Title

: The effect of temperature on membranes

Name Date Class

: Yii Seng Ong : 15 August 2011 : 12M15

Student ID : 2011200378 Name of lecturer : Madam Ida Muryany binti Md. Yasin

Objective The purpose of this practical is to use beetroot to examine the effect of temperature on permeability of cell membrane and relate the effects observed to membrane structure. This practical also aimed at highlighting experimental and investigative skills of students. Introduction 1. Beetroot Beetroot, also known as the table beet, garden beet, red beet or informally simply as beet, is one of the many cultivated varieties of beets and arguably the most commonly encountered variety in North America, Central America and Britain. Beetroot contains betalains. Betalains are the red pigments of beetroot, Beta vulgaris. Betalains are made up of red betacyanins and yellow betaxanthins. Betacyanines include about 90% of beetroot betalaines. The betalains are watersoluble and exist as internal salts in the sap vacuoles of plant cells and, by means of the properties of the tonoplast and cell membrane, does not leak into the cytosol or the extra-cellular sap of the beetroot. In order for the betalain to leave the cell, it needs to pass through two different membranes: the membrane bounding the vacuole and the membrane enclosing the cell. When beetroot is cut, the cells are sliced and naturally red pigment spills out. If the slices were thinner, thus providing a larger surface area it therefore speeds up the pigment leakage. But if the membrane is destroyed and the phospholipid bilayer and possibly proteins are altered, more red pigment, betalains leaks out by means of diffusion. The red pigment serves as a marker for scientists who want to isolate intact vacuoles.

Figure 1 : Raw beetroot which is the main material used in this experiment In this experiment, the effect of temperature on the permeability of plasma membrane is being investigated. By using the cell of the beetroot, we can experimentally investigate the approximate temperature at which the proteins are

serious denatured. It is because once the membrane proteins have been denatured, the integrity of the lipid bilayer of the plasma membrane may also be compromised. Escape of the vacuole contents will indicate this has happened. In this experiment, the cells of beetroot contain an intensely red, watersoluble pigment in their interior. It is found in the large central vacuole of each cell. The pigment may escape in sufficient quantities to be detected in the aqueous medium around the tissue. This occurs if harmful external conditions for example when heat are applied to the beetroot tissue. 2. Spectrophotometer

Figure 2 : Spectrophotometer which is the main apparatus used in this experiment

A spectrophotometer is a photometer (a device for measuring light intensity) that can measure intensity as a function of the light source wavelength. Important features of spectrophotometers are spectral bandwidth and linear range of absorption or reflectance measurement. A spectrophotometer is commonly used for the measurement of transmittance or reflectance of solutions, transparent or opaque solids, such as polished glass, or gases. The light transmittance of a solution can be tested using spectrophotometer. In this experiment, a spectrophotometer is used to investigate the effect of temperature on permeability of membrane by measuring the absorbency of red pigment escaped from the beetroot section in a solution. The amount of light that passes through the solution is indicative of the concentration of red pigments that do not allow light to pass through. The spectrophotometer is precise, reliable and accurate compared to a natural eye test comparison or a colour chart comparison.

3. Function and structure of cell membrane Cell membrane is a biological plasma membrane that separates the intracellular components of the cell from the extracellular environment. It also

separate compartments inside the cell to protect important processes and events. The cell membrane is a semi-permeable membrane which is selectively-permeable to ions and organic molecules and able to regulate the movement of substances in and out of cells, thus facilitating the transport of materials needed for survival. The movement of substances across the cell membrane can be either active or passive. It is important in maintaining a homeostatic environment in the cell to keep us healthy and alive. The plasma membrane also serves as the attachment surface for the extracellular matrix and other cells to help grouping cells together to form tissue. It also plays a role in anchoring the cytoskeleton to provide shape to the cell.

