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ALBG10

TITLE : The Effect of Temperature on Membranes


NAME : NUR SHUHADA BINTI MOHD FIRDAUS WONG I/C NUMBER : 930409125468

GROUP : ALM12M16 STUDENT ID NUMBER: 2011889202

DATE OF PRACTICAL : 2 AUGUST 2011 LECTURERS NAME : SIR WILLIAM NGU

Title
The effect of temperature on membranes.

Objective
To investigate the effect of temperature on membrane structure.

Introduction
A recipe for cooked beetroot usually recommends that you do not cut the stalk or remove the skin.If you ignore these warnings,you are likely to end up red-handed.Beetroot contains red pigment called betalains,located within the cell vacuole.Normally these pigments cannot pass through cell membranes but they leak out when the beetroot is cooked.Why does this happen? The red pigment contained in beetroots is called betalains and this is what gives the beetroot its dark colour.The betalains is a group of nitrogen containing pigments which are normally yellow, orange, pink, red and purple. They are found in the vacuole and thus are used as markers for scientists who wish to extract intact vacuoles from plants for research. To extract the pigment, the membranes must be disrupted. This can be done by heat shock, by detergents or by solvents (e.g. ethanol or acidified methanol). When you heat a beetroot, you disrupt the cell membranes.
betalains

http://www.nandyala.org/mahanandi/images/beetrootcurry/DSCN1269.JPG

Let's look at the cell membrane and see how that membrane keeps all of the pieces inside. When you think about a membrane, imagine it is like a big plastic bag with some tiny holes. That bag holds all of the cell pieces and fluids inside the cell and keeps any nasty things outside the cell. The holes are there to let some things move in and out of the cell.1 All living cells have a plasma membrane that encloses their contents. In prokaryotes and plants, the membrane is the inner layer of protection surrounded by a rigid cell wall. These membranes also regulate the passage of molecules in and out of the cells.

http://upload.wikimedia.org/wikipedia/commons/e/ee/CellMembraneDrawing.jpg

The plasma membrane is the structure that maintains the integrity of the cell.It is also the barrier across which all substances entering and leaving the cell must pass.It is made of almost entirely protein and lipid,together with a small variable amount of carbohydrate.The cell membrane is described as a fluid mosaic for two reasons.First,the protein part of the cell membrane was once thought to be an even layer spread over the outside and inside of the phospholipid. It is now thought to be unevenly distributed and scattered,more like a mosaic than a layer.Second,the phospholipid part is fluid,with its molecules constantly moving about.2
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http://www.biology4kids.com/files/cell_membrane.html Clegg, C J. Edexcel Biology for AS. London: Hodder Education, 2008.

The cell membrane is actually a double membrane or we called as phospholipid bilayer. It is made up out of two layers of phospholipid molecules.Phospholipid membranes have two main functions which are controlling the transport of materials from one side to other.In chloroplasts and mitochondrion,they keep pigments and carrier molecules in fixed positions. Phospholipid bilayer contains many different types of molecules and they each have different parts to play in the overall structure and function of the membrane, these molecules are Phospholipids,Cholestrol,Glycolipids and Glycoprotein. Phospholipids forms a bilayer which is the basic structure of the membrane, they have non polar tails which means that it is difficult for polar molecules or ions to get passed them .It can be said they are barrier to most water soluble substances. It has hydrophilic (water-loving) properties.For example,hydrogen bonds readily formed between the phosphate head and the water molecules.The remainder of the phospholipid comprises two long,fatty acid residues consisting of hydrocarbon chains.These tails have hydrophobic (water-hating) properties.Therefore,phospholipid are unusual in being partly hydrophilic and partly hydrophobic.2

http://4.bp.blogspot.com/_rBYpndaJ_ak/TLYrm_wLzZI/AAAAAAAAARQ/usJ7Fipbj8U/s1600/Lipid+Bilayer+Basic+Stru cture.jpg

