Title The effect of temperature on permeability of membranes Objective To investigate the effect of temperature on permeability of membrane Problem statement

When beetroot is put into hot water, the water will turn red. This involves the disruption of the cell membrane of beetroot and the leakage of the red pigments that give the solution its colour. Protein is very sensitive towards high temperature, so the permeability of plasma membrane changes according to the temperature of water. Hence, an experiment is conducted to study the effect of temperature towards the permeability of plasma membrane. Hypothesis General hypothesis: The higher the temperature, the higher the permeability of plasma membrane. Null hypothesis: The higher the temperature, the lower the permeability of plasma membrane.

Introduction Plasma membrane Plasma membrane is a structure that surrounds all living cells. It controls substances moving into and out of the cell and is also responsible for other properties of the cell. Other membranes surrounding nucleus and other organelles are almost similar to plasma membrane. Cell membrane is composed mainly of phospholipids and proteins and consists of a small amount of carbohydrates which are arranged in a fluid mosaic structure as shown in the figure below.

Figure 1:Fluid mosaic structure of cell membrane In the fluid mosaic model, the membrane is a fluid structure with a mosaic of various proteins embedded in or attached to a double layer (bilayer) of phospholipids. The phospholipids form a thin, flexible sheet while carbohydrate extends out of the protein. Membranes are not static sheets of molecules locked rigidly in a place. A membrane is hold together primarily by hydrophobic interactions, which are much weaker than covalent bonds. Most of the lipids and some of the proteins can shift about laterally. It is rare for a molecule to flip-flop transversely across the membrane, switching from one phospholipid layer to another. The lateral movement of phospholipids within the membrane is rapid. Proteins are much larger than lipids and move more slowly but some membrane proteins do drift. Some membrane protein move in highly directed manner. However, many other membrane proteins stay attached to the cytoskeleton or the extracellular matrix.

A membrane remains fluid as temperature decreases until eventually it settle into a closely packed arrangement and the membrane solidifies. The temperature a which it solidify depends on what lipid it is made of. The membrane remains fluid if it is made on unsaturated hydrocarbon tail. When a membrane solidify, their permeability change and enzymatic proteins in the membrane may become inactive if their activity requires them to move within the membrane. However, membranes that are too fluid cannot support the protein function. Phospholipids are arranged in bilayer. Their polar hydrophilic glycerol head faces outwards and their hydrophobic fatty acid tails facing each other in the middle of the bilayer. This hydrophobic layer act as barrier to all except for the smallest molecules which isolates the two sides of the membrane. Distinctive kinds of membranes may contain phospholipids of different fatty acid that affect the strength and flexibility of the membrane. Animal cell membranes also contains cholesterol linking the fatty acids together thus stabilizing and strengthening the plasma membrane. The proteins usually span from one side of the phospholipid bilayer to the other(integral proteins), but can also sit on the one of the surface(peripheral protein). The proteins have hydrophilic amino acid on the outside of the membrane and hydrophobic amino acid in contact with fatty chains inside the membrane. Protein comprises approximately 50% of the mass of the membrane and responsible for the most of the membrane properties. Proteins that span the membrane are usually involved in transporting substances across the membrane. The proteins inside the surface of cell membranes are often attached to the cytoskeleton and are involved in maintaining the cells shape, or in cell motility. They may also be enzymes which catalyse the reaction in the cytoplasm. Proteins on the outside surface of cell membranes can act as receptors by having a specific binding site where hormones or other chemicals can bind. This binding then triggers other events in the cell. They may also be involved in cell signaling and cell recognition or they may be enzymes, such as maltase in the small intestine. Carbohydrates are found on the outer surface of all eukaryotic cell membranes and are attached to the membrane proteins or sometimes to the phospholipids. Proteins with carbohydrates attached are called glycoproteins while phospholipids with carbohydrates attached are called glycolipids. The carbohydrates are short polysaccharides composed of variety of distinctive monosaccharide and form a cell

coat or glycocalyx outside the cell membrane. The glycocalyx is involved in protection and cell recognition, and antigens such as the ABO antigens on the blood cells are usually cell-surface glycoproteins.

Beetroot The beetroot(Beta vulgaris), which is also often referred to simply as beet, is dark purple globe shaped vegetable as shown in the figure below.

Figure 2: Beetroot The beetroot is also referred to as table beet, garden beet, and red beet, and is just one among a variety of beets cultivated across the world. The beetroot is however the most popular and widely consumed of all. The beetroot has been cultivated and consumed since ancient times and is still an important ingredient in diets across the world. Beetroot contains sodium, potassium, phosphorus, calcium, iodine, iron, copper, vitamins B1, B2, B3, B6 and C. The beetroot has a characteristic of deep purple red colour that derives from betalain pigments. Betalains are alkaloid pigments that are found in some families of plants belonging to the order Caryophyllales, but in no other plants. Betalains are not found in plants containing anthocyanin pigments(pigment which can be found in red or purple vegetables and fruits)-structurally they are unrelated as often thought. They also have been found in some fungi i.e; Fly Agaric. They can be divided into betacyanins and betaxanthins based upon their molecular structure. Betacyanins generally appear red to red violet in color(absorb in the 535-550nm range). Their basic structure is as shown in figure 3 below. Betaxanthins generally appear yellow in

colour(absorb in the 475-480 range). They cause colour in both flowers, fruits and sometimes vegetative organs. They are water soluble and can be found in vacuole.

