Beetroot Experiment. The purpose of this experiment is to determine the effect of temperature on the permeability of cell membranes

by nealparkes (student)

2.1 Effect of Temperature on Membrane Permeability

Abstract

The purpose of this experiment is to determine the effect of temperature on the permeability of cell membranes. As beetroot cell contain a coloured pigment within the cell vacuole called betalain. The leakage of the coloured pigment betalain out of the cell will act as a maker to determine how permeable the cell membrane is at a particular time. To determine the permeability of the membrane uncooked beetroot cylinders will be bathed in water and exposed to different temperatures. The water in which the beetroot has been bathed in will then be measured for is absorbency. Here I demonstrate that there is a positive correlation between temperature and membrane permeability.

Introduction

Membranes consist of a phospholipid bilayer; this is two layers of phospholipids facing in opposite directions to each other. A phospholipid molecule is made up of a phosphate head connected to a lipid tail3. The phospholipid head orientates itself so that faces the external environment or inwards into the cytoplasm. The non-polar tails are hydrophobic and as a result face inwards to the centre of the plasma membrane4.

Figure 1 shows the phospholipid bilayer arranged so that the hydrophobic (water hating) tails are orientated in the middle and the hydrophilic (water loving) heads are facing outwards from the cell1

The Plasma membrane is not rigid and in fact the phospholipid molecules are fluid in that they can move laterally and diffuse, the addition of cholesterol to the cell membrane improves the fluidity if the membrane.

In addition to the phospholipids found in the membrane there is a large amount of protein present6. There are two major groups of proteins found associated with the plasma membrane. There are the proteins which are adjacent to the membrane known as peripheral proteins. The second group is the integral proteins which are embedded into the membrane7.

Figure 2 shows the complex structure that is the plasma membrane. The image shows the arrangement of membrane proteins present within the phospholipid bilayer2

The plasma membrane is a complex and important structure whose major function is to determine which substances enter and leave the cell. Each protein channel will only let a specific molecule through, this is essential for controlling what goes in and out of the cell.

Cytoplasm

How would temperature affect the membrane?

The purpose of the cell plasma membrane is to selectively control the movement of substances in and out of the cell. As the temperature increases both the lipids and the proteins are affected differently. The fatty acid tails of the phospholipid molecules begin to melt as the temperature increases3. This means the phospholipid molecules become more fluid as there is more freedom to move. As the fluidity increases the selective permeability of the membrane is reduced, allowing molecules in and out of the cell that would have otherwise been prevented from doing so4. Both the peripheral and transmembrane proteins can become denatured as the temperature changes. At about 20°C proteins start to become denatured loosing their 3-dimensional shape. As the temperature increases above this membrane channel proteins begin to loose shape, preventing the normal movement of substances in and out of the cell. Above 60°C proteins become totally denatured. Cholesterol also plays a major role in controlling the fluidity of the membrane and its overall stability5. At these temperatures cholesterol melts leading to reduced stability of the membrane.

The colour of Beetroot

The red/purple colour of a beetroot cell is a result of a pigment held in the vacuole. If cell surface membrane ruptures or there is damage to the cell, the pigment escapes from the cells like a dye. The integrity of the plasma membrane of beetroot cells can be measured by manipulating this process. The red/purple pigments are called betalain pigments, named after the red beetroot (beta vulgaris), and they replace anthocyanin in plants.

Predictions-

I predict that there will be a positive correlation between temperature and the permeability of the plasma membrane. At temperatures below 20°C I predict that there will be little movement of the betalain pigment across the plasma membrane as its movement will be selectively controlled by transport proteins. As the temperature rises to 20°C, membrane proteins will begin to denature, slightly increasing the fluidity of the plasma membrane and allowing more of the betalain pigment to cross the membrane. At 40°C there will be greater breakdown of the proteins, this will lead to more gaps between the proteins and the lipids allowing for more molecules to transverse the membrane. At 60°C the huge amounts of the proteins in the membrane will have denatured leading to a greater fluidity in the membrane lipids. In this situation betalain will easily be able to leave the cell in greater quantities. At 80°C the phospholipid tails and the cholesterol molecules will start to melt. As the phospholipids and cholesterol maintain the structural integrity and fluidity of the membrane it will begin to fall apart. There will be no inhibition of what can cross the membrane and therefore anything will be able to leave and enter the cell. Betalain will be diffuse freely from the cell. At 100°C the membrane would have completely fallen apart and the Betalain will diffuse into the water down a concentration gradient.