Cholestrol will help to get the fluidity of the membrane regular and it will prevent it from becoming too rigid or too fluid. Cholesterol is used also for the mechanical stability of membranes.This is very important because without this membranes would break rapidly and cells would burst. Meanwhile,Proteins act as carriers and also channels for ions and glucose. Proteins also act as a receptor for hormones.This will detect which hormone it is and what it does. Glycolipids and Glycoprotein are only found on the exterior surface of the cell membrane, they are formed by
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Clegg, C J. Edexcel Biology for AS. London: Hodder Education, 2008.

carbohydrates attaching to lipids to form Glycolipids and to proteins to form glycoproteins, they act as receptors and aid in recognition of cells. Diffusion is the movement of molecules from a higher concentration gradient to a lower concentration gradient. This also means that,movement will occur until the two gradients have reached equilibrium, the state in which the molecules are evenly distribute. Diffusion can occur between two separate parts of fluid across a membrane. 2

http://dickinsonn.ism-online.org/files/2009/11/626px

Many molecules that cells require are too large to pass between phospholipids molecules. They have also been charged and therefore unable to pass through the hydrophobic region in the centre of the bilayer. Protein molecules exist in membranes to facilitate the diffusion of these substances.Channel proteins are transmembrane proteins that form tunnels,or pores, through the bilayer for water soluble molecules. Some channels are open all the time, others open when triggered by the presence of a chemical such as hormone.On the other hand,Carrier proteins change shape to help move molecules into or out the cell. Molecules bind to the protein which stimulates the protein to change its overall shape, so allowing the molecules to diffuse through membrane. 2

http://t2.gstatic.com/images?q=tbn:ANd9GcSvNT84e_qi5iTnQ0KJ8TkFjfXtVfrodVM1gi9cIbJ17MTDkcU

Some substances required by cells are in a lower concentration outside the cell than inside. Cells cannot obtain the substances by diffusion. Carrier proteins similar to those used in facilitated diffusion 2
Factors Affecting the Rate of Permeability in a Cell Membrane." 123HelpMe.com. 06 Aug 2011 http://www.123HelpMe.com/view.asp?

move ions across membranes but against the concentration gradient.. Active transport is also used to pump molecules and ions out of the cell.2
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id=148801

A spectrophotometer is a photometer (a device for measuring light intensity) that can measure intensity as a function of the light source wavelength.It is commonly used for the measurement of transmittance or reflectance of solutions, transparent or opaque solids, such as polished glass, or gases. However they can also be designed to measure the diffusivity on any of the listed light ranges that usually cover around 200nm - 2500nm .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.3

Spectrophotometer

The rate of membrane permeability is dependent on a many factors. The molecular size of the solute will affect the rate of diffusion as the permeability mostly decreases with increasing size.Moreover, The pH and temperature also have great effect on the permeability of the plasma membrane and in the experiment I will test how temperature has an effect on the rate of permeability.2
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Factors Affecting the Rate of Permeability in a Cell Membrane." 123HelpMe.com. 06 Aug 2011 http://www.123HelpMe.com/view.asp?

id=148801 3 http://en.wikipedia.org/wiki/Spectrophotometry

Problem statement

What is the effect of temperature on membrane structure?

Hypothesis
The higher the temperature,the higher the permeability of the plasma membrane.
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Variables
Fixed variables : 1 cm length of beetroot sections,diameter of beetroot, volume of distilled water,cork borer Size 4. Manipulated variable : Temperature of water bath (C) Responding variable : Absorbency reading

Apparatus
a) Size 4 cork borer

h) i) j) k) l) m) n)

Boiling tube rack Thermometer (one per water bath) Cuvettes Small measuring cylinders Pipette for measuring 2 cm3 4 boiling tubes Spectrophotometer

b) White tile c) Knife d) Ruler


e) Plastic beaker about 250 cm3

f) Forceps g) Water baths at 35C,45C,55C and 65 C

Materials
Raw beetroot and distilled water

Procedure
1. Sections from a single beetroot were cut using a size 4 cork borer on a white tile.
2. The beetroot sections were left in a beaker of distileed water for 15 minutes to remove excess dye that

leaked out from the cells which were damaged when the beetroot was being cut.