Figure 3: basic structure of betacyanins Beetroot pigment is used commercially as a food dye. It changes color when heated so it can be used in ice creams, sweets and other confectionary and also known as cheap and has no allergic side effects. When beetroot is heated, the molecules start to spin and vibrate faster. The water expand too. This will have a disruptive effect on any membrane in its way. To make things worse, lipids become more fluid as temperature goes up so the membrane becomes more fragile. Protein, if heated too much also will untangle and break apart. When this happens to the proteins spanning a lipid membrane, they will form holes that will destroy the delicate structure. Hence, any pigments in the innermost compartment will spill out.

Materials Raw beetroot, distilled water, water bath at 25C, 35C, 45C, 55C and 65C, tissue and labeling stickers

Apparatus A size 4 cork borer, white tile, knife, ruler, forceps, boiling tube rack, 6 boiling tubes, thermometer, spectrophotometer, cuvettes, stopwatch, pipette, small measuring cylinder, beaker and waterproof marking pen.

Variables Manipulated variable: Temperature of water bath (C) Responding variable: Absorbency readings (optical density) Fixed variables: Diameter of beetroot, Length of beetroot, Volume of distilled water in the boiling tube

Procedure 1. Sections from a single beetroot were cut into sections into a size 4 cork borer on a white tile. Six slices from these sections were cut using a knife into 1 cm length each, using a ruler. 2. Six labeled boiling tubes were filled with 5cm3 distilled water using a measuring cylinder. 3. Four of the six boiling tubes were placed in water baths of different temperature, namely 35C, 45C, 55C, 65C. The other two which were labeled 0C was put in a beaker filled with ice in the freeze and 25C was left in a beaker of water in room temperature. 4. The boiling tubes were left in their respective water baths and freeze for 5 minutes in order for the distilled water in the boiling tubes to achieve the required temperature. 5. After 5 minutes, one slice of beetroot was placed into each of the boiling tubes using forceps. 6. The beetroot slices were left in the respective water baths for 30 minutes. 7. After 30 minutes, the beetroot slices were removed from the boiling tubes. 8. The boiling tubes were shaken to disperse the dye in the solution. 9. Seven cuvettes were prepared, and were filled in using pipette with the solutions: a. Cuvette A : 2cm2 of distilled water b. Cuvette B : 2cm2 of the solution immersed in the beaker filled with ice in the freeze c. Cuvette C : 2cm2 of the solution immersed in water bath of 25C d. Cuvette D : 2cm2 of the solution immersed in water bath of 35C e. Cuvette E : 2cm2 of the solution immersed in water bath of 45C f. Cuvette F : 2cm2 of the solution immersed in water bath of 55C g. Cuvette G : 2cm2 of the solution immersed in water bath of 65C

10. A spectrophotometer was switched on and it was set to read % absorbance.

11. Cuvette A(with distilled water) was placed into the spectrophotometer, and the spectrophotometer was adjusted to read zero absorbance for distilled water. The setting of the spectrophotometer was not altered anymore during the experiment. 12. Cuvette B was then placed into the spectrophotometer, and the reading for absorbency was taken. These steps were repeated for cuvettes C, D, E and F. 13. The readings for absorbency of each solution were recorded in Table 1. 14. The whole experiment was repeated twice in order to get more accurate and reliable results.

Safety Precautions 1.Beetroots contain betalains or red pigments which are used as dyes. When handling the beetroot, be careful not to spill beetroot juice on clothes as it will stain very badly. Proper attire that is lab coat and covered shoes must also be wore during the whole experiment. 2.Knife is very sharp and dangerous. When handling this apparatus, extra precaution should be taken. Same goes with the lids of the water bath. This is to prevent scalding. A boiling tube holder should also be used when removing boiling tubes from their water baths to prevent scalding. 3.It is necessary for the distilled water in the boiling tubes to be put into their respective water baths for 5 minutes before adding beetroot slices. This is to ensure that the distilled water achieved the required temperature so the results will be more accurate. The volume of distilled water in the boiling tubes should also be accurate, so that the effect of osmosis can be ignored. 4.Besides that, it is necessary to shake the tubes before getting 2 cm of the solution to be tested in the spectrophotometer. This is to disperse the dye in the solution so the distribution of the dye is uniform throughout the solution hence a more valiable and valid results are obtained. 5.The cuvettes should be clean and dry. It is necessary to ensure that there are no water droplets on the outer surface or inner surface as this will affect the refraction of light, therefore affecting the readings of the spectrophotometer. The outer surface of the cuvettes must also be wiped with tissue to ensure that there are no fingerprints or scratches as these will also affect the refraction of light.

