NAME: NADHIRAH RAZANAH BINTI SHAHROL

CLASS: 12 M 12

STUDENT ID: 2011276022

TITLE OF : THE EFFECT OF TEMPERATURE ON EXPERIMENT PERMEABILITY OF PLASMA MEMBRANE DATE OF : 3RD AUGUST 2011EXPERIMENT

NAME OF LECTURER: MR. MANOHARAN

ABSTRACT

This experiments title is The effect of temperature on the permeability of plasma membrane. Our objective for carrying this experiment is to investigate the effect of high temperature, room temperature and lower temperature on the plasma membranes permeability and thus relating it with the effects observed on the structure of the membrane. This experiment is being conducted using beetroot plant as it contains red pigments, known as betalains that is located within the vacuole of the beetroot cell. This will be discussed later on the following topic in this report.

Our experiment begins when we started to prepare six sections of beetroot by using a cork borer which then became cylinder in shape. After they were cuts into the same length that is 1 cm, they were being immersed in boiling tubes containing water bath of different temperatures, which were 5C, 25C, 35C, 45C, 55C and 65C. Soon, they were transferred into distilled water for 15 minutes before about 3 cm of each solution is taken and being transferred into cuvettes, the container that is used to be put into spectrophotometer so that their reading of light absorbency can be measured properly.

We will found out from this experiment that the higher temperature of water bath in which the section was being immersed in, the higher the reading shown on the spectrophotometer will be. So, the highest reading indicates the highest concentration of the solution and also the highest percentage of absorbency. Hence, we can conclude that the higher the temperature, the higher the permeability of the plasma membrane will be.

INTRODUCTIONPlasma MembraneAll living cells, prokaryotic and eukaryotic, have a plasma membrane that encloses their contents and serves as a semi-porous barrier to the outside environment. The membrane acts as a boundary, holding the cell constituents together and keeping other substances from entering. The plasma membrane is permeable to specific molecules, however, and allows nutrients and other essential elements to enter the cell and waste materials to leave the cell. Small molecules, such as oxygen, carbon dioxide, and water, are able to pass freely across the membrane, but the passage of larger molecules, such as amino acids and sugars, is carefully regulated.

According to the accepted current theory, known as thefluid mosaic model, which is proposed by S.G Singer and G. Nicholson1, the plasma membrane is composed of a double layer (bilayer) of lipids, oily substances found in all cells (see Figure 1). Most of the lipids in the bilayer can be more precisely described as phospholipids, that is, lipids that feature a phosphate group at one end of each molecule.

1 S.G Singer and G. Nicholson, from University of California Phospholipids are characteristically hydrophilic("water-loving") at their phosphate ends andhydrophobic("water-fearing") along their lipid tail regions. In each layer of a plasma membrane, the hydrophobic lipid tails are oriented inwards and the hydrophilic phosphate groups are aligned so they face outwards, either toward the aqueous cytosol of the cell or the outside environment. Phospholipids tend to spontaneously aggregate by this mechanism whenever they are exposed to water.Within the phospholipid bilayer of the plasma membrane, many diverse proteins are embedded, while other proteins simply adhere to the surfaces of the bilayer. Some of these proteins, primarily those that are at least partially exposed on the external side of the membrane, have carbohydrates attached to their outer surfaces and are, therefore, referred to asglycoproteins. The positioning of proteins along the plasma membrane is related in part to the organization of the filaments that comprise the cytoskeleton, which help anchor them in place. The arrangement of proteins also involves the hydrophobic and hydrophilic regions found on the surfaces of the proteins: hydrophobic regions associate with the hydrophobic interior of the plasma membrane and hydrophilic regions extend past the surface of the membrane into either the inside of the cell or the outer environment.Plasma membrane proteins function in several different ways. Many of the proteins play a role in the selective transport of certain substances across the phospholipid bilayer, either acting as channels or active transport molecules. Others function as receptors, which bind information-providing molecules, such as hormones, and transmit corresponding signals based on the obtained information to the interior of the cell. Membrane proteins may also exhibit enzymatic activity, catalyzing various reactions related to the plasma membrane. In prokaryotes and plants, the plasma membrane is an inner layer of protection since a rigid cell wall forms the outside boundary for their cells. The cell wall has pores that allow materials to enter and leave the cell, but they are not very selective about what passes through. The plasma membrane, which lines the cell wall, provides the final filter between the cell interior and the environment.22 http://micro.magnet.fsu.edu/cells/plasmamembrane/plasmamembrane.html