Figure 3 : fluid mosaic model of cell membrane of S.J. Singer and Garth Nicolson 1972 According to the fluid mosaic model of S. J. Singer and Garth Nicolson 1972, the biological membranes can be considered as a two-dimensional liquid where all lipid and protein molecules diffuse more or less easily. The cell membrane consists primarily of a thin layer of amphipathic phospholipids which have hydrophobic tail and hydrophilic head. They are spontaneously arranged so that the hydrophobic "tail" facing each other and are protected from the surrounding polar fluid while the hydrophilic "head" regions to associate with the cytosolic and extracellular faces of the resulting bilayer. This forms a continuous, spherical phospholipid bilayer. The arrangement of hydrophilic heads and hydrophobic tails of the phospholipid bilayer generally allows for the passive diffusion of hydrophobic molecules but prevents polar solutes such as amino acids, nucleic acids, carbohydrates, proteins, and ions from diffusing across the membrane. Proteins also can be found embedded in the bilayer. They may pass through the bilayer as transmembrane protein or they may be inserted at the cytoplasmic or exterior face. The proteins that are on the outer edge or on the surface of the membrane have carbohydrates molecules attached, usually short sugar chains. These are called glycoproteins. The carbohydrates part of the glycoproteins is important in cell recognition, which is the ability of cells in the body to tell whether or not a cell is from another individual or another organism (invading pathogens), for example, in the immune system. Some substances, particularly carbohydrates and ions, are transported across the membrane via the proteins. Whereas some substances,

including water, are transported directly through the lipid layer. The presence of proteins such as transmembrane protein complexes affords the cell membrane the ability to regulate the movement of substances across the membrane.

Figure 4: model of cell membrane

4. Permeability of membrane The permeability of a membrane is the ease of molecules to pass through it. Permeability depends mainly on the electric charge of the molecule and to a lesser extent the molar mass of the molecule. Electrically neutral and small molecules pass the membrane easier than charged, large ones. However, the permeability of plasma membrane can be altered due to various reasons and one of it is temperature which is experimented in this experiment.

Problem statement What is the effect of temperature on permeability of beetroot cell membrane? Hypothesis The permeability of cell membrane increases as the temperature increases. Variables

Variables Manipulated variable:

How the variables are conducted

Temperature

The boiling tubes that contain beetroot sections are submerged separately in water baths with different temperature : in water bath of room temperature that is 25 oC , in water bath of 35 oC, 45 oC, 55oC and 65 oC.

Responding variable: The reading of colorimeter ( The unit of absorbency of red pigment) Constant variable: Time taken for the beetroot to submerge in the water bath of different temperatures. Apparatus Size 4 cork borer, white tile, knife, ruler, 250 cm3 of plastic beaker, forceps , boiling tube rack, boiling tubes, thermometer ( one per water bath), cuvettes, stopwatch, distilled water, small measuring cylinders, waterproof marking pen, pipette for measuring 2 cm3, spectrophotometer Materials Raw beetroot, water baths at 25 oC, 35 oC, 45 oC, 55oC and 65 oC, distilled water Pipette 2cm3 of water in the boiling tube, which has become red in colour due to beetroot pigment escaped from the tissue, into a cuvette. After placing the cuvette into colorimeter, read and record the reading shown.

During the experiment, by using a stopwatch, make sure that all the beetroot sections are submerged in their respective water bath of different temperatures for exactly 1 minute.

Procedure 1. A single beetroot section is prepared using a size 4 cork borer. Five sections of beetroot with a uniform length of 1cm are then cut from that single section. 2. Five labelled boiling tubes each containing 5cm3 distilled water are placed into water baths at 25 oC, 35 oC, 45 oC, 55oC and 65 oC. Those boiling tubes are left for 5 minutes until the distilled water inside each boiling tubes reaches the required temperature. 3. All the five beetroot sections are placed into each boiling tubes respectively and then they are left for 30 minutes in the water bath. 4. The beetroot sections are removed and the tubes are shaked to disperse the dye in the water. 5. 2 cm3 of distilled water is transferred to a cuvette suing a pipette. The cuvette is then placed into the spectrophotometer, making sure that the lights shines through the smooth sides. 6. The spectrophotometer is adjusted to read zero absorbance for clear water. The setting is not altered during the experiment. 7. The dye solution from boiling tube labelled 25 oC is transferred into a cuvette and placed into the spectrophotometer. The reading of absorbency is taken for three times to have the average reading. 8. Step 7 is repeated for other temperatures. 9. All the data recorded is tabulated. 10. A graph of unit of absorbency of red pigment against temperature of water bath is plotted.

