Permeability of Cell Membrane in Beetroot Cells
Biology Assessment Practical Isabelle Litwin
Title:
Permeability of Cell Membrane in Beetroot Cells
Aim:
To find out how temperature affects the cell membrane permeability of beetroot cells
Theory/ Background:
Permeability of a cell to solutes in an aqueous medium depends upon the physical and chemical make-up of the membrane. The maintenance of the living cell depends upon the continued presence and functioning of a selectively permeable membrane. If the membrane is altered in any way, this may have an effect on its permeability and therefore the properties of the cell of which it is a part. Irreversible changes in the permeability of the membrane usually lead to the death of the cell.
The cell membrane provides a boundary between the cell's inner environment and its surrounding outer environment. It allows different substances to pass through at varying rates, this is described are differentially permeable. It controls things such as sugar concentration, ion concentration and pH levels. If the pH levels were to be affected, the enzymes would not function efficiently thus allowing chemical reactions to take place.
Proteins in the membrane vary in size and occurrence. Some penetrate all the way through called intrinsic proteins (transmembrane); some only penetrate half way called extrinsic proteins. Transmembrane proteins have hydrophilic and hydrophobic regions which cause the proteins to align in a specific way in the membrane. Proteins with carbohydrate side chains, called glyco-proteins, act as receptor sites and help the protein to align with other cells.
Cholesterol molecules are located in different places in the membrane; they control the fluidity of the membrane. If there are o lot of cholesterol molecules then the membrane will be rigid, less molecules will mean a more fluid membrane. Without the cholesterol molecules, the membrane breaks down and the cell bursts.
There are many things that can affect what is transported in and out of the cell: temperature, pH, other chemicals (solvents) can be main factors affecting this.
An excessive pH range (very acidic or very alkaline solution) could damage the ionic bonds in the proteins present in the membrane, therefore causing the shape of the protein to change preventing the membrane from functioning properly.
Chemicals such as organic solvents e.g. ethanol, cause the membrane to dissolve effectively killing the cell as everything in the cell is released this is because the molecule is turned inside out therefore exposing the hydrophobic interactions which will readily dissolve in them.
Substances travel in and out of the cell in various ways:
a) Passive diffusion this is when molecules in a liquid or gas move from an area of their higher concentration to an area of their lower concentration down a concentration gradient.
b) Facilitated diffusion happens through hydrophilic pores created by protein molecules; these channels allow some molecules through in preference to others.
c) (a) Endocytosis involves engulfing a foreign material, it happens in two ways:
(i) phagocytosis- 'cell eating'
(ii) pinocytosis- 'cell drinking'
(b) Exocytosis is the removal of waste materials from the cell.
d) Osmosis is the net movement of water molecules from an area of their higher concentration to an area of their lower concentration through a differentially permeable membrane.
e) Active transport is what brings things into the cell against the concentration gradient via a carrier protein.
The pigment in the beetroot cells is called anthocyanin. It is contained in the vacuole of the cell so when the cell is ruptured, the contents diffuse out. The pigment leakage is caused by diffusion. The more pigment that leaks out this indicates a greater the rate of diffusion.
Temperature has a great effect on how substances are transported in and out of cells. As the membrane is called differentially permeable, it only lets certain substances through. Those that cannot pass through, pass through protein channels. If the temperature is increased dramatically, then this will start to affect the bonds holding the proteins together because the bonds vibrate so rapidly that they break.
Preparation for the Practical:
We had previously done an experiment investigating the effect of temperature on enzymes. As enzymes are proteins, we could use what we found out in that practical to ...
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Temperature has a great effect on how substances are transported in and out of cells. As the membrane is called differentially permeable, it only lets certain substances through. Those that cannot pass through, pass through protein channels. If the temperature is increased dramatically, then this will start to affect the bonds holding the proteins together because the bonds vibrate so rapidly that they break.
