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 LECTURER’S NAME : SIR WILLIAM NGU 0
ALBG10TITLE :
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
LECTURER’S NAME : SIR WILLIAM NGU
0
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.
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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
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1http://www.biology4kids.com/files/cell_membrane.html2Clegg, C J. Edexcel Biology for AS. London: Hodder Education, 2008.
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
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
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
3
http://4.bp.blogspot.com/_rBYpndaJ_ak/TLYrm_wLzZI/AAAAAAAAARQ/usJ7Fipbj8U/s1600/Lipid+Bilayer+Basic+Structure.jpg
2Clegg, 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
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
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http://dickinsonn.ism-online.org/files/2009/11/626px
http://t2.gstatic.com/images?q=tbn:ANd9GcSvNT84e_qi5iTnQ0KJ8TkFjfXtVfrodVM1gi9-cIbJ17MTDkcU
2 Factors Affecting the Rate of Permeability in a Cell Membrane." 123HelpMe.com. 06 Aug 2011 http://www.123HelpMe.com/view.asp?id=148801
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
move ions across membranes but against the concentration gradient.. Active transport is also used to
pump molecules and ions out of the cell.2
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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2 Factors Affecting the Rate of Permeability in a Cell Membrane." 123HelpMe.com. 06 Aug 2011 http://www.123HelpMe.com/view.asp?id=1488013 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.
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
b) White tile
c) Knife
d) Ruler
e) Plastic beaker about 250 cm3
f) Forceps
g) Water baths at 35°C,45°C,55°C and 65 °C
Materials
Raw beetroot and distilled water
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h) Boiling tube rack
i) Thermometer (one per water bath)
j) Cuvettes
k) Small measuring cylinders
l) Pipette for measuring 2 cm3
m) 4 boiling tubes
n) Spectrophotometer
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 35°C , 45°C , 55°C and 65°C 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 35°C.
c) Cuvette C : 3cm3 of the solution immersed in water bath of 45°C.
d) Cuvette D : 3cm3 of the solution immersed in water bath of 55°C.
e) Cuvette E : 3cm3 of the solution immersed in water bath of 65°C.
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.
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12. 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.
13. The readings for absorbency of each solution were recorded in Table 1.
14. 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-prac-large.jpg
Results :
Temperature(°C)
Absorbency (arbitrary unit) Average absorbency
(arbitrary unit) 1 2 3
35.0 0.158 0.159 0.159 0.159
45.0 0.227 0.226 0.226 0.226
55.0 0.518 0.518 0.518 0.518
65.0 0.579 0.578 0.578 0.578
Table 1
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30 35 40 45 50 55 60 65 700
0.1
0.2
0.3
0.4
0.5
0.6
Temperature, °C
Abs
orba
nce
Read
ing,
ari
bitr
ary
unit
(a. u
.)
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Graph of the absorbance reading of the dye solution (a. u.) against
Temperature (°C)
Discussion
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 40°C, absorbency increases
more rapidly. When temperature rose from 60°C to 70°C, 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 ºC - 70 ºC. 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.
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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
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.
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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.
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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4 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
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)
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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.pdfc) http://www.newworldencyclopedia.org/entry/Beet d) http://2.bp.blogspot.com/_qE8rf3Kb4fI/Rp6q0RaA7sI/AAAAAAAAAZo/0VDkTCx_4tc/s400/
threebeets2.jpge) 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=121435g) http://en.wikipedia.org/wiki/Spectrophotometry h) http://www.courseworkbank.info/courseworkbank.info.php?
f=R0NTRS9CaW9sb2d5L01vdmVtZW50IG9mIFBpZ21lbnQgdGhyb3VnaCBDZWxsIE1lbWJyYW5lcy5wZGY=
i) http://www.slideshare.net/wkkok1957/absorbance-of-anthocyanin-in-beetroot-experiment/ 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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