and a reading is taken for optical density. The reading is repeated twice for every temperature in order to get the average values for the readings. 10. All the results are tabulated accordingly. 11. The results are then used to plot a graph of absorbance against temperature
SAFETY PRECAUTIONS During the experiment, caution is advised for the following: 1. The raw beetroot sample is handled carefully so to avoid the red dye in beetroot from spilling on the skin and/or clothes. 2. The knife is used with great caution as the sharp edges can bring serious injury to the user if it is not handled properly. 3. The readings for the absorbance of the solutions are taken thrice to obtain the average reading. 4. After setting the spectrophotometer to read zero absorbance for clear water, the setting is not adjusted again after that. This is to ensure that the readings are close to the actual value. 5. The cuvettes are cleaned with distilled water first before they are filled with the solutions from the water baths. They are also wiped with lint-free tissue paper to ensure that they are completely free from dust and water droplets. 6. The boiling tubes are handled carefully as they are extremely fragile.
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RESULTS OPTICAL DENSITY (OD)
Temperature (˚C) 1
2
3
Average
0
0.162
0.164
0.153
0.160
25
0.242
0.242
0.241
0.242
35
0.275
0.275
0.274
0.275
45
0.356
0.355
0.350
0.354
55
0.484
0.486
0.475
0.482
65
0.564
0.564
0.565
0.564
Table 1: The optical density of the solutions according to the temperature of the water baths
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Graph of optical density against temperature 0.8
0.7
0.6 65, 0.564
0.5 )
55, 0.482
D O ( y t i s n0.4 e d l a c i t p O
45, 0.354
0.3 35, 0.275 25, 0.242 0.2 0, 0.16
0.1
0 0
10
20
30
40
50
60
70
Temperature (oC)
Graph 1
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DISCUSSION Table 1 shows the results for the experiment. On the other hand, Graph 1 shows the relation between absorbance of light with temperature in Celsius. o
The solution placed in the 0 C water bath has a very pale pink colour, indicating that the o amount of pigments in the solution is the least. Second, the solution placed in the 25 C water bath has o a light pink, while the solution placed in the 35 C water bath is pink, indicating that there is an o increase in the amount of betalains in the solution. At 45 C, the solution collected is red, while the o o solutions immersed in the 55 C and 65 C water baths are dark red and blood red respectively. From the results obtained, a trend can be observed where as the temperature of the water bath increases; the permeability of the plasma membrane also increases, shown by the increasing optical density of the water samples obtained. This is also seen in the solutions collected from the water bath o whereas the solution left in 0 C water bath has a pale pink colour and the colour becomes darker and redder as the temperature of the water bath increases.
Diagram 3: The cuvettes, placed side by side in the order of increasing temperature of water baths they were immersed in. The cuvette on the far left side contains distilled water. (Image source: Author’s own photo)
Besides, as the temperature increases, the average kinetic energy of water molecules also increases. Therefore, the collision between water molecules and the pigments in beetroot become more frequent, causing the solution to turn darker, hence increasing the optical density for the solution. The phospholipid membrane and the vacuole membrane are also disrupted due to the high temperature, allowing more pigments inside the vacuole to leak into the water. This causes the optical density of the o solution to increase linearly until 35 C. o
After 35 C, the slope of the graph becomes steeper, showing that the rate of change of absorbance is higher than at temperatures lower than that. This is because at temperatures higher than o 35 C, proteins in the plasma membrane start to denature. As this happens, holes are formed in the phospholipid bilayer, allowing more pigments in the beetroot cells to spill out into the water. Besides, the structure of the phospholipid bilayer starts to change into a more fluid state from a tightly packed structure, allowing the betalain molecules to diffuse This occurrence can also be seen in solid butter
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when it is heated, where the butter melts into an oily fluid. This confirms the reason why the graph o starts to become steeper after 35 C. o
However, after 55 C, the slope of the graph starts to become less steep again. This indicates that there is a small amount of betalains left in the cell vacuoles. It is speculated that temperature is not the only factor that causes changes in the plasma membrane structure. The concentration of ethanol, a type of alcohol, in a solution containing beetroot samples can also influence the permeability of the plasma membrane. To do this, beetroot samples are immersed into solutions containing different concentrations of ethanol at the same temperature. The samples are then analysed for its optical density using a spectrophotometer.
SOURCE OF ERROR During the experiment, the results obtained have slightly deviated from the expected values. One of the factors that have caused this is the fact that the time for all the boiling tubes to be immersed in their respective water baths is not exactly the same. For instance, during the process of collecting the boiling tubes from the water baths, some of the boiling tubes are left longer than the others inside the water baths. This has caused the optical density of some of the solutions to be higher than the exact value. Nevertheless, the deviation in the readings is not very high to make the results to be invalid, so the readings are still acceptable. Other than that, during the experiment, the beetroot samples were not immersed in distilled water first to remove the excess red dye from the samples. As a result, the solutions contain more pigment than the desired results. However, this was done for all beetroot samples, so the relative difference in the optical density of the solutions is still close to the true value.
CONCLUSION From the experiment, it is confirmed that temperature plays a major role in causing the structure of the plasma membrane to change into a more permeable membrane. It is seen that as the temperature increases, the optical density of the solution increases, meaning that the permeability of the membrane increases as well. The high temperature disrupts the phospholipid bilayer, causing to become more permeable while the proteins embedded in the membrane are denatured, allowing the passage of more pigments from inside the cell to the water outside. Therefore, the hypothesis is accepted.
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REFERENCES 1. Neil A. Campbell, Jane B. Reece et al. (2008). Biology. Eighth Edition. San Francisco (CA): Pearson Benjamin Cummings. p.125 2. Ibid. 3. Ibid. p. 99 4. http://www.ncbi.nlm.nih.gov/books/NBK9898/ th 5. Eldra P.Solomon, Linda R. Berg, Diana W. Martin. (2008). Biology- International Edition. 9 Edition. Canada: Brooks/Cole Cengage Learning. p. 108 6. Ibid. p. 114 7. Robinson, Trevor (1963). The Organic Constituents of Higher Plants. Minneapolis: Burgess Publishing. p. 292 8. http://www.elmhurst.edu/~chm/vchembook/568denaturation.html 9. http://www.nist.gov/pml/div685/grp03/spectrophotometry.cfm
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