Biology Laboratory Report- Plasma Membrane
Third Lab Report. Hope this benefits someone....
BIOLOGY LABORATORY REPORT
THE EFFECT OF TEMPERATURE ON MEMBRANES
NOR HUSNA BINTI MOHD ROSLI
11 SC 3
SUBMISSION DATE :
MISS ZAKIAH BINTI ZAKARIA 9 SEPTEMBER 2013
OBJECTIVE To investigate the effect of temperature on permeability of beetroot membrane. INTRODUCTION
Beetroot, or scientifically-known as Beta vulgaris have betalain pigment (or well-known as anthocyanin). Beetroot is one of the famous ingredients in the secret recipes of United Kingdom. It is a delicious yet healthy vegetable to be consumed by our body. At the newly production of beetroot, the people only eat their roots and their only greatly used as medicine for health treatment. Since the world’s development, beetroot is used for preparing healthy dishes, still as health treatment but they are used as ingredients in their recipes. Beetroot have sodium, potassium, phosphorus, calcium, iodine, iron, copper and vitamin B1, B2, B3, B6 and C. Betalain that is from beetroot pigment is in deep purple root colour. Betalains are alkaloid pigments that are found in some families of plant belonging to the order Caryophyllales, but not in no other plants. In addition, betalains are not available in plants that contains anthocyanin pigments which is the pigment that can be found in red or purple vegetable and fruits, which is structurally related. Betalain also found in some fungi, for example Fly Agaric. They can be separated into betacyanins and betaxanthins based upon their molecular structure. Betacyanins usually appear red to red violet (absorb in 535-550nm range). Betaxanthins officially appear as yellow in colour which they absorb in 475-480 range. They had caused presence of colour in both flowers, fruits and sometimes vegetative organs and also are water soluble and can be found in vacuole. If the beetroot is heated, the molecules starts spinning and vibrating faster. The water will expand too. This officially may disrupt the membrane. What make it worse is, lipids become
more fluid when the temperature rises then making the membrane more fragile. Protein at the phospholipid bilayer will untangle too and break apart. If this happens, they will make holes that will destroy the original structure of plasma membrane. Lastly, any pigments in the intracellular environment of the cell will leaks. Health Benefits From Beetroot Raw beetroot (100g) provides: Energy Protein Carbohydrate of which sugar of which starch Fat of which saturates Fibre Sodium Equivalent as salt
g g g g g g g g g
Per 100g 162 kJ / 38 kcal 1.7 7.6 7.0 0.6 0.1 0.0 1.9 0.1 0.17
GDA* 2% 4% 3% 8% 0% 0% 8% 3%
*Recommended Daily amount (GDA) average adult (woman) Source: McCance & Widdowson 6th edn.
Reduces blood pressure and risk of heart attack and stroke *approved in Journal of Cambridge.
“A cup of beetroot juice a day, or a generous helping of green vegetables, may help lower blood pressure, a new British study finds.”
Source- The Star newspaper; published on 17 April2013. 2.
Powerful antioxidant properties
Contains folic acid for normal tissue growth
Reduces risk of osteoporosis
Stabilises blood sugar
Treats anaemia and fatigue
Helps slow progression of dementia
Plasma membrane act as protective layer of a cell. It provides a protective barrier which also separates the internal layer of a cell from the extracellular environment. It is a selectively permeable barrier that does controls the movement of molecule into our out of the cell. It is called as semi-permeable or partially permeable to the molecules or ions. This means it only allows certain molecules or ions to pass through which is small and uncharged molecules. Plasma membrane is made of composition of phospholipids, glycolipid, cholesterol, glycoprotein and carbohydrate chains. This made highly composition of it makes its structure scientifically called as ‘fluid mosaic model’. According to fluid mosaic model of SJ Singer and GL Nicholson (1972), which then replaced the earlier model of Davson and Danielli, this membrane can be considered to have two dimension of liquid, which lipid and protein can diffuse more or less easily. Although the lipid bilayers that form the basis of the membrane form two dimension of liquid itself, the membrane contains higher amount of protein, which mainly made its structure. Examples of these structures are protein-protein complexes, pickets and fences formed by actin-based cytoskeleton, and potentially lipid rafts.