Aim

The aim of this study is to determine the effect that temperature has on the structural integrity of the plasma membrane. By observing the amount of the betalain pigment released from beetroot cells as temperature increases.

Hypothesis

From the information that I have gathered from the literature I believe that the following will occur:

As the temperature increases there will be greater permeability of the plasma membrane and more movement of Betalain out of the cell. Leading to a higher reading on the colorimeter.

Null Hypothesis

The null hypothesis is therefore:

That as the temperature increases there will be ...

This is a preview of the whole essay

Aim

Hypothesis

From the information that I have gathered from the literature I believe that the following will occur:

As the temperature increases there will be greater permeability of the plasma membrane and more movement of Betalain out of the cell. Leading to a higher reading on the colorimeter.

Null Hypothesis

The null hypothesis is therefore:

That as the temperature increases there will be no significant movement of the Betalain across the plasma membrane as compared to the control.

Variables

Dependent variable = temperature.

The purpose of this study is to investigate the effect of temperature on the plasma membrane. In order to carry out this investigation, the experiment will be repeated at several temperatures to discover at which intervals the plasma membrane begins to break down.

Independent variable = Betalain pigment as measured by colorimeter

This is the variable that I am trying to measure in order to determine the structural integrity of the plasma membrane.

Constant variables

In order to make this a fare teat and to ensure that we are measuring the effect of temperature alone on the release of the pigment from the cell. There are certain factors that we need to keep constant.

The first is the size of the beetroot, as we are ultimately measuring the diffusion of the pigment out of the cell. We need to ensure that the surface area for diffusion is the same. If different sized samples of beetroot are used, there is the potential that more pigment will diffuse out of the larger sample solely due to the surface contact with the water and not as a result of temperature alone.

The second is the direction in with the beetroot is cut from the stalk. The cells that make up a beetroot stalk will be orientated in a particular manner. Cutting the stalk into different segments at different orientations could mean that more cells are exposed to the outside in one segment and the others again affecting the amount of pigment released into the water.

The third is the washing of the beetroot segments. Cutting the segments with a knife and there subsequent handing in the process will cause damage to the cell (especially those nearest to the site of cutting. To ensure that all pigment in the water at the end of the experiment is due to temperature alone, all beetroot segments must be washed thoroughly and for the same amount of time.

The fourth is the time of removal of the beetroot segments. To ensure that temperature is the sole factor in this temperature it is important that all segments are removed from the water at the same time (or as near to it). If one segment was allowed to incubate in the water for longer than the others, there is greater time for diffusion of the beetroot pigment into the water.

The fifth is the volume of water used in which the beetroot segment is placed and heated. If the volume of water is changed this will either concentrate or dilute the amount of Betalain pigment released from the beetroot cells. Then when it comes to measuring the amount of pigment released by the use of a colorimeter the value will be different.

The water is also another variable which we have controlled as regular tap water has certain impurities and a marginally lower pH than pure water which, if the water was slightly more acidic, could have a small effect on the results, therefore for all the areas of the experiment, distilled water is used as it has a neutral pH of 7. Distilled water also accounts for the water potential (ψ) as tap water will have impurities and will have a lower water potential than distilled, if this were the case during the experiment the rate of diffusion may have been affected.

Although temperature is the dependent variable in this experiment, this is a concern with fluctuations in it. The temperature of the water bath will be either cooling or heating up to room temperature. This experiment is carried out to determine at what temperature the membrane breaks down. It is integral to the results of the experiment that the water baths are constantly monitored to ensure that the temperatures do not fluctuate too widely from the expected temperature.

Repeating this experiment twice will hopefully help to remove the errors/anomalies and variations in the experiment.

Method

The method was carried out as described in the NEC handout

Apparatus

As described in the NEC handout

Risk assessment

There are several risks associated with this experiment I have listed them below and ranked there probability and severity. (Highest being 10 lowest being 1)

How the probability of these risks can be reduced

Being cut by glass/scalpel- be extra aware of these dangerous objects, if glass is smashed get it cleared up safely immediately. Use a suitable cutting surface and cutting mat when using the scalpel and put a protective cover over the blade when walking with it.

Being scolded- take care when around hot water baths and know where nearest cold tap is and make it easily accessible. Use tongs when collecting test tubes from the water baths.

Slipping/tripping – before I start I will check the room and check there are not any obstructions on the floor (i.e. bags etc). If water is spilt on the floor I will make sure it is cleared up immediately so the floor is not slippery.

