Investigating the effect of temperature on the permeability of cell surface membranes

Background knowledge of membranes-

Membranes are made up of a phospholipid bilayer; a phospholipid is simply a phosphate group (AKA the head) connected to a lipid (AKA the tail). The polar heads are hydrophilic and point outwards to the external environment or inwards to the cytoplasm. The non-polar tails on the other hand are hydrophobic, this means they do not like water; this is why they point inwards. The diagram below shows how a phospholipid bilayer behaves in water (notice the lipid tails are pointing inwards, away from the water).

The membrane is not quite as simple as this though, it also has other components to it. There are also proteins in the bilayer which can allow certain molecules in and out of the cell. Small molecules such as the respiratory gases (Oxygen and Carbon dioxide) pass through the membrane by diffusion, in between the phospholipids. Larger molecules however, do not fit in between these gaps. Instead, they pass through channels created by the proteins in the bilayer. This process can be active, which means it requires ATP (active transport) or it can be passive, which means no energy is required (facilitated diffusion). Even though water molecules are very polar, they too can diffuse rapidly through the phospholipids because they are small enough. Large polar molecules on the other hand, can not diffuse through the bilayer (i.e. glucose and amino acids). These molecules must diffuse through the hydrophilic channels created by the proteins in the bilayer.

Each protein channel will only let a specific molecule through, this is essential for controlling what goes in and out of the cell.

As you can see from the picture above, the membrane is quite complex. This is called a fluid mosaic model, this shows all the components known of a membrane. Using this model makes it easier to study the permeability of membranes.

How would temperature affect the membrane?

Temperature will affect each component in the membrane differently. The proteins in the membrane can be found in just one layer or span the whole bilayer. If the temperature is raised enough, the protein could become denatured and break down. If the protein spans the whole bilayer, a gap would be left where the protein used to be. If this was to happen, anything could enter or leave the cell. Proteins start to denature at about 20°C. This is where proteins will start to lose their shape, in transport proteins; this will prevent the passageway from letting the specific molecule through, and would let others pass through. Most of the proteins will be completely denatured at a temperature of 60°C. The cholesterol in the membrane controls the fluidity of the membrane (to prevent the membrane from becoming too rigid or too fluid) and it plays an important part in the mechanical stability of the cell. The hydrophobic region of cholesterol helps prevent ions from passing through the membrane. If the cholesterol was heated too much it would melt. Cholesterol and the lipid tails of the phospholipids would start to melt at 80°C. This would make the membrane too fluid. The lipid tails on the phospholipids would also be affected by the temperature and the membrane would lose its structure. Without cholesterol the membrane would become to fluid and may burst.

Predictions-

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Predictions-

The cell I will be using will be beetroot (Beta Vulgaris Craca); this is because of the red dye inside the cell (Betalain). This red dye will make it easier to measure the permeability. To measure the permeability I will use a colorimeter which measures the percentage of light absorbed or transmitted through a sample depending on the setting. The darker the colour is, the greater the absorption percentage will be. I predict that as the temperature increases, the permeability will also increase. I believe this because the components of the membrane will start to break down, allowing substances to pass through the membrane uncontrolled. At 0°C I predict that nothing will happen as their will be very little (if any) movement in the cell. When the temperature is increased to 20°C, I predict that the dye will start to leak out a little; I think this because the proteins will start to lose their specific shapes and start to let the dye escape. At 40°C more of the proteins will start to denature, which will make more gaps in the bilayer, letting more of the Betalain out. At 60°C the vast majority of the proteins in the membrane will be denatured which will leave lots of gaps in the membrane. The Betalain in the large beetroot vacuole will now leave the cell quite quickly. 80°C is where the phospholipid tails and the cholesterol molecules will start to melt. As the phospholipids are the main parts of the membrane bilayer, if they melt, the whole membrane will start to lose its structure and start to fall apart. Without the cells membrane, anything will be able to leave and enter the cell, so the Betalain will be able to diffuse freely from the cell. If the cholesterol melts, the membrane would lose control of the fluidity of itself and the membrane would burst. At 100°C the membrane would have completely fallen apart and the Betalain will diffuse into the water where there is a lower concentration.

The experiment

Method-

I am going to test the effect of temperature on membrane permeability by using beetroot. I am using this because as the permeability increases, the cells will start to leak, I will see this as the purple dye (Betalain) starts to come out of the cell.

I must make sure that the test is fair by using the same beetroot for all of the samples. I am going to use a scalpel and a ruler so that the samples are all the same size, I must also make sure that I rinse the beetroot with cold water to ensure that there is no dye on the surface already. I will place the samples in test tubes of cold water (once they are rinsed) and then put the test tubes in water baths at different temperatures. There will be 5cm3 of water in each test tube, this will be enough to submerge the beetroot sample but will not dilute the dye too much. To get a good set of results, I will be testing at 6 temperatures. The 6 temperatures are 0°C, 20°C, 40°C, 60°C, 80°C and 100°C. I have explained why these temperatures are significant in my predictions. I will repeat the test 5 times for each temperature; I feel this would be enough to get accurate results. If there were any anomalies I could simply disregard them and still have a fairly accurate average.

