Effect of temperature on membranes

Active transport involves a carrier protein as energy from ATP is used to change the shape of the carrier to bind to the specific molecules, release the transported molecules and return it to its original shape to transport more molecules. The breakdown of ATP into adenosine diphosphate is catalyzed by the enzyme ATPase. Energy is released by the breaking of the bond between the adenosine diphosphate and a phosphate group.

Beetroot

Image 2: A bunch of fresh beetroots

Beetroot is commonly known as table beet, garden beet, red beet and botanically-known as Beta vulgaris (a cultivated variety of beetroot). The wild seabeet which it origins from is a native of coastlines from India to Britain and is the ancestor of all cultivated forms of beet. Sea beet was first domesticated in the eastern Mediterranean and Middle East – although it was only the leaves that were eaten at that time. The beetroot is known for many health benefits and has been used in traditional medicine to treat a variety of ailments for a long time. It is claimed that the beetroot aids tissue growth, lowers blood pressure, reduces risk of osteoporosis, prevents certain cancers, lowers cholesterol, is high in powerful antioxidants and is able to treat anaemia and fatigue.

The beetroot has a spherical shape with a cylinder stalk on the top of it and is available in many varieties. Beetroot provides a rich source of carbohydrates, protein, and has high levels of important vitamins, minerals and micronutrients. Beetroot is also a great source of, magnesium, potassium, iron folic, acid, zinc, calcium, phosphorus, sodium, niacin, biotin, betanin and beta-carotene. It also contains the important vitamins such as vitamins A, B6 and C, antioxidants and fibres.

The red pigment contained in beetroots which is known as betalain is what gives the beetroot its dark red colour. Betalain is a group of nitrogen containing pigment that is normally yellow, orange, pink, red and purple of which the most studied pigment is a red pigment known as betanin. Unlike antocyanins, betalain are not pH indicators and do not change colour when the pH changes. In the beetroot cells, betalain are usually stored in the vacuole and thus serve as a marker for researchers who intend to isolate vacuoles as the red colourings make it easier to distinguish the vacuoles from the other contents of the cells. The red-violet vesicles obtained from the protoplasts of beetroots are intact beet vacuoles. In order for betalain pigment to exit the cell, the pigment molecules would have to pass through two different membranes which are the tonoplast and the cell surface membrane. To obtain the pigment, these membranes must be disrupted. In living tissue of beetroot, a leakage of the betalain pigment can occur if the tissue is treated with high heat, acid or solvents such as ethanol. Freezing beetroot tissues kills them and afterwards the pigments will leak out. The rate of pigment leakage depends on the amount of surface area of the cut beetroot exposed. Thinner slices have a larger surface area thus speeding up pigment leakage.

Betalains are alkaloid pigments that are found in some families of plants belonging only to the order Caryophyllales. Betalains are not found in plants containing anthocyanin pigments as they are not related structurally. Betalains have also been found in some fungi. Betalains can be divided into betacyanins and betaxanthins based upon their molecular structure. These pigment found in beetroot is used commercially as a food dye to its wide availability, hence cheap cost and that it is a natural source of food colouring and thus, has no known side-effects.

Spectrophotometer

A spectrophotometer is used in the study of spectrophotometry. Spectrophotometry is the quantifiable study of electromagnetic spectra. A spectrophotometer consists of two instruments which are the spectrometer to produce light of any selected colour (wavelength) and a photometer for measuring the intensity of light. These two instruments are arranged in such that a liquid in a cuvette can be placed between the beam of light from the spectrometer and the receiving end that is the photometer to form a spectrophotometer. The amount of light that passes through the liquid in the cuvette is then measured by the photometer. A voltage signal is sent from the photometer to a display device. The magnitude of this signal is dependent on the amount of light that passes through the liquid in the cuvette.

One of the most common uses of a spectrophotometer is the measurement of light absorption. If intensity of colour is linked to the concentration of a substance in solution, then that concentration of that substance can be measured by determining the extent of absorption of light at an appropriate wavelength.

Image 3: Spectrophotometer

Problem Statement:

What is the effect of temperature on the permeability of cell membrane?

Hypothesis:

As temperature increase, the absorbance reading increases indicating a greater permeability of the cell membrane

Variables:

Manipulated variable: Temperature

Responding variable: Absorbance reading which indicates the permeability of cell membrane

Constant variable: pH of solution, size and surface area of the beetroot, amount of distilled water used in the boiling tube and time left in the water bath

Materials:

Beetroot, distilled water

Apparatus:

Cork borer, white tile, ruler, spectrophotometer, cuvettes, KimWipes, stopwatch, water baths of different temperatures, thermometer, boiling tubes, boiling tube rack, measuring cylinder, forceps, tongs, knife, 250ml beaker  

Technique:

Sections of the beetroot were immersed in 5cm3 of distilled water in different temperatures for 30 minutes. The beetroot sections were then removed and the surrounding solutions are measured for their absorbencies using a spectrophotometer.