Preparation for the Practical:
We had previously done an experiment investigating the effect of temperature on enzymes. As enzymes are proteins, we could use what we found out in that practical to aid us in preparing for this practical. As the temperature increased, the rate of reaction also increased until it reached a point.
Once it hit a temperature of about 60?C the rate decreased as the shape of the enzyme's active site was changed and damaged irreparably, this was due to Denaturation.
We could apply this to this practical, making it easier to predict what would happen and at what temperature the permeability of the membrane would be the greatest.
The temperatures would range from a low temperature through to a very high temperature. This would give a rough idea of how the pigment leakage is affected by the heat.
The things we thought would help us were tested in the pilot experiment we carried out. This enabled us to decide whether the temperature range was adequate, whether the size of the beetroot was too large or too small, whether the filter gave a reliable reading, and whether the intervals between the samples was long enough.
From the results we collected, we could work out what factors could make the practical more successful or what could make the practical more difficult or less accurate.
Prediction:
I predict that the greater the increase in temperature, the more pigment will leak out and the more intense the colour of the pigment will be.
Hypothesis:
Temperature has an effect on the shape of many biological substances e.g. membranes, and proteins. In cell membranes, the greater the increase in temperature, the greater the increase in flexibility of the membrane due to weakened bonds, however, once the temperature has exceeded 60?C, the structure of the membrane, as in proteins, is affected.
Denaturation occurs once the temperature has increased to such a high level, therefore more pigment will leak out.
Apparatus:
* Scalpel
* Beetroot
* Beakers
* Ruler/pencil/pen/rubber
* Bunsen burners
* Tripods
* Colorimeter/Cuvettes
* Timer
* Sieve
* Water (tap and distilled)
* Thermometers
* Cutting tile
* Freezing salts/ice
* Pipettes
* Heat proof mats
* Tripod gauzes
* Size 5 borer
* Glass stirring rods
* Matches
* Splint
* Tweezers
Actual Practical:
Method:
. Set up two beakers with 250ml of water at 10?C using ice and freezing salts to keep the temperature down to 10 and at 20?C (below room temperature).
2. Set up three water baths with 250ml of water at 40?C, 60?C and 80?C.
3. Keep an eye on the temperatures by constantly checking the temperature on the thermometer, when they get close to the one you want, remove and replace occasionally to keep at the same temperature.
4. Rinse 10 test tubes with distilled water, pour 10ml of distilled water into each test tube and place two test tubes into each of the beakers.
5. Using the borer (size 5), bore five beetroot pieces.
6. Using a ruler and a scalpel, cut 10 beetroot pieces to lengths of 2cm.
7. Place the 2cm pieces of beetroot into a sieve and rinse under cold, running water to remove excess pigment that was released during-free hand sectioning.
8. Place two pieces of beetroot into each test tube and start the timer once all the pieces have been placed onto the test tubes.
9. Take a sample (2ml) using a pipette every 2 minutes from the beakers at 10? and 80?, put into a cuvette and place each sample in front of the beaker it was taken from.
0. After 12 minutes take two samples from each of the beakers at 20?C, 40?C and 60?C and one sample from the beakers at 10?C and 80?C.
1. Transport all the cuvettes in the order they were collected in a stable carrier to the colorimeter for testing.
2. Pour distilled water into a cuvette as a reference and place into colorimeter and press the reference key, this allows the colorimeter to have something to compare the absorption of the sample to.
3. Place the 520 yellow filter into the slot, this is because yellow is the complementary colour of purple, which is the colour of the pigment of the beetroot.
4. Remove the reference and place into the slot each cuvette in turn, firstly the cuvettes for 10?C, 20?C and so on.
5. Record the results for each reading in a table.
Justifications:
The temperature of the water baths ranges from 10?C to 80?C as I thought it would give a good range of results which would show what happens to the absorbency with an increase in temperature.
Filter number 520 is a yellow filter which is the complementary colour of purple. As the colour of the pigment is a mauve/purple colour, it seemed the obvious one to pick as it would not absorb the colour of the solution.