Beetroot Also called beet, the beetroot is a firm, clean globe shaped vegetable with no soft wet areas. If still attached, it should have fresh, clean young leaves. The beetroot is characterised by dark purple skin and a distinctive purple flesh. During BC, the leaves of the beetroot were only eaten. Today, the root is used more often than the leaves since it stays fresher longer. Some beetroots are cultivated for distilling and the sugar industry. They are used as vegetable and as food colouring. Containing the powerful antioxidant betalains, which gives beetroot its deep red hue, this vegetable purifies the blood and has anti-carcinogenic properties. Research shows it boosts the bodys natural defence in the liver, regenerating immune cells. Also contains silica, vital for healthy skin, fingernails, ligaments, tendons and bones. Beet is believed to be native of the Mediterranean region of Europe and probably Western Asia. It has been used as vegetable for the last 2000 years, even by early Greeks and Romans. It was so appreciated by ancients that it was offered on silver to Apollo in his temple at Delphi. Beetroot contains sodium, potassium, phosphorus, calcium, iodine, iron, copper, Vitamins B1, B2, B3, B6 and C. Each capsule provides approximately 1- 2 mg of elemental iron.3 3 http://fruitsnvegetables.com/beetroot.htmlThe idea in this experiment is that the pigment is contained within a membrane and when that membrane breaks down it releases the pigment. Membranes are made of protein structures and so the structure is broken down by heat or ph. Take a good look at the membrane structure shown before.The pigments are actually as had been mentioned before, betalains pigments, named after the Red beet (Beta vulgaris). They replace anthocyanins in plants of the order Caryophyllales (Cacti, beets & Co., bougainvillaea, phytolacca, large-flowered purslane etc. and also in some fungi such as fly agaric). Their function is not really known, but it is guessed that when present in the flowers they serve to attract pollinating insects and when present in seeds they may attract birds for the dispersal of the seeds. In cultivated species man may have arbitrarily selected for more coloured lines, because they are attractive to look at (red beet!), or the colour may have co-segregated with another useful trait. (For example, the colour may be genetically linked with a characteristic that improves flavour - by selecting varieties for flavour, man may have also selected for colour by chance.) There is no indication that betalains protect plants against pathogens (fungal, bacterial or viral) or herbivores, and they do not absorb UV light. Unlike anthocyanins, betalains are not pH indicators, i.e. they do not change colour when the pH is lowered. Betalains are in the vacuole. They serve as markers for people who want to isolate intact vacuoles; the red-violet vesicles you can get from protoplasts are intact beet vacuoles. So betalain pigments have to cross 2 membranes. In living tissue (beet slices) you can get leakage by a heat shock, by acid treatment or of course by incubating them in acidified 80 % methanol. Thinner slices have a larger surface, thus speeding up pigment leakage. Putting beet slices in the deep-freeze of course kills them, and afterwards the pigments will leak out. A pH of about 3 - 4 stabilises the pigments and protects against oxidation. Pigment extracts must be protected from direct sun light and should be kept in a cool and dark place.4

4 http://www.studyzones.comBeet pigments are unstable at higher temperature, but the chemistry depends on the pH and composition of the solution, oxygen concentration, how long the solution is boiled for example. Generally, plant extracts are complex, i.e. they contain phenolics and lots of reactive substances in addition to what you are looking at, and they just turn brown (eg at 80C).4Dr Alexander Ferenczi5 has been using raw beets to cure cancer, and nothing else since the late 1950s. The only problem is the same one as you get with heroic injections of vitamin C. If you eat too much raw beetroot, it can kill the cancer faster than your liver can dispose of the waste products. So if you have a large cancer, start with small quantities of beetroot, and gradually increase them until you start to feel unwell, and then back off the amount. You may get a fever if your liver is being asked to work too hard. You could probably get the same results with vitamin C injections, but having to increase the quantity gradually would mean multiple injections, whereas raw beetroot tastes good6. Spectrophotometer