The sample

I will cut my samples all from the same beetroot. When I am cutting the beetroot I will cut a 1cm3 sample. These samples will be large enough to produce enough dye but still small enough to easily fit into the test tube.

How I would carry out the experiment

Once I have all of my samples of beetroot placed into the test tubes of water, I will place them in water baths that will be set at the 5 temperatures I am testing. The samples will be left in the water baths for 30 minutes; this will be enough time for the sample to get to the temperature of water. I must make sure that all of the samples are in the water for a fair result. After the 30 minutes, I will place the samples in a colorimeter. Colorimeters transmit light at the sample and calculate the percentage of the light absorbed. So the darker colours will have higher absorption percentage.

Results

The results I will record will be the temperature and the percentage absorption. I will put these results into a table like the one below

I will repeat each temperature 5 times and calculate the average. Once I have these results I will then plot them onto a graph. I predict that as the temperature increases, the more dye will be released so the % absorption will also increase. I think the graph will look similar to the one below:

I will plot a line of best fit on the graph so I can see the pattern between the temperature and how much Betalain is secreted.

The possible variable that may affect my results and how I plan to reduce the effect

Room temperature- the sample may start releasing dye before the test starts, this will particularly affect the results for the 0°C tests. To reduce this I will make sure the environment is as cool as possible to minimize the effect, to do this I could use air conditioning or do the test near a window as it will be near enough freezing outside (the test will be conducted on the 9th December 2006)

Time left in the water bath- to regulate this I will simply use a stopwatch to time 30 minutes for each sample.

Size of sample- it is important to make sure the size of the sample is consistent to give fair results; I will do this by using a ruler and taking care when I am cutting the samples.

Volume of water in test tube- this is important because the water will dilute the Betalain, I need to make sure that this is the same in all tests because if it is more dilute, more light will be let through.

The colorimeter- I must make sure I use the same colorimeter for each test, individual colorimeters may give slightly different results.

To get fair results I will closely monitor all the variables and make sure they are all kept consistent for all tests.

Risk assessment

I have assessed all of the possible risks and given them a probability and severity (highest being 10 lowest being 1)

How the probability of these risks can be reduced

Being cut by glass/scalpel- be extra aware of these dangerous objects, if glass is smashed get it cleared up safely immediately. Use a suitable cutting surface and cutting mat when using the scalpel and put a protective cover over the blade when walking with it.

Being scolded- take care when around hot water baths and know where nearest cold tap is and make it easily accessible. Use tongs when collecting test tubes from the water baths.

Slipping/tripping – before I start I will check the room and check there are not any obstructions on the floor (i.e. bags etc). If water is spilt on the floor I will make sure it is cleared up immediately so the floor is not slippery.

Results Analysis

This table shows the percentage of light transmitted through the solution of water and Betalain in the test tube as measured by the colorimeter. The results can be seen in graph form on the left.

As you can see in the graph, as the temperature increases, the transmission decreases, this means that the solution was darker so less light could get through. The reason the solutions are darker is because of Betalain released from the Beetroots vacuole. More Betalain is released as the temperature increases. This is due to the cell membranes proteins denaturing and the lipids and cholesterols melting. This is what I predicted before the experiment. Test 2 of 8˚C was quite high in comparison with tests 1 and 3, this is not a problem though as it is not significantly higher and I have averaged them out.

Evaluation

The practical was carried out on the 9th of December 2005, at the beginning of the practical session there was a shortage of equipment so I did not get started straight away. Firstly, I made sure that all of my test tubes were clean to make all tests as fair as possible. When I put the samples in the colorimeter, I used the same test tube and cleaned it after each sample. By using the same test tube in the colorimeter, I ensured that there were no differences in results because of the test tube (for example, some test tubes may be slightly thicker than others). Another measure I took to make sure there were no unfair results was to use the same beetroot for all of my samples; if I did not do this it would have been unfair because different plants may have a higher concentration of Betalain in their vacuoles. The water I put the samples in was distilled water, this was to make sure that the water concentration was 100% at the beginning of the experiment and make sure there were no other chemicals in it.

For the first test at 65˚C the % transmission read 0%, I consider this result as an anomaly. I believe this because the other 2 results were 22% and 25%, the solution was clearly letting some light pass through as I could see this. This may have been down to human error or an error in the colorimeter which I believe is more probable.

As a result of this, I disregarded the anomaly and just done the average of the 2nd and 3rd tests.

Doing 3 repetitions of each test may not have given me the most accurate result, I may have got a more accurate result if I done more repetitions (e.g. five). If I had repeated the test more, the anomaly would not have affected the result as much, and my results would have been more accurate.

Even with the anomalous result, I still feel my results are accurate enough. The predictions I made before the experiment were correct according to the results, and the scientific background knowledge fits in with the results too.

Investigating the effect of temperature on the permeability of cell surface membranes

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