Procedure:

1. Sections or a single beetroot are cut using a cork borer. Seven 1cm length slices from those sections are measured out using a ruler and cut using a knife on a white tile.
2. The sections are placed in a beaker of distilled water to wash away the excess dye.
3. 5cm3 of distilled water is measured out using a measuring cylinder and poured into seven boiling tubes.
4. The boiling tubes are placed into water baths at 0°C, 24°C, 35°C, 45°C, 55°C and 65°C for 5 minutes until the distilled water in the boiling tube reaches the required temperature.
5. A section of beetroot is immersed into each of the boiling tubes.
6. The stopwatch is activated. The boiling tubes are left in each water bath for 30 minutes.
7. The boiling tubes are then removed from their respective water baths and the beetroot sections are removed. The boiling tubes are shaken to disperse the dye in the solution.
8. The spectrophotometer is switched on and the setting to read absorbance is chosen.
9. Distilled water is poured into a cuvette until it is ¾ filled.
10. The cuvette is placed into the spectrophotometer and the spectrophometer is adjusted to read zero absorbance for clear water.
11. The dye solution in the water bath at 0°C is poured into a cuvette until it is ¾ filled.
12. The cuvette is placed into the spectrophotometer and the reading for the absorbency is taken. This step is repeated two times and the average of the results is obtained.
13. Steps 11 and 12 are repeated for dye solutions in the water bath at  24°C, 35°C, 45°C, 55°C and 65°C using a clean cuvette for each solution.
14. The results are tabulated and a graph of absorbance reading against temperature is plotted (Graph 1).

Results:

Table 1: Absorbance readings of dye solutions in water baths of different temperatures

Graph 1: Graph of absorbance reading against temperature

Discussion:

        From Table 1 and Graph 1, it can be observed that as the temperature increases, the percentage absorbency increases. The intensity of the red dye is measured using spectrophotometer. The reading of the spectrophotometer will be higher if the intensity of the red dye is higher. A higher reading shows that more pigment particles are present in the solutions surrounding the beetroot sections which reflect the permeability of the cell membrane.

        According to the data obtained the absorbance readings of the dye solutions increase as the temperature increases. This shows a positive correlation between the two variables. From Graph 1, it is noticed that the graph can be divided into 3 sections. Firstly, there is a gradual increase in absorbance reading as the temperature increases from 0°C to 45°C followed by a sharp increase in the absorbance reading of the dye solutions between 45°C and 55°C. After 55°C, the absorbance reading increases slightly to reach the maximum absorbance reading obtained which is 0.403AU.

        In the first section of Graph 1, the absorbance reading increases gradually as the temperature increases. As soon as the sample of beetroot is placed into the distilled water diffusion occurs naturally, which is the net movement of molecules from a region of high concentration in the (beetroot sections) to a region of low concentration (distilled water surrounding beetroot sections) and the red pigment from the beetroot cells colours the distilled water red. At 0°C, the absorbance reading is 0.010AU and at 45°C, the absorbance reading is 0.119AU. In this range of temperature, the absorbance reading is relatively small indicating little leakage of the red pigment molecules. This indicates that the cell membrane is still intact. The phospholipid bilayer which makes up the cell membrane is not too fluid and the transport proteins in the bilayer are still functioning. The 3D shape of the protein which is the key for the functionality of the protein is unchanged. More pigment leaks out as the temperature increases as the rate of diffusion is affected by temperature. As the temperature increases, the rate of diffusion increases. The molecules in the cell possess more kinetic energy and move faster through the cell membrane through simple diffusion and facilitated diffusion.

        In the second section of Graph 1, between 45°C and 55°C, there is higher pigment leakage. At this temperature range, the phospholipid layer is disrupted as the increase in temperature causes the phospholipids to move further apart. This may create gaps in the phospholipid layer that separates the contents of the cell and the solution surrounding the beetroot sections. The cell membrane is no longer intact.