3. After that,four slices from these sections were cut using a knife into 1 cm in length each,measured

with a ruler.
4. Four labelled boiling tubes were filled with 5cm3 of distilled water and were placed in water baths of

different temperature,namely 35C , 45C , 55C and 65C for 5 minutes. 5. After 5 minutes,the one beetroot slice was placed in each boiling tube using a pair of 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. Five cuvettes were prepared,and were filled in with the solutions:
a) Cuvette A : 3cm3 of distilled water b) Cuvette B : 3cm3 of the solution immersed in water bath of 35C. c) Cuvette C : 3cm3 of the solution immersed in water bath of 45C. d) Cuvette D : 3cm3 of the solution immersed in water bath of 55C. e) Cuvette E : 3cm3 of the solution immersed in water bath of 65C.

1. A spectrophotometer was switched on and it was set to read % absorbance. 2. 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.

3. Cuvette B was then placed into the spectrophotometer and the reading for absorbency was taken.

This step was repeated for cuvettes C,D and E.


4. The readings for absorbency of each solution were recorded in Table 1.

5. The whole experiment was repeated twice in oreder to get more accurate and reliable results.
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http://vcebiology.edublogs.org/files/2010/03/beetroot-praclarge.jpg

Results :
Absorbency (arbitrary unit) 1 2 3
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Average absorbency

Temperature(C) 35.0 45.0 55.0 65.0 0.158 0.227 0.518 0.579 0.159 0.226 0.518 0.578 Table 1 0.159 0.226 0.518 0.578

(arbitrary unit) 0.159 0.226 0.518 0.578

Graph of the absorbance reading of the dye solution (a. u.) against Temperature (C)

Discussion

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From the graph,we can see that as the temperature increases,the absorbency increases.The lowest reading is 0.159 and the highest reading is 0.578. By observing the trends of the graphs that were produced from the spectrophotometer, it is clear to see that the data supports the hypothesis that temperature does in fact have an effect on the rate of diffusion of betanin molecules in beetroot to water. That the higher the temperature the more betanin molecules will diffuse out. At the beginning, absorbency increases with temperature slowly.As temperature rises above 40C, absorbency increases more rapidly. When temperature rose from 60C to 70C, absorbency has roughly leveled and may have reached a constant.The proteins in the membranes which are the phospholipids, cholesterol begin to denature when the temperature increases from 60 C - 70 C. The higher temperature breaks down the phospholipids, cholesterol and proteins in the membrane and so more dye is released. Basically, it is the input of kinetic energy due to the build up of temperature that increases the rate of diffusion. This in turn will damage and denature the plasma membrane causing substances contained within the membrane to leak out. It is the breakdown of phospholipids in the membrane, which cause gaps to appear allowing red pigment to pass through. As the red pigment particles move faster, they diffuse out of the membrane at a faster rate, increasing more as the temperature increases. Therefore,as the temperature increase,intensity of solution increases. There are some sources of error and limitations in the experiment.First of all, The newly cut beetroot slices were not put in distilled water long enough, so excess dye was not washed away thoroughly. Some pigment may have leaked out during the experiment due to broken vacuole.This can be overcomed by leaving the beaker of distilled water and beetroot slices overnight before the experiment to make sure that all excess dye is washed away.

Other than that,there could be a difference in the sizes of the beetroot slices.Not all the beetroot slices were cut into the same size.This would affect the surface area of the beetroot slices,which would directly influence the rate of diffusion.This could mean that some beetroot slices could diffuse at an
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increased or decreased rate,which would consequently alter the amount of red pigment diffused into the water,and so affect the final result.To overcome this,a micrometer screw gauge can be used instead of a plain ruler. Furthermore,the temperature of the water bath may not be constant.This is because the lids of the water baths were always being lifted.When the lids are being lifted,the temperature of the water baths will change,causing some heat lost to the surroundings.This will cause the kinetic energy of the pigment and the membrane molecules to be affected,therefore affecting the end results. Besides that,if the cuvettes did come into contact with the skin or any dirt particles in the air,these particles could adhere to the side of the cuvette and so affect and alter the amount of light transmitting through the cuvette,thus altering the reading.Therefore, cuvette face should be thoroughly cleaned after every use. In addition, make sure to place the cuvette without touching the face which the light is to pass through. There are limitations in this experiment as the storage condition and age of the beetroot are something that we cannot control and avoid of.The beetroots provided in the experiment were from unknown sources and this might cause them to vary from each other.For example,a more agy beetroot will have less red pigments compared to the young one and this may cause an unfair result. In addition, breeds of beetroot that are not the typical deep red, such as Burpee's golden and albina vereduna, have a greater or lesser distribution of the two betalains pigments.