I chose a size 5 borer as it was not too narrow and not too wide; it was also easier to fit into the test tubes. The pieces were cut to 2cm in length to ensure that they fit into the test tube horizontally as well as vertically. Two would be put into each test tube as one might not release a lot of pigment.
The Bunsen burner has to be removed every so often to prevent the water baths from boiling and exceeding the desired temperature.
I chose to do the extremes in temperature the whole way through to see how they varied from the highest to the lowest, as the temperatures in the middle would not vary too much.
The samples would be taken every two minutes to get good range of results to allow me to find out what I am looking for.
Tweezers would be used to prevent any of the pigment staining the clothes or the skin during the transfer from the sieve to the test tubes.
Variables:
* The temperature has to be kept at the chosen temperatures to make sure that the results are as accurate as possible, and so that unnecessary damage is not caused to the membrane. Thermometers in each water bath will make checking the temperature a lot easier
* The size of the beetroot pieces has to be as similar as possible, therefore using a ruler for measuring the length is essential.
* The amount of distilled water in each test tube has to be the same otherwise the accuracy of the practical will be wrong from the start. Therefore a measuring cylinder will be used to measure out equal amounts into each test tube.
* The timing must be as accurate and as close to 2 minutes as possible for this I will be using a timer.
Results:
Temperature(?C)
0?
20?
40?
60?
80?
Time (mins)
ABSORBANCY(in arbitary units)
2
0.00
-
-
-
0.26
4
0.00
-
-
-
0.39
6
0.00
-
-
-
0.58
8
0.03
-
-
-
0.68
0
0.06
-
-
-
0.72
2
0.02
0.02
0.03
0.01
0.03
0.37
0.55
0.57
AVERAGE ABSORBTION
0.02
0.025
0.02
0.46
0.57
Risk Assessment:
A major risk in doing this practical was that we were using water baths; therefore care was needed to prevent any spillages of the water which in some cases reached temperatures as high as 80?C, which could cause serious burns. Also during the heating of the water baths, the tripods and gauzes would become quite hot, so care should be taken when working near them. When the baths are not being heated, then flame colour should be turned to yellow and moved away from the baths. Do not leave water baths unattended, especially if they are being heated.
The area in which is being worked in must be clear of any mess and spillages to prevent any accidents from occurring.
When boring the beetroot, make sure the borer is positioned vertically to prevent the beetroot from sliding off the mat. Also, the beetroot should not be held in the hand when boring, as when the borer is pushed in, a nasty injury could be sustained.
When cutting the beetroot into pieces of desired length, caution should be taken when using the scalpel, as it is a lot sharper and more precise then using a knife and could cause severe cuts, also, fingers should not be too close to where you are cutting.
A lab coat and goggles should be worn at all times. The beetroot's pigment is a crimson/mauve colour and stains deeply if it comes into contact with material; therefore the lab coat prevents the pigment from staining your clothes. The pigment that seeps out of the ruptured cells is called anthocyanin. Hair should also be tied back to prevent any risk of it setting alight or just getting in the way of the practical.
Analysis:
From the results collected it is clear that temperature has an effect on the cell membrane as at low temperatures there was very little or no pigment released (0.02 absorbency at 10?C) and as the temperature increased, the amount pigment leakage also increased (0.57 absorbency at 80?C). This could be explained by the increased heat having an effect on the bonds in the proteins in the membrane.
The hydrogen bonds break which damages the tertiary structure of the protein and instead of keeping its normal globular shape, it reverts to a more fibrous form, this is called Denaturation. Therefore instead of being globular they have been reduced to their primary structure, they become long and thin, although there is some secondary structure, there is a gap in the membrane which allows the pigment to seep out of the cell. The amount leaked out depends on how badly the temperature has affected the hydrogen bonds in the proteins.
The cholesterol molecules are also affected as they control the fluidity of the membrane, the increased heat causes the membrane to become more fluid and flexible, therefore making it more likely for the pigment to leak out through larger gaps in the membrane. Without cholesterol molecules, the membrane soon breaks down making the membrane unstable and the cell bursts open.
