Study the effects of temperature on the movement of the red pigment Anthocyanin through the vacuole of a beetroot.

Hypothesis:

In this experiment I aim to study the effects of temperature on the movement of the red pigment Anthocyanin through the vacuole of a beetroot. Under normal conditions, the movement of Anthocyanin across the plasma membrane is zero.

As temperatures increase until around 40°C, there will be no change in the amount of Anthocyanin movement across the vacuole membrane. However, as temperature increases past around 45°C, Anthocyanin will start to ‘leak’ out of the vacuole. This is because the 4 bonds holding the all the membrane proteins in its tertiary structure will be broken. These bonds are the Hydrogen bonds, Peptide, disulphide bonds and hydrophobic interactions. They bonds break due to the greater kinetic energy (k.e) found in the membranes bi-lipid layer. This in turn leads to the fluid mosaic model becoming more animated and so the Hydrogen bonds break, this in turn breaks the other bonds holding the channel proteins tertiary structure together and so the protein unravels or denatured. This in effect leads to large gaps in the plasma membrane where the channel protein used to be. I predict that the denaturation will therefore begin at around 50ºC and continue till around 80ºC

These resulting holes are large enough to allow the several Anthocyanin molecules to pass through. I therefore predict that concentration of Anthocyanin in the surrounding water will be quite low at around 45-50°C. However, as temperatures increase, the k.e will give the fluid mosaic model more movement and this will pull the gap even wider, allowing more molecules to pass at any one time. This I believe will start at around 60°C and continue until around 80°C.

At such temperatures I predict that the concentration of Anthocyanin in the water will increase. This is because not only are the gaps in the plasma membrane of the vacuole larger, but also because the Anthocyanin will have a greater kinetic energy. This aids the diffusion process and so concentration will increase.

However, as temperatures exceed around °C, I expect the diffusion rates will start to stabilize, finally stabilising at around 80°C. This means that the increasing rate of diffusion will fall to a constantly stable level where there is no change in the rate of osmosis. I think this because the proteins have been fully broken down at an earlier temperature and resulting gap in the plasma membrane extended to its fullest extent and the k.e can no longer influence the membrane. My null hypothesis is that a changing temperature will not effect the movement of Anthocyanin across the vacuole.

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I have chosen to record data starting at 30°C increasing in 10°C increments. I chose to start at 30°C as I can accurately measuring the point at which the proteins start the denaturation process. Between 40°C and 70°C, I will measure the transmission every 5°C increase. I will do this as it allows me to measure the breakdown of proteins with differing temperature levels

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While I am conducting this experiment, along with validity and reliability of data, safety is a prime factor that I will take into consideration. When I am cutting beetroot using a scalpel, I will make sure that I am cutting away from my body. Also when I am heating the boiling tubes in the water bath I will place them with the top facing towards the wall as if it were to overheat then the steam would be facing towards the window instead of flesh.

Apparatus:

Beetroot sample, cork borer, scalpel, 100cm³ beaker, Bunsen burner, heatproof mat, tripod, gauze, test tubes x 9, syringe, thermostatic waterbath, stop clock, colorimeter, cuvettes x 9

Plan:

I will firstly cut a beetroot sample using a cork borer, using such as it ensures all the beetroot samples are the same diameter. I will then cut them to an equal length of 3cm using a scalpel. After I have cut the samples, I will wash them in cold water for about 10 seconds to ensure that no loose Anthocyanin leaks out of the cut vacuoles at low temperature, falsifying my results.

I will obtain a beaker of 100cm³ of distilled water to ensure that all impurities in the water are removed that could affect the results. I will the place 5cm³ of the water into a test tube using a syringe. I will use a syringe, as it is the most accurate way of measuring volumes. I will then place the sample into a waterbath along with 2 other prepared samples.

I have chose to use a water bath as using this method it will ensure that the samples remain at a set temperature and I can test more than one sample at a time. I will set the first temperature at 30°C. When the test tubes have reached the set temperature I will place the samples in and start the stop clock. I will record the time I entered the samples into the water leave them in the water for a period of 2 minutes. When this time has elapsed, I will remove the samples from the test tubes and place the test tubes in a rack to cool. After I have finished with the final sample, I will test the water from the first sample in a colorimeter.