Safety Precautions
Using the cork borer and knife to cut the beetroot also gives the problem of cutting yourself with them. To prevent this keep your free hand as far away from the knife as possible.. Also remember to use a white tile as this prevents causing damage to the table.
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Beetroot contains betalains.It can stain clothes and skin very badly. Always ensure that you wear proper fitting clothes and nothing that is too loose. You should also wear your lab coat to avoid spilling anything on your clothes. Sandals and open toed slippers should be avoided.Wearing rubber gloves may also help this purpose as they give an extra layer for the skin from being stained while cutting the beetroot. Furthermore,avoid touching hot objects with bare hands.Be careful when handling and transferring the test tubes into water bath.Use test tube holder to avoid scalding. Glass objects take a long time to cool, so allow plenty of time to cool before touching them.

Further studies
Beet tissue will be used as a model to investigate how various alcohols affect cell membrane integrity. Beet cells contain a red pigment called betacyanin located in the tonoplast. Betacyanin, a water-soluble pigment, cannot pass across the membrane of the tonoplast or cell membrane of the beet cells as long as these membranes remain intact 4. If however, these cells are exposed to changes in a lipid soluble solvent like ethanol, the integrity of the cell membrane becomes compromised. As a result, betacyanin can leak out of the cells and into the surrounding water. The extent of the damage to the cell membrane is directly related to the intensity of red color that appears in the water surrounding the beet 4. The intensity of the red color can be quantitatively assessed using a spectrophotometer. The intensity of color should be proportional to the amount of damage sustained by the beet cell membrane.4
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Beetroot cell membrane permeability experiment. (2003, February 22). In WriteWork.com. Retrieved 11:28, August 02, 2011, from http://www.writework.com/essay/beetroot-cell-membrane-permeability-experiment

Conclusion
The hypothesis is accepted.The higher the temperature,the higher the permeability of membrane.Increase in temperature increases the kinetic energy of molecules, hence raises the rate of diffusion.Proteins in the cell membrane are very susceptible to temperature changes. A further increase
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in temperature above the optimum point will change their shapes and denature them. The cell membrane is now damaged, unable to properly control transport across it.Colour leakage increases significantly. When the cell membrane becomes fully permeable, the red pigment in the vacuole of the beetroot cell can move freely from the cell out to the water, hence rate of colour leak reaches a constant.

(2803 words)

References
a) http://www.biology4kids.com/files/cell_membrane.html b) http://www.sonalkhirwadkar.co.uk/base/files/archive/de_lisle/a_levels/biology/cell_ membrane_permeability.pdf c) http://www.newworldencyclopedia.org/entry/Beet 14

d) http://2.bp.blogspot.com/_qE8rf3Kb4fI/Rp6q0RaA7sI/AAAAAAAAAZo/0VDkTCx_4tc/ s400/threebeets2.jpg e) http://academic.brooklyn.cuny.edu/biology/bio4fv/page f) "The Effect of Temperature on the Cell Membrane of Beetroot Cells." 123HelpMe.com. 02 Aug 2011 http://www.123HelpMe.com/view.asp?id=121435 g) http://en.wikipedia.org/wiki/Spectrophotometry h) http://www.courseworkbank.info/courseworkbank.info.php? f=R0NTRS9CaW9sb2d5L01vdmVtZW50IG9mIFBpZ21lbnQgdGhyb3VnaCBDZWxsIE 1lbWJyYW5lcy5wZGY= i) http://www.slideshare.net/wkkok1957/absorbance-of-anthocyanin-in-beetrootexperiment/ j) Clegg, C J. Edexcel Biology for AS. London: Hodder Education, 2008. k) Fullick,Ann.Edexcel AS Biology.Essex:Pearson Education Limited,2008.

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