Results Temperature of water bath/ oC 25 35 45 55 1 0.138 0.141 0.238 0.424 Absorbency unit /A 2 3 0.137 0.137 0.142 0.239 0.423 0.142 0.237 0.424 mean 0.137 0.142 0.238 0.424

65 0.469 0.469 0.469 0.469 Table 1 : Data collected from spectrophotometer in respective to different Temperature of water bath

0.5 0.45 0.4

0.469 0.424

Absorbency unit of red pigment/A

0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 25 35 45 55 65 0.137 0.142 0.238

Temperarure of water

bath/oC

Graph 1 : Graph of absorbency unit of red pigment against temperature of water bath

Discussion The objective of this experiment is to find out what is the effect o temperature to permeability of cell membrane of beetroot. So, in order to do this , the surrounding temperature or the temperature of water bath is modified. So, when we alter the temperature, it will done something to the plasma membrane of beetroot. Based on this experiment, we can see that the colour intensity of red dye is increasing or become darker. The basic principle of this experiment is, when we heat the water bath to certain temperature, the kinetic energy of water molecule will become higher. This kinetic energy from the water will also transferred into the water molecule in the vacuole as it is in contact. After as the kinetic energy increase, the molecule will move faster and they will collide more frequent with each other. Until a certain stage, these high energy particles will collide with the cell membrane or phospholipid bilayer and thus

penetrate the layer. As the result, the red stain particle called betalain will leak out to the outer of the beetroot cell. Table 1 shows the result obtained when boiling tube is immersed in various temperature and then left for 15 minutes. Table 1 also show the general trend of data with increasing temperature. The same thing also goes with the graph. In Graph 1, we can see that the reading of spectrophotometer increase as the temperature increase. In order to understand this trend of data we should understand the basic principle in this experiment. The spectrophotometer is used to measure the percentage absorbance of light by the solutions where the beetroot cylinders are immersed into after the heat treatment. The higher the spectrophotometer reading brings about a higher percentage absorbance and so the higher is the amount of red dye present in the solution because there is more red dye in the solution to absorb the light transmitted from the spectrophotometer. The increase in amount of red dye in the solution is related to the increase of the permeability of beetroot membrane. Hence, an increase in the reading of spectrophotometer indicates that the permeability of the beetroot membrane increases. It is accurately drawn in the graph that when the beetroot cylinder is immersed into distilled water at room temperature which is 25 oC, the spectrophotometer shows the reading of 0.137 A. At this temperature, the colour of stain is not so visible. Only pale pink colour visible at this temperature. Presumable, we understand that, the betalain leak out from the cell membrane or phospholipid bilayer when the plasma membrane is denatured or the plasma protein damage. But then, why there is also the pale pink colour at this room temperature. Why do the pigments also being released when the beetroot is at room temperature? This may be due to the pigments leakage when we wash the beetroot before the experiment. The leakage is not only occurs in once, but it may be continuously leak at once we start the experiment. For the next temperature, there is no much high increase in spectrophotometer reading. When the temperature of water bath is 35 oC, the reading of spectrophotometer is 0.142A. Similarly, when the temperature of water is 45 oC, the reading of spectrophotometer is 0.238A. This reading only show a slight increase compared to the previous one. There is no significant change when the temperature is increase by 10 oC, but the increase is also noticeable and can be considered. But, when the temperature is increase more, that is from 45 oC to 55 oC, there is a sudden and drastic change to the spectrophotometer reading , that is from 0.238A to 0.424A although the temperature is also increase at a constant rate that is 10 oC also. Why did this thing occur? Probably, the assumption that we can make about this result is, when the temperature of water bath increase beyond 45 oC, the membrane proteins in the cell membrane start to break entirely and expelled all the substance inside the cell, especially betalain, the red pigment. To relate this statement to a matter of fact, the below explanations about working principle of phospholipid bilayer should be considered.

In order to function correctly, the cells should be able to control transport across partially permeable cell surface membrane. So normally, betalain in the cell vacuole cannot pass through that membrane. In addition, betalains also should pass through two layers of membranes. One is from the membrane that bounding the vacuole and the other is the membrane that enclosing the cell.

Diagram 5: Effect of temperature on the packing of the hydrocarbons

At normal room temperature, the phospholipid bilayer is in a gel state and tightly packed (refer to Diagram 5). At higher temperatures, the bilayer actually melts and the interior is fluid allowing the lipid molecules to move around, rotate, exchange places. As more phospholipids move around, more betalains are able to leak out. The cellulose cell wall on the outside of the cell is fully permeable and so no barrier to the egress of the pigment. Consequently at higher temperature, the red dye in the distilled water where the beetroot cylinder is placed is more concentrated. From 25-45 oC, the temperature increase gradually and steadily, which this means that the absorbency unit increases slowly. This shows that there is only a small amount of betalains present in the distilled water after it is immersed in that range of water bath. This increase is not so significant because 45 oC is not high enough to break the bond and the order of arrangement of phospholipid bilayer. The increase in heat energy only increases the kinetic energy of the phospholipid molecules to move around, rotate and exchange places which at the same time increases the rate of random movement of betalains out through the cell membrane