Results

A mathematical calculation can be carried out on this experiment to see if there is a correlation between the temperature and one of the readings in the results; transmission or absorbency. If there is then it is possible to accept or reject our original hypothesis. For this calculation I will be using transmission, as it is an easier figure to use being larger, the average data will be applied to the calculation to get a more accurate result. The hypotheses remain the same from the main experiment found earlier in the report.

Data Table: Spearman's Rank Correlation

Calculate the coefficient (R) using the formula below. The answer will always be between 1.0 (a perfect positive correlation) and -1.0 (a perfect negative correlation).

When written in mathematical notation the Spearman Rank formula looks like this:

Now to put all these values into the formula.

d² = 2 multiplying this by 6 gives 12
n=6, therefore n3 – n = 63-6 = 216 – 6 = 210
R = 1 - (12/210) which gives a value for R:

= 1 - 0.057143

= 0.942857

The R value of 0.94 suggests a fairly strong positive relationship.

A further technique is now required to test the significance of the relationship.

The R value of 0.94 must be looked up on the Spearman Rank significance table below as follows:

Degrees of freedom = number of pairs -2 = 6-2 = 4

The results show that there is a significance of 5%, therefore there we can say with a confidence of 95% of the hypothesis and we can therefore reject the null hypothesis. There we can say that as the temperature is increased the permeability of membrane increases.

Conclusion

The above result supports my original hypothesis and so gives a 95% probability to reject the null hypothesis. This shows that there is a very strong positive relationship between temperature and transmission, which the graph of the average results also shows and further supports the original hypothesis, that the increase in temperature will damage the beetroot membranes and cause them to rupture, leaking of Betalain.

Discussion

The results show that at a temperature of below 15°C there is more Betalain pigment in the water than at a temperature of above 15°C this is at first a surprising result. However if one looks at the science behind this then the results can be explained easily.

This effect could be a result of what is called freezing injury. At colder than normal temperatures the plasma membrane undergoes contraction. It is thought that intrinsic proteins which span the two sides of the plasma membrane may become damaged and break down. These broken protein channels can then become a source of leakage of molecules from the cytoplasm. It is also thought that the membrane lipids can under go rearrangement into non layered structures. This would certainly explain why there is more pigment found in the water at lower temperatures.

As the temperature rises towards 15°C the membrane becomes more fluid there is less damage of the membrane due to freezing injury. The membrane is more stable as it is operating at a temperature more conducive to its normal environment. The membrane will have more integrity and will leak less. This would help to explain why there is a drop in the movement of the pigment out of the cell.

As the temperature rises towards 50°C there is greater movement of the pigment out of the cell. This is due to the structure of the proteins that make up a high proportional of the plasma membrane. Increase the temperature leads to an increase in the kinetic energy of molecules, especially those molecules held together by weak molecular bonds. The relatively weak forces holding the different parts of the polypeptide chains together (such as hydrogen bonds, sulphur bridges and ionic bonds) become disrupted easily leading to deformation of the tertiary protein structure of the protein. This leads to molecules that would have otherwise been prevented from leaving the cell a means of escape. The betalain molecules move across the partially permeable membrane by osmosis- the betalain molecules move by diffusion from an area where they are more highly concentrated to an area where they are at a lower concentration, along a concentration gradient.

Above temperatures of 50°C there is a marked increase in the amount of Betalain seen in the water. The explanation for this rapid movement of Betalain from the cell is most because protein's denature and tertiary structure unravels (the strong covalent bonds between the R groups of amino acids in the polypeptide chains are destroyed). At temperatures exceeding 60°C proteins are almost entirely denatured and have lost their biological function. This may help to suggest why the gradient of the graph becomes steep up to 60°C.

However at temperatures above 60°C there is greater release of the pigment out of the cell than at temperatures below 60°C. I have already suggested that at this temperature all proteins are denatured. This effect must therefore be a result of other intrinsic factors in the membrane. Apart from protein phospholipids make up a large proportion of the membrane structure. The structure of the membranes is made up of a phospholipid bilayer; each phospholipid consists of a hydrophobic tail and a hydrophilic head.