Whilst the completed samples are waiting to be tested in the colorimeter I will increase the set temperature the waterbath at a higher temperature, when this temperature has been achieved I will add the next samples. For the finished samples I will drain 4cm³ of the water from the test tubes into a cuvette using a syringe. I will use a syringe, as it is the most accurate way of measuring volumes. I will set the colorimeter to the green filter as this has the most effective wavelength of light for measuring the transition of a red pigment.

I will then calibrate the colorimeter by pressing the test button on clear distilled water. I will then test each of the samples. I will record the results into a table for comparison

Method:

Set the colorimeter to the green filter
Cut the beetroot using the cork borer, set the water bath to 30ºC
Cut the beetroot cylinder to 3cm³ along the along its length. Wash the sample.
Boil 100cm³ in a beaker using a Bunsen burner. Place 5cm³ of the water into the test tubes and place into the waterbath when it has reached target temperature, add the beetroot sample
Start the stop clock and when the timer 1:30 minutes, remove the beetroot and allow 30 minutes to pass before testing.
Whilst waiting for the 30 minutes to pass, increase the set temperature on the waterbath and add the next sample.
Calibrate the colorimeter by taking a reading using distilled water
Place 4cm³ of the sample water into the cuvette. Place with the arrow on the cuvette facing inwards.
Take a reading ensuring the setting is on transition, not absorbance.
Repeat above with samples mixed with beetroot at different temperature.

Results:

n.b: The bold number in the bracket indicates the filter used on the colorimeter. (3) Indicates a green filter whereas (4) indicates a green/blue filter. The red numbers indicate anomalies. I have excluded these from the mean calculations. I have excluded them as they may falsify the averages.

Graph:

Analysis:

Conclusion:

In general my results show a decline in the light transmission, therefore the amount of Anthocyanin present in the in the water as increases as the temperature decreases. The most marked increases in concentration are from 55ºC to 60ºC whereby the light transition fell by 29%. The reason 50ºC had a smaller amount of Anthocyanin present is that maybe the proteins were beginning to be denatured, but that there was not enough k.e to transport it across the membrane. My hypothesis was therefore correct when I predicted that the amount of pigment would increase with increased temperatures.

However the temperature I specified for the increase to occur was too low. In my hypothesis I had predicted that the denaturation of proteins in the bi-lipid was to be completed by 70ºC at 80ºC Anthocyanin was still leaving the vacuole. This is to due to the greater k.e of the Anthocyanin molecules and of the vacuole membrane. With the evidence from the T-test showing that between 55ºC and 60ºC light transmission decreased doubled, I can now reject my null hypothesis as the results did indeed show that the amount of pigment did increase with increased temperatures

The denaturation process appears to start at around 45ºC, at the temperature that I predicted. I based the denaturation process on the denaturation of enzymes in animal cells. Therefore I expected there to be several anomalies. I think that around 70ºC the denaturation of the proteins was fully completed, as the light transition difference is very small.

Evaluation:

There are several large issues to take into consideration when considering the validity of the data collected in this experiment. The most major of the issues is that we are working under the assumption that all cells in the beetroot have the same amount of Anthocyanin contained in them. Those samples taken from the edges of the beetroot for example may not have the same Anthocyanin content as those from the centre. However, to avoid this issue to a degree, all the samples I used in my experiment were taken from the same beetroot.

As the plan presented here is not the plan used in the final experiment, the differences between the methods may have altered the findings. In my experiment I planned to use a water bath, however in the plan I was given it required the use of a Bunsen burner to heat the water. This meant that the water might not have been kept at a constant temperature. This would have invalidated my findings and falsified my conclusions.

Another issue is that the cuvettes that have been used may have been old or stained. This may of given the impression of more Anthocyanin present than there actually was as some of the light may have been blocked by the stains on the cuvettes.

Also as collective members doing the same experiment as me have pooled the results, there may have been discrepancies between the methods. As different people used different apparatus, a faulty piece of apparatus can force me to reject their results. This would not be the case if all the experiments had conducted using the same pieces of apparatus, as all the results would have the same characteristics