        

        Besides that, at this temperature range, the bonds important in holding the polypeptide chains together in forming the 3D structure of a protein are weakened and eventually broken as the temperature increases over 40°C. As temperature rises, the molecules in the molecule begin to vibrate more and more as the heat provides more energy, when heat reaches a certain extent they vibrate so violently that the bonds are broken. As the bonds are broken, the 3D shape of the protein is altered and the specific shape of the active site is lost causing the transport proteins in the bilayer to lose their function. The protein is said to have been denatured. When the proteins in the bilayer undergo denaturation, gaps form in the cell membrane and destroy the delicate its delicate structure. The cell membrane is no longer intact and this allows the more red pigment molecules from to spill out from the cells through the increasingly porous cell membrane. The red pigment colours the clear distilled water red with higher intensity as the temperature increases.

        

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Evaluation:

        One of the precautions of the experiment is that a cork borer is used to cut sections from the beetroots that diameter of the sections of beetroot is uniformed. For the same reason, a ruler was used to measure the length of the beetroot slice before cutting it with a knife. This ensures that all the beetroot sections immersed in distilled water in the boiling tube are of the same length and width and thus contain same about of pigment molecules, thus causing the absorbance reading obtained to be more accurate. Parallax error was avoided when measuring the beetroot sections on the ruler or using a measuring cylinder by reading the scale with eyes perpendicular to it.

        The beetroot sections were immersed in distilled water after it was cut to remove the excess dye which may have leaked out of the beetroot cells that were damaged by the cutting initially. A tissue paper could have been used to wipe the surface of the beetroot sections dry before immersing placing them into the boiling tube to remove excess liquid to obtain a more accurate result. Another precaution is to use equal amounts of distilled water in the boiling tubes by measuring out 5cm3 using a measuring cylinder before pouring it into the boiling tubes.  This is to ensure the accuracy of the absorbance readings obtained.

        

        The boiling tubes was also placed in their respective water baths for 5 minutes before a section of beetroot was placed in them so that the temperature of the distilled water in the boiling tubes would be the same as the temperature of the water baths it is placed in. The boiling tubes were also shaken before the dye solution was placed into a cuvette to ensure that the dye is evenly distributed throughout the solution.

        The spectrophotometer was calibrated before the absorbance readings of the dye solutions were taken to ensure that the absorbance reading for clear distilled water is 0AU. This was done by inserting a cuvette ¾ filled with distilled water into the spectrophotometer and adjusting the instrument to read zero absorbance. As the spectrophotometer is a highly sensitive instrument, specially designed tissue wipes known as KimWipes were used to wipe the clear sides of the cuvette clean before placing the cuvette into the spectrophotometer. This is to remove liquid or residue on the outer surface of the cuvette that may affect the absorbance reading of the dye solution contained in it. For the same reason, it was ensured that the dye solution in the cuvette does not contain any bubbles. The absorbance reading for each temperature was repeated two times and the average absorbance reading for each temperature was calculated to ensure the reliability of the results obtained.

         

Safety Precautions:

A lab coat should be worn at all times in the lab to prevent clothes from any stains if any spills occur. Throughout the experiment, care must be taken to ensure that beetroot juice does not spill on the skin or clothes as it will stain very badly. Closed shoes should be worn in the lab to provide adequate protection in case of any mishaps.  When cutting the beetroot sections using the knife and cork borer, both these apparatus should be handled with care. Tongs should be used to remove the boiling tube from water baths with higher temperatures to prevent scalding.

Conclusion:

It can be concluded that as the temperature increase, the absorbance reading increases indicating a greater permeability of the cell membrane. The hypothesis is accepted.

References:

Cell membrane

1. GAN W. Y., 2008. Success Biology SPM. Malaysia: Oxford Fajar
2. FULLICK A., 2008. Edexcel AS Biology. China: Pearson
3. Image of lipid bilayer of the cell membrane. [jpeg] Available at:  [Accessed on 3 October 2010]
4. HOH Y.K., Longman AS Level Course in Biology: Core Syllabus Volume 1. Singapore: 2004

Beetroot

1. Image of beetroot. [jpeg] Available at:  [Accessed on 3 October 2010]
2. LoveBeetroot.co.uk. History of Beetroot. Available at:  [Accessed on 3 October 2010]
3. LoveBeetroot.co.uk. Healthy Facts. Available at:  [Accessed on 3 October 2010]
4. BiologyMad.com. Beetroot Pigments. Available at:  [ Accessed on 3 October 2010]

Spectrophotometer

1. Wikipedia. Spectrophotometry. Available at:  [Accessed on 3 October 2010]
2. Ruf. Rice.edu. Spectrophotometry.Available at:  [Accessed on 3 October 2010]
3. Image of a spectrophotometer. [jpeg] Available at:  [Accessed on 3 October 2010]

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