Plasma membrane mainly consist of uncounted amount of phospholipids which made up of lipid. The combination of phospholipids form phospholipid bilayer, which the phospholipids each made up of two structure – hydrophilic head group and hydrophobic tail. Hydrophilic head group, which is water-loving and interact to water molecules structured by glycerol, phosphate and choline. The hydropobic tails, which are water-hating provide a hydrophobic environment within the bilayer. In addition, phospholipid not just made up by lipid, but its structure is also strengthened by the tough structure of cholesterol. Cholestrol is mainly at the polar head of phospholipid and exposed to extracellular environment/ water. As we all know, plasma membranes are mostly permeable to small and uncharged molecules. Polar molecules, mostly ions, can pass slowly through the membrane due to their inherent charge.
Besides that, plasma membranes also made up with help of proteins. Protein acts as a platform which can transfer charged yet bigger molecules or ions for the cell. These proteins function as a pore or carrier of that molecules, which generally known as pore protein and carrier
protein. Pore protein is an unchanged structure which can transfer small molecules and charged ions through the cell. Carrier protein is a structure of protein that can change shape to carry large molecules inside the cell. The types of diffusion that mainly occur in these proteins are facilitated diffusion which are then separated to passive transport and active transport . Passive transport is diffusion from higher concentration region to lower concentration region through carrier protein whereas active transport is diffusion from lower concentration region to higher concentration region with the help of energy (from ATP molecule). This is mainly occurs in pore protein.
PROBLEM STATEMENT How does the temperature affect the reading of colorimeter? HYPOTHESIS When the temperature increases, more dye is released from the beetroot into the distilled water. The high temperature may distort the active site , thus may denature the structure of fluid mosaic model of plasma membrane of the beetroot. The higher the temperature, the more the plasma membrane will be damaged. Thus, higher reading of colorimeter is obtained. VARIABLES TYPE OF VARIABLES Manipulated variable: Temperature of solution (°C) Responding Variable: The reading of colorimeter Constant Variable: Size of beetroot (
WAY TO CONTROL THE VARIABLES Use water bath at different temperature, which is 30°C, 40°C, 50°C and 60°C. Measure the temperature using thermometer. Repeat the reading of colorimeter and calculate the average of the reading to make sure that accurate reading is obtained. Measure using a ruler and cut 1
beetroot using a knife
APPARATUS Medium-sized cork borer, white tile, knife, ruler, forcep, 2 boiling tube racks, 12 boiling tubes, thermometer, colorimeter, 12 cuvettes, watch and 100
measuring cylinder, test
tube tongs MATERIALS Raw beetroot, water baths at 40°C, 50°C, 60°C and distilled water. TECHNIQUE Put each cuvette with 3
of beetroot solution into colorimeter and read the reading which
indicates the of absorbance of beetroot dye into distilled water. The reading is tabulated into a table and a graph is obtained. PROCEDURE 1. White tile, medium-sized cork borer and knife was prepared. The cork borer is pushed inside the beetroot and then withdrawed. The cylinder of beetroot inside the corkborer is pulled out and placed onto the white tile. 2. By using knife and ruler, measure 1
of beetroot cylinder and cut it carefully to
small cylinder. The action is repeated to produce 12 small cylinder of beetroot for each boiling tubes.
of distilled water is measured using 100
of measuring cylinder and poured
into 12 boiling tube each. 4. 9 boiling tubes are put into the water bath which 3 test tubes for each different temperature of 40°C, 50°C and 60°C.
Another 3 test tube are put in a test tube rack and placed at the particular side of laboratory as the room temperature is considered as 30°C. Use thermometer to ensure an accurate temperature. The boiling tubes are leave for 5 minutes. 5. Using test tube tongs, the boiling tube is then removed from the water bath. Put a piece of beetroot in each boiling tubes using a forcep. Step 4 is repeated by leaving it for 30 minutes. 6. The boiling tubes of 40 °C are diverted from water bath. 3
of the solution is
measured using a measuring cylinder. It is then filled into three cuvette.
7. Step 6 is repeated by using solution of 30 °C, 50°C and 60 °C. 8. The colorimeter is adjusted and re-zero by using 3
of distilled water in a cuvette.