5 Dr Alexander Farenczi, MD of Csoma, Hungary6 http://healthforu.info/health/healthfood/Beets-Health-Food.php Thespectrophotometeris an instrument which measures the amount of light of a specified wavelength which passes through a medium. According to Beer's law, the amount of light absorbed by a medium is proportional to the concentration of the absorbing material or solute present. Thus the concentration of a coloured solute in a solution may be determined in the lab by measuring the absorbency of light at a given wavelength. Wavelength (often abbreviated as lambda) is measured in nm. The spectrophotometer allows selection of a wavelength pass through the solution.Usually, the wavelength chose is the one which corresponds to the absorption maximum of the solute. Absorbency is indicated with a capital A.At the spectrophotometer, you should have two cuvettes in a plastic rack. Solutions which are to be read are poured into cuvettes which are inserted into the machine. One should be marked "B"for the blank and one "S" for your sample.A wipette should be available to polish them before insertion into the cuvette chamber.Cuvettes are carefully manufactured for their optical uniformity and are quite expensive. They should be handled with care so that they do not get scratched, and stored separate from standard test tubes. Try not to touch them except at the top of the tube to prevent finger smudges which alter the reading. For experiments in which minor imprecision is acceptable, clean, unscratched 13 x 100 mm test tubes may be used7.An example of an experiment in which spectrophotometry is used is the determination of the equilibrium constant of a solution. A certain chemical reaction within a solution may occur in a forward and reverse direction where reactants form products and products break down into reactants. At some point, this chemical reaction will reach a point of balance called an equilibrium point. In order to determine the respective concentrations of reactants and products at this point, the light transmittance of the solution can be tested using spectrophotometry. The amount of light that passes through the solution is indicative of the concentration of certain chemicals that do not allow light to pass through.87 biology.clc.uc.edu/fankhauser/Labs/.../Spectrophotometer.htm8 http://www.answers.com/topic/spectrophotometryPROBLEM STATEMENTWhat is the effect of different temperatures on the permeability of the plasma membrane?OBJECTIVEAt the end of the experiment, I have learnt :1. How to use beetroot to examine the effect of temperature on the cell membranes and relate the effects observed to the membrane structure. 2. A better understanding on the structure and characteristics of cell membranes.3. How the membrane structure will change under different conditions.4. How to use a cork borer in taking out the cylinder shape of beetroot.5. About the importance of spectrophotometer and how to handle it.6. That the cuvette must be in a perfect condition because even a single scratch on it could affect the result greatly.AIMTo determine the effect of varying temperature on the membrane of the beetroot cell.EXPERIMENTAL HYPOTHESISTemperature or heat has a significant effect on the quantity of pigment that diffuse out of beetroot cell.NULL HYPOTHESISTemperature or heat has no significant effect on the permeability of beetroot cell surface membranes. The permeability is the same irrespective of the temperature.

VARIABLES

VariableWays of handling

Manipulated variable :Temperature of treatmentDiffuse the beetroot in variety temperature of water bath.

Responding variable :Intensity of pigmentMeasure the concentration of distilled water after being immersed by beetroot cylinder using spectrophotometer.

Constant variable : Type of plant used

Diameter of 6 beetroot cylinder Length of 6 beetroot cylinder

Volume of distilled water in the test tubes

Time used to immerse beetroot cylinder in water bath Time used to immerse beetroot cylinder in distilled water Type of spectrophotometer For every temperature, the same plant is used that is beetroot. Every section of beetroot is taken out using the same cork borer. Every section of beetroot is cut into the same length that is 1 cm. Knife is used to cut and ruler is used to measure. 15 ml of distilled water is used for each test tube. Volume of water is measured using measuring cylinder.

For every temperature of water bath, time used to immerse the beetroot is 1 minute.

Beetroot cylinders are then being transferred into test tubes containing same amount of distilled water. They are immersed for 15 minutes. The reading is taken using the same spectrophotometer.

APPARATUS AND MATERIALS

Six test tubes labelled 5C, 25C, 35C, 45C, 55C and 65C, cork borer, spectrometer, white tile, knife, ruler, forceps, beakers, boiling tube rack, boiling tubes, thermometer, spectrophotometer, plastic cuvettes, water bath, stopwatch, small measuring cylinder, distilled water, water bath at 5C, 25C, 35C, 45C, 55C and 65C.

