The purpose of a cell membrane is to control the transport of substances moving into and out of a cell. The membrane is an extremely thin layer (8 to 10 manometers (nm)) thick, which is partially permeable. It consists mostly of lipids and proteins. The lipids found in cell membranes belong to a class known as triglycerides, so called because they have one molecule of glycerol chemically linked to three molecules of fatty acids. The majority belong to one subgroup of triglycerides known as phospholipids.
Despite their many differences in appearance and function, all cells
have a surrounding membrane enclosing a water-rich substance called
the cytoplasm.
In the cells of a beetroot plant, a substance called betalain is
contained within the plasma membrane. It is betalain which gives
the beetroot its characteristic red colour. If a cell is
damaged in a beetroot plant and the membrane is broken, the
betalain leaks from the cells like