Lipids have an optimum transition temperature at which they remain stable, this is about 60°C and once this temperature had been passed the lipids simply melt. The breakdown of the lipid bilayer happens in the same manner as the destruction of the protein channels. The increase in temperature causes an increase in kinetic energy of the fatty acid molecules that make up the phospholipid molecules. This increase in kinetic energy causes the fluidity of the membrane to increase. As the kinetic energy increase with temperature more of the phospholipid molecules melt and the integrity of the plasma membrane breaks down leading to maximum leakage into the water.

The reason why the curve starts to flatten out above 80°C is because there is maximum damage of the membrane. All of the protein and most of the phospholipids have broken down. By this point no more Betalain can leave the cell as it apposed osmotically by the concentration of Betalain inside and outside of the cell.

The presence of the glycolipids, glycoproteins also have an effect on the results; as they all cease to function at higher temperatures. As they convey structural advantages to the membrane their loss would weaken the structure appreciably. As the glycolipids and glycoproteins break down the hydrogen bonds formed with the external solution would also disappear. Thus, allowing greater diffusion of Betalain out of the cell.

Evaluation

Although the results of this experiment do seem match with that of the literature, I do believe that there are numerous sources of error that may have affected the accuracy of it.

Firstly there was a slight delay in placing all of the beetroot segments into their respective containers. As I had access to only one timing device, all of the experiments were done at the same time. Although the delay was only seconds it could still have had a bearing on the amount of Betalain leaking from the cell. If I were to do this experiment again I could have staggered the starting time so that each experiment was carried out with a one minute delay. This would have given me adequate time to place the segments into the water bath and would have been one less error in the experiment

When washing the beetroot segments it is difficult to know whether you have removed all of the excess Betalain. Drying the beetroot with the kitchen paper although done carefully could have done some damage to the cell membranes and as a result more Betalain would be reduced. I do think that as all of the segments were washed for the same period of time and dried under the same conditions that this error would be small.

I believe that to make more accurate predictions about the exact moment the protein or phospholipid molecules break down. It would have been better to conduct this experiment with more temperature intervals (say every 10°C). In this current experiment the gaps between the temperature intervals are too large and are not at really intervals. This makes any predictions on the results less accurate as you are using quite large temperature ranges.

I would have liked to repeat each of the temperature results a third time as I believe that an N=2, is not very significant to base any finding on. I think a third set of results would have been more significantly valid.

A major limitation is the actual manner in which the segments are obtained. Although I was careful in trying to obtain segments of the same size and cross sectional area they will never actually be the same. If the surface area of the segments differ only slightly this will have an effect (if only slightly) on the amount of Betalain that can leave the cell through diffusion.

It would also have been a good idea to actually check this effect in other cells, from other species and plants and possibly animal cells (although the availability of such may have been limited and the practical methods to identify breakdown of the plasma membrane been difficult). I believe that having results form other species of plants would dramatically improve the accuracy of any conclusions made.

Overall I believe that the results of this experiment are valid but there are several practical errors that limit the accuracy of the conclusions based upon it.

Cold-induced leakage of amylase from the zymogen granule and sealing of its membrane by specific lipids

http://biologyatsmc.wikispaces.com/Cell+Membrane
http://www.colorado.edu/intphys/Class/IPHY3730/03plasmamembrane.html
Kimball's Biology Pages, Cell Membranes
Alberts B, Johnson A, Lewis J, et al. (2002). Molecular Biology of the Cell (4th Ed.). New York: Garland Science. ISBN 0-8153-3218-1. http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=mboc4.section.1864.
Singer SJ, Nicolson GL (Feb 1972). "The fluid mosaic model of the structure of cell membranes". Science 175 (23): 720–31. doi:10.1126/science.175.4023.720. PMID 4333397. http://www.sciencemag.org/cgi/content/abstract/175/4023/720.
Lodish H, Berk A, Zipursky LS, et al. (2004). Molecular Cell Biology (4th ed.). New York: Scientific American Books. ISBN 0716731363.
Jesse Gray, Shana Groeschler, Tony Le, Zara Gonzalez (2002). "Membrane Structure" (SWF). Davidson College. http://www.bio.davidson.edu/people/macampbell/111/memb-swf/membranes.swf. Retrieved 2007-01-11.

UCI 205340050507X Candidate Number 0507 NEC student number SS121598Page

Teacher Reviews

Here's what a teacher thought of this essay

Adam Roberts

17th of Sep 2013

**** This is a good account of the investigation. The discussion and evaluation are of a high standard with good use of A level terminology. In other places, however, there could be more attention to detail and more detailed explanations. The author does not seem to be sure how to identify independent and dependent variables.

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