9. Cuvette containing beetroot dye of 30°C is put inside the colorimeter.
The reading of colorimeter which is the reading of absorbency is recorded and tabulated in Table 1. The action is repeated using beetroot dye from 40°C, 50°C and 60°C . 10. The data is plotted in a Graph 1.
Reading of colorimeter (AU)
DISCUSSION The experiment is conducted to investigate the effect of temperature on permeability of the membranes. From this experiment, we make the different temperature as variables that can be manipulated whereas the reading of colorimeter as the responding variable. From the table, we can say that the lowest temperature of water bath, which is 40°C (we considered this as room temperature) have lowest reading of colorimeter which is 0.213 whereas, the lowest highest temperature of water bath, which is 60 °C has highest reading of colorimeter which is 0.512. From the graph, we know that the higher the temperature, the higher the reading of colorimeter which indicates the absorbency of the solution. This means that the permeability of plasma membrane is higher yet more beetroot dye is released into the distilled water. From temperature of 30°C to 40°C, the reading of colorimeter slightly decrease. This is due to human error as the lid of water bath of beetroot solution 40°C is frequently opened and closed. The heat was released to surrounding which make the reading of temperature inaccurate and may probably affected the reading of colorimeter. From 40°C to 50°C the reading of colorimeter jumps and increase in a higher rate. This is because the high temperature of water bath destroyed the structure of proteins in a cell membrane. The higher temperature of water bath makes the red dye in the beetroot cell gains more kinetic energy, thus vibrates at the faster rate. This also the cause which lead the diffusion of beetroot dye out of the cell into the cell environment or surrounding of the cell membrane. On the other side, we know that phospholipid bilayer is also made up of proteins. The proteins which are formed by amino acid is held by hydrogen bond and disulphide bridge. This hydrogen bond became weak when in contact with high temperature. The disulphide bridge, which also known as SS-bond is a type of covalent bond and have low boiling point, so its structure can be easily ruptured or destroyed when exposed to higher temperature. This can lead to the damage of the vacuoles and leaking in the cell membrane. The dye inside the damaged vacuoles diffused out of the cell membrane and leaks to the surrounding of the cell. This diffusion moves down the concentration gradient which is from reagion of higher concentration to lower concentration region. It also didn’t need the help of energy (ATP). At the temperature between 50°C and 60°C, the reading of the colorimeter becomes increase in a lower rate than before. This shows that leaking of the beetroot pigment still happens but the
gradient is not as steep as the reading of colorimeter of 40°C to 50°C. This is because some of the denatured proteins before block the holes made by the first low flow of beetroot leakage from the cell into the surrounding. When you heat a beetroot, you destroyed the cell membrane. The membrane is made up of phospholipid bilayer. These are formed by the two parts of phospholipid which is the hydrophilic head (water-loving) and the hydrophobic tail (water-hating). The hydrophilic head is more exposed to water while the hydrophobic tail will remains inside the intracellular environment of the cell. They form sack in a cell. One protects all around the outer layer of the cell to form phospholipid bilayer while some of them forms vacuole. When you heat something, automatically you give it an energy. Molecules at the surrounding starts to spin and vibrates faster. The water also will expand. This will give the membrane structure a disruptive effect. To make things worse, lipids become more fluid as the temperature rises so that the membranes become more fragile. Proteins remarkably is a machine. Proteins formed of coild and folded strings of amino acid and held by hydrogen bonds and disulphide bridges. If we overheating it, they will break apart and became untangled. If this happens at the proteins spanning a lipid membrane, they will form holes that will destroy its delicate strucure. That will just make any pigments of the cell spills out. SAFETY PRECAUTIONS We must wear a lab coat when conducting the experiment as the beetroot dye can badly give stains to our clothes. We also have to use the knife and the cork borer with great care to avoid any injuries to ourselves. Besides, when handling the water bath, we must make sure that the distance of ourselves and the container of the water bath is far enough. We also must use test tube tongs to insert the boiling tube into the water bath. Make sure the is no direct contact with the water bath as it can scald our skin. When taking the measurement of distilled water and the length of beetroot, we must make sure that the reading is taken accurately. Next, it is crucial put distilled water for 5 minutes in each water bath with different temperature before adding beetroot slices. This is to make sure