PROCEDURES

1. A few sections are cut from a single beetroot using a cork borer. Six slices of 1 cm length are cut from these sections. 2. Six labelled test tubes each containing 15 ml of distilled water are placed on test tube rack.3. Beetroot cylinders are being immersed into water baths at 5 C, 25 C, 35 C, 45 C, 55 C and 65 C for 1 minute. Each beetroot is immersed in water bath of temperature 35 C, 45 C, 55 C and 65 C, using boiling tube. As to get 25 C in temperature, a small cube of ice is put into a beaker containing water bath, and the beetroot is immersed in it. Whereas in order to get 5 C, a lot of ice cubes are used to be put into water bath in a beaker. 4. After that 1 minute, each beetroot is transferred into six test tubes containing distilled water which have already been labelled according to the temperature tested on it. They are left there for 15 minutes. 5. Next, the spectrophotometer is switched on. It is set to read % absorbance.6. The spectrophotometer is adjusted to read zero absorbance for clear water. The setting must not be altered again during the experiment.7. 3 cm of the dye solution is placed into a spectrophotometer cuvette and the reading for absorbency is taken. The readings are taken for all the temperatures.

SAFETY MEASURES & RISK ASSESSMENT

1. Lab coat should be wear throughout the experiment. Careful not to spill beetroot juice on skin or clothing as it will stain very badly. Wear a lab coat throughout the experiment. Care should be taken so as not to spill the beetroot juice on the skin or clothing as it will stain very badly.2. As to avoid scalding, be careful when working with hot water.3. Be careful when using the knife to cut the beetroot and a cork borer to take out the beetroot cylinder. An egg slicer or a mini plastic mitre block can be used to replace knife to minimize the risk of getting cuts.4. All fragile glassware must be handle with care so that they are not broken. 5. When handling hot boiling tubes use forceps instead of bare hands.6. Students have to avoid wearing slippers or sandals in the lab when using glassware or chemicals.Students should never go barefoot in the laboratory. Instead of all that, they should wear fully-covered shoes as a prevention from any spillage of chemicals or breaking of apparatus. 7. Work area should be cleared of any materials that are not needed for performing the experiment. This is to provide an ample space for students to move about and hence, reduce chances of sustaining any injuries.8. Listen to the lecturers instruction before using the spectrophotometer so that students can use it in a correct way.

RESULTSTreatment temperature of beetroot / CAbsorbency / %

50.030

250.047

350.067

450.159

550.494

650.607

Graph of percentage absorbance (%) against temperature (C)

DISCUSSIONIt can be seen from the result that during the increment of the temperature of water bath, the absorbency percentage of the dye solution also increases. This means that the permeability of the beetroot cell surface membrane increases when the temperature of the water bath increases. This event causes more betalains leak out from the beetroot and thus, a higher concentration of dye solution is produced. In finding of the true reason behind all this, we must know the structure of cell membrane very well. A lot of different kind of molecules present in it for example, cholesterol, phospholipids, proteins, glycolipids, and glycoproteins. These molecules act as an aid in maintaining the cell membranes normal function. The property that makes it very difficult for polar molecules and ions to pass through the membrane is because of the hydrophobic fatty acid tails of phospholipids. Hence, the place where water-soluble molecules normally choose to pass through the membrane is via protein. Temperature does strongly affect the proteins present in the cell membrane. The shape of the proteins could be permanently changed as the temperature of the surroundings increase, which then leads them to become denatured. This thus makes the cytoplasm and other substances in the membrane to leak out from beetroot cylinder. Hence, the steady increment in the amount of leakage of the red pigments from the cells as the temperature increase causes the increase in the solutions concentration. More protein would change in shape as the temperature increase has become the reason why the absorbance percentage increases when the beetroot is being heated at an increasingly high temperature. This is parallel to the research that I have done which is stated in page 6. At low temperatures which are 5 C and 25 C, the shape of membranes protein does not change so much thus the leakage also did not occur much. Apart from that, the temperatures that need to be heated that are 35 C, 45 C, 55 C, and 65 C, denaturation of protein occurs more thus causes a higher diffusion rate of red pigments into distilled water. Hence, this explains the higher reading in spectrophotometer which remarks the higher percentage absorbency for those solutions of these temperatures. In addition, there is another explanation for this. For the membrane, there is a limit in temperature which is supposedly can be withstand. As long as the temperature reached is within the limit, the permeability of membrane should be fine and not be affected. However, when this limit is exceeded, water within the beetroot will expand, exerting a high pressure on the membrane. The higher the temperature, the more the molecules in the beetroot cell vibrate and thus destroy the structure of the beetroot cell surface membrane, resulting in a leakage of beetroot juice into the distilled water. These results are reliable as they have been compared with other classmates, but it might be more accurate if I could take an average reading within a limited amount of time. However, some errors that might occur during the experiment is that I havent rinsed the beetroot after taking out the cylinder using cork borer and even after being cut into same length for all sections. These might affect the result as there will be excess dye to the solution because of this, causing the reading to be inaccurate. Other than that, the age of beetroot that is already old might not be a good one to be used in the experiment as their membrane is already not strong enough to withstand temperature thus pigment will leaks out easily.