The process by which the pigment leaks out is called diffusion. Heat affects the rate of diffusion in the way that the greater the increase in heat, the greater the energy of the molecules. The more energy the molecules have the more they move around, this is how the pigment leaked out.
At lower temperatures, the molecules were moving around slowly so there was very little leakage, however, when the temperature increased so did the velocity of the molecules which increased the amount of pigment leakage.
The heat affects not only the proteins in the membrane but also the membrane itself, as the heat increases the membrane becomes a lot more fluid which may allow some of the pigment to escape. The gradients in graph A show this effect as the gradient for 80?C is a lot steeper than that for 10?C which shows that the pigment leaked out more at the higher temperature.
Graph B shows the average absorbencies for each sample at the different temperatures. The lower temperatures were not affected as expected, the pigment did not leak out as the membrane and the proteins were intact. Once the temperature reached highs of 60?C and higher, the shape and consistency of the membrane changed causing the anthocyanin to leak out of the disfigured cell.
Evaluation:
Looking at graph B point three (40?C) looks to be anomalous as it does not follow the trend of the rest of the graph, if it was to follow the trend the point would have an absorbency of approximately 0.2. However, it can be accounted for, as 40?C like lower temperatures, does not affect the proteins. The bonds are still stable and intact therefore it won't have as much variation in the absorbency of light through the sample. Other than that average, the other average results follow the predicted pattern of increasing the absorbency.
My results back up my prediction as absorbency increased with temperature.
The individual readings were as expected, the absorbency increased with temperature and duration of the experiment. However, the last readings after 12 minutes for 10?C and 80?C showed to be anomalous as the absorbency decreased. The result for 80?C may be explained by the possible evaporation of water in the test tube due to the high temperature, causing the solution's concentration to increase, decreasing the absorbency. This could be prevented by using a bung in the test tubes.
Inaccuracies/Improvements:
Many inaccuracies could have occurred during the course of the experiment. One being the uneven spread of pigmentation in the beetroot, some areas were much darker than in other areas, this could have an effect on the amount of pigment which was released by the cell. This factor is not counted as human error as there is nothing we can do to prevent this from happening. Also if different pieces of beetroot were used, it could affect the results obtained.
This could be overcome by using the same beetroot through out the experiment, that way we know that if the pieces are from the same beetroot, then the colouration of the pigment will be similar in all the pieces. If they were from different pieces of beetroot then the intensity of the pigment released may cause inaccuracies to occur.
The beetroot may not have rinsed properly therefore the pigment may have leaked out prematurely in effect causing the percentage transmission to be less than would have been otherwise. Rinsing the beetroot thoroughly would make the first reading more accurate. We could prevent this inaccuracy from happening by blotting the beetroot before transferring it into the test tubes.
The surface area of the beetroot may also play a part in the accuracy of the results, as varying sizes in area would have produce a varying amount of pigment leakage. The larger the surface area, the more pigment will leak out. This could be due to free hand sectioning. A way to avoid the variation could be to use a device which has cutting utensils at a set distance which would produce pieces that were of equal length.
Temperature was another aspect that could have caused results to be inaccurate as the temperature was very hard to keep the same, as it was very hard to stop the fluctuation from occurring. The baths may have been heated too much, or they may have had time to cool down, in effect, affecting the amount of pigment leaking out of the cell due to the increased fluidity of the membrane.
Inconsistent stirring could have lead to uneven spread of the pigment, therefore when a sample was taken out for testing; the percentage transmission was greater than would have been otherwise. One way of overcoming this would be to stir regularly to ensure even spread of pigment.
Bibliography:
* Letts A-Level Biology Study Guide Glenn Toole and Susan Toole
* Understanding Biology Glenn Toole and Susan Toole
* Biology 1 OCR/Cambridge
* www.google.com
* www.cell-adhesion.net
* www.yahoo.com
* This document was downloaded from Coursework.Info - The UK's Coursework Database
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