that the water bath have reached the exact temperature before inserting the beetroot pieces and the results become more accurate. Besides that, we also have to shake the beetroot solution before carefully pouring it into a cuvette. This is to make sure that the distribution of dye in the solution equally distributed in order to have a more accurate reading of colorimeter. Hence, a more valid and reliable results can be obtained. Last but not least, we must make sure that the surfaces of the cuvettes are dry enough and clean. This is also to make sure that there is no droplets of solution at the surfaces as it may cause refraction of light when determining the absorbency which can affect the data. The outer surfaces of cuvettes should be wiped using a dry and clean tissue to make sure that there is no fingerprints or scratches as they also can affect the reading due to the refraction of light. LIMITATION Limitations are factors that can't be controlled in an experiment. It also may affect the realibility and validity of the results. There are many types of beetroot and certain beetroot may only gives out little amount of red dye while some of it may gives out high amount of red dye. The size of beetroot that we cut before may have slightly difference in its length. When we cut the pieces of beetroot which is 1 cm each using a ruler, parallax error most probably have occurred due to the human mistakes. The red pigment from the beetroot may not evenly distributed. This is something that we cannot avoid, thus one of the way to control the effect of it to the reading of colorimeter is we take the cylinder of beetroot from almost the same region. This can produce only slight difference to the concentration of red dye in a beetroot. Besides that, there might be some scratches at the surface of the cuvette when trying to take the reading of colorimeter. The colorimeter is a very sensitive device. There should not be any scratches or fingerprints at the surface of cuvette as this may affect the reading of the colorimeter. The scratches and fingerprints may make the results differ from what it should be as the light passing through it can be diffracted thus affect the reading of colorimeter. Lastly, the temperature of water bath cannot be constant as the lid of the water bath were always being lifted. This can cause heat to loss to the surrounding. The situation can be seen when conducting the experiment using 40 °C of water bath. As a result, the reading of colorimeter become slightly decreased from what it should be.
FURTHER STUDY There are any other ways to know what other effect will affect the permeability of cell membrane. The first effect is the pH of the solute. The level of pH did affect the pigment of the beetroot. The dye of the beetroot (betalain) leaks and diffuse out from the cell membrane due to the low or high level of pH. The pH level is truly one of the factor that affect the permeability of the membrane. The protein will be denatured because of the high alkaline properties and acidic properties in a cell. Thus, this will cause the breakdown of lipid bilayer. For example, we can use alcohol as a substance to investigate the effect of it to the plasma membrane. The higher the amount of alcohol used against the membrane, the leakage of the red-coloured-dye from beetroot pieces into the surrounding. Higher amount of alcohol means the concentration of alcohol increases. This cause the membrane to rupture and lost its structure. This is because alcohol is a fat-solvent. Thus, it will dissolve the cell membrane as cell membrane is made up of lipid. CONCLUSION If the cell membrane is exposed to high temperature, the cell membrane of the cell will be broken down as protein and the lipid bilayer is denatured. Thus, this results to higher permeability of the substance. The hypothesis is accepted.
REFERENCES Ditte A Hobbs, Nedi Kaffa, Trevor W George, Lisa Methven and Julie A Lovegrove. British Journal of Nutrition/ Volume 108/ Issue 11/ December 2012, pp 2066-2074. CopyrightAuthors 2012. DOI: http://dx.doi.org/10.1017/S0007114512000190(AboutDOI), Published online: 14 March 2012 Cooper GM. The Cell: A Molecular Approach. 2nd Edition, Sunderland (MA): Sinauer Associates, 2000 http://www.thestar.com.my/Lifestyle/Health/2013/04/17/Beetroot-and-green-veg-found-toreduce-blood-pressure.aspx http://www.lovebeetroot.co.uk/ http://www.ncbi.nlm.nih.gov/books/NBK9898/ http://www.shmoop.com/biology-cells/plasma-membrane.html https://www.boundless.com/biology/the-plasma-membrane/membrane-overview/introductionto-plasma-membrane/ http://www.youtube.com/watch?v=faXBWOETTb4