.

CONCLUSIONThe result shows that the higher the temperature of the water bath in which the beetroot is being immersed in, the higher the permeability of the beetroot cell surface membrane. Temperature does have significant effect on the permeability of plasma membrane. The experimental hypothesis is accepted.

LIMITATIONS / ANOMALOUS RESULTSOne of the errors that might affect the accuracy of the results is that the beetroot sections used are not of the same size or length. It is because the length of the sections is not measured correctly before cutting, resulting in some sections to be longer or shorter than the others. Besides, the surface area of the beetroot sections might not be the same due to carelessness while cutting the beetroot. Some are being cut straight while some are being cut with an angle, which means that the surface is a bit slant. Besides that, the surface area for beetroot that is being cut into cylinder using cork borer might be small. Apart from that, an old beetroot is also not good for the experiment. These cause the results to be less accurate and reliable. Besides that, the volume of the distilled water in which the beetroot sections are placed might be different. If this happens, the concentration of the pigment will be greatly altered, causing a failure in comparing the results accurately and drawing a fair conclusion. Apart from that, the results will also be affected if the beetroot sections are not washed out before putting it in water treatment. And if being washed, some beetroot cylinders are washed less than others. This may cause excess dye to remain in the sections, which will change the concentration of the solution later on. Moreover, another error is that the dye is not dispersed evenly in the solution, causing some area of the solution to be more concentrated than other areas, influencing the accuracy of the readings of the colorimeter.In addition, there may be fingerprints or scratch on the clear side of the cuvettes caused by improper cleaning before putting the cuvettes into the spectrophotometer. The prints or scratch may absorb light itself, leading to inaccurate readings on the spectrophotometer and making the results unreliable. The results may also be affected if there are scratches on the cuvettes. And last but not least, the reading from spectrophotometer is only taken once. Every machine does not always give the accurate result straight on spot. If this occurs, then it means we cannot rely on the result.

SUGGESTIONS FOR IMPROVEMENT

To improve the accuracy of this experiment, the sections used ought to be of the same size. This can be done by using the same-sized cork borer when cutting the beetroot. Besides that, the person conducting the experiment has to be precise and meticulous when cutting the beetroot into sections to produce sections of the same length as well as surface area. Apart from that, to be more accurate, maybe we can cut it into cubes instead of cylinder and the beetroot used should be fresh enough.Next, the volume of water that the beetroot sections are placed should be the same for each sets of the experiment. The measuring cylinder should be used to measure the exact amount of water to ensure accuracy. The volume of water has to be measured from where the meniscus sits. In other words, our eye vision must be perpendicular to the scale of the measuring cylinder to avoid parallax error. The beetroot sections should be washed, and in the same amount of time. They must be rinsed in the water and be removed at the same time, too. The beetroot sections need to be kept in the water baths for the same amount of time too, which is 30 minutes. To overcome the problem of uneven dispersion of dye in the solutions, the solutions ought to be stirred using a glass rod first before they are being placed in the cuvettes for readings to be taken.Next, the smooth side of the cuvettes must be cleaned or wiped using wipette before inserting them into the spectrophotometer. This is to clear the cuvettes of any finger prints that may affect the light absorbency. Do not label or clean the cuvettes using brush as these actions will form scratches on the surface of the cuvettes.Lastly, the reading should be taken three times and then calculate the average out of it to increase the accuracy out of it. Sometimes, we do not have enough time to do that so, one other way is that we can compare our result with other classmates or partners.

FURTHER WORK1) Experiment on the effect of different concentration of alcohol on plasma membrane.2) Experiment on the effect of pressure on plasma membrane of beetroot plant. BIBLIOGRAPHYBooks:Edexcel AS Biology, 2008, Ann Fullick, Pearson Education Limited

Websites: Plasma membrane, http://micro.magnet.fsu.edu/cells/plasmamembrane/plasmamembrane.html

Beetroot, http://fruitsnvegetables.com/beetroot.html http://healthforu.info/health/healthfood/Beets-Health-Food.phphttp://www.studyzones.com

Spectrophotometer, biology.clc.uc.edu/fankhauser/Labs/.../Spectrophotometer.htm http://www.answers.com/topic/spectrophotometry

( 4317 words)

[Type text]Page 4

15