From 45-65 oC, there is a sharp and sudden increase in the reading of spectrophotometer which can be shown by the steep climb on the Graph 1. It means that the amount of betalains suddenly increases and the percentage absorbance is also increases. At this temperature actually, most of enzymes are denatured and the same thing goes with plasma membrane. Besides, the permeability of the cell membrane is even higher due to more and more plasma proteins have been denatured, in addition to the melting of the bilayer because of the violent movement of the phospholipid molecules. By exposing a cell membrane to alcohol, it will affect its permeability. Therefore, an increase in alcohol concentration is accompanied with an increase of permeability of cell membrane. But by increasing the concentration of alcohol beyond a certain point it may have no more effect on the cell membrane because the alcohol would have broken down the cell membrane completely. This is due to the effect of alcohol on the lipids in the membrane. Lipids are essential to the structure of the cell membrane as they control the substances that enter and leave the cell. Alcohol could also destroy the membrane proteins by denaturing the threedimensional shape of proteins due to change in pH caused by alcohol. The alcohol could also destroy the hydrogen bonds that hold the protein structure. Therefore, by gradually adding alcohol to the beetroot, the red pigment is allowed to escape from the cell due to the damage of cell membrane by alcohol. When the concentration of alcohol increases, the permeability of cell membrane will also increase but only until a maximum point which can be shown through the graph 2 below. The unit of absorbency of red pigment indicates the permeability of cell membrane.

Absorbency unit of red pigment/A

Alcohol concentration/% Graph 2 : Graph of absorbency unit of red pigment against alcohol concentration

Evaluation In the experiment, there are some limiting factors and sources of error that might have affected the final results obtained. Thus, certain improvements to the techniques should have taken to enhance the accuracy of the methods, reliability and validity of results. Firstly, the beetroot sections used are not precisely in exactly the same size as all the sections were cut using a knife with the help of a ruler. This may exposed to many errors in manual judgment. If beetroot sections used were not in the same size, the surface area to volume ratio and the distance of diffusion are not identical. This will affect the rate of diffusion and cause inaccuracy in the results obtained. This can be overcome by using an electronic balance to measure the mass of the beetroot sections to ensure they have the same surface area to volume ratio or using a different piece of equipment rather than knife such as mini plastic mitre block or something similar to an egg slice. Other than that, during the experiment, sometimes water droplets are present on the glass surface of cuvettes. Thus, the solution transferred into the cuvette may be diluted and this will reduce the actual colour intensity of the solution. This can be improved by getting rid of the water inside first before the cuvettes are used. The uneven dispersion of dye in the water in the boiling tubes may affect the reading of absorbency. Thus, the distilled water in the boiling tube, which has become coloured due to pigment escaped from the tissue, should be stir with the glass rod or shaked for a certain number of times to ensure an even dispersion of dye from the beetroot red pigments. There will be a random effect on the result if the beetroot sections are not rinsed first before placed into the boiling tubes because some of the beetroot sections may have juice inside. The effect can be prevented by washing all the beetroot sections for the same amount of time. We should avoid touching the cuvettes too often in order to reduce grease deposit on the glass surface which might affect the spectrophotometer reading. The outer surface of the cuvettes must be dried in order to increase the accuracy of the readings. This is because the presence of water might have caused the reflection within spectrophotometer. In this experiment, a big beetroot is used to obtain all the sections of beetroot from the same beetroot fruit. This will increase the accuracy of the results obtained since all the sections used were came from the same source. Besides, the beetroot fruit given was not cooked. This will further increase the validity of the results. Lastly, the electronic water baths were used to maintain the temperature of the water instead of heating it with Bunsen burner. This can prevent the fluctuation in temperature of water.

Safety precautions 1. The cover of the electronic water bath is closed when it is not in used to ensure the temperature within the water bath is maintained throughout the whole experiment. 2. Care should be taken during the extract of the beetroot cylinder. This is because the beetroot juice might spill and stain on our lab coat. 3. Care must be taken when handling the water bath to prevent scalding. Further work 1. To investigate the effects of alcohol concentration on permeability of membrane. Conclusion The permeability of the cell membrane increases as the temperature increases. The reading of the spectrophotometer increases when the temperature increases as more red pigment is released from the beetroot section. The hypothesis is accepted.

Bibliography www.biology-online.org http://www-saps.plantsci.cam.ac.uk/records/rec82.htm www.mrothery.co.uk/cells/resources/betacyanin.doc http://www.biologymad.com/resources/BEETROOT%20PIGMENT2.doc Biology ninth-edition Campbell