Results
Temperature (C) 0 25 35 45 55 65 Absorbency (optical density) Reading 1 0.136 0.174 0.489 0.461 0.558 0.577 Reading 2 0.139 0.174 0.357 0.516 0.557 0.577 Reading 3 0.140 0.209 0.493 0.516 0.557 0.577 Average absorbency (optical density) 0.138 0.186 0.446 0.498 0.557 0.577

Table 1: Absorbency of solutions read from a spectrophotometer

Average absorbency versus temperature

Average absorbency (optical density) 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0 10 20 30 40 Temperature(C) 50 60 70

Figure 4: Graph of average absorbency against temperature of water bath

Discussion A spectrophotometer consists of two instruments, namely a spectrometer for producing light of any selected colour (wavelength), and a photometer for measuring the intensity of light. The instruments are arranged so that liquid in a cuvette can be placed between the spectrometer beam and the photometer. The amount of light passing through the tube is measured by the photometer. When white light is directed at a coloured solution some wavelengths are absorbed. The transmitted light is now coloured because it is missing some wavelengths of white light. For example, a substance which absorbs blue and green light appears yellow-red. This means that the higher the absorbance, the higher the colour intensity of the solution.

From the graph, we know that, the absorbency increases with the increase of temperature. The lowest average absorbency reading is 0.138(optical density) which is at 0C temperature and the highest average absorbency reading is 0.577 (optical density) which is at 65C. The graph shows that as the temperature increase, the reading of spectrophotometer also increases but not uniformly. As can be seen on the graph the reading of spectrophotometer increases slightly at 0C to 25C. However the steepest line from 25C to 35C show that the increase of the spectrophotometer reading is the highest in this temperature. From 35C to 65C, there is a slight increase in the reading of spectrophotometer. When temperature increases, the intensity of the solution increases as there are more pigments released into the solution. This shows that when temperature increases, the permeability of the membrane also increases. When temperature increase, the pigments and the molecules that makes up membranes gain more kinetic energy. The molecules then become less stable and form holes which allow some substances to be able to escape through the holes. The pigments, on the other hand able to diffuse out of the cell with the high kinetic energy. Other than that, the membrane proteins may also denature at high temperature. At high temperature, all the bonds that hold the basic structure of proteins are disrupted. This includes disulphides bridges, ionic bonds, Van der Waals forces and the hydrogen bonds. As a result, the proteins change shape and lose their function as the function of protein are determined by its structure. As such, the membrane is no longer stable and substances such as pigments or betalains can leak out of the cell, into the solution. If the different temperature in this experiment is replaced with the different concentration of alcohol, the same graph will be obtained (graph 2)

Absorbency

Alcohol concentration Graph 2 : Graph of average absorbency against the alcohol concentration. As the alcohol concentration increase, the absorbency reading also increase which indicates that the permeability of membranes also increase with the increase of alcohol concentration. This is because alcohol is an organic solvent. The phospholipid which is the major component in the membrane will dissolve in the alcohol and form holes. This make the membranes are more permeable and more red pigments leak out into the solution. As such the colour intensity increase and the average absorbency reading also increase

Conclusion From this experiment, we can conclude that temperature affects the cell membrane. As the temperature increases, the permeability of the membrane increases. Membrane structures are also affected due to the increase in temperature which increases the kinetic energy of the membrane molecules. In addition, membrane structures are also affected as the proteins are slowly undergoing denaturation along with the increase of the temperature. Hence, the hypothesis is accepted : the higher the temperature, the higher the permeability of the membrane.

Limitations There are some limitation in the experiment which might affect the validity and the realibility of the results. First of all, the red pigments in the slices of beetroot may not be evenly distributed. As this is something that cannot be avoid, we can only minimize the errors caused by this limitation by using the slices of beetroot from the same region so that the distribution of the red pigments are about the same.

Other than that, parallax error may occur when we were cutting the beetroot slices into 1 cm of length each by putting ruler at the side. As a result, the length of the beetroot slices might differ from one another. Hence, we should be more precise when cutting the beetroot slices. Besides that, the temperature of the water baths may not be constant since the lids of the water baths were always being lifted. As the lids are lifted, some heat may lost to the surrounding and this will affect the kinetic energy of the pigment and the molecules and thus affecting the results.

References 1.www.biologymad.com/resources/BEETROOT%20PIGMENT2.doc 2.http://www.diethealthclub.com/health-food/beetroot-health-benefits.html 3.http://www.biologymad.com/cells/cellmembrane.htm 4.http://www.madsci.org/FAQs/anthocyanins.html 5.http://www.ruf.rice.edu/~bioslabs/methods/protein/spectrophotometer.html 6.http://www.scienceteacherprogram.org/chemistry/Tehilla97.html 7.Jane B.Reece, 2011,Campbell Biology ninth edition, United State of America, Pearson International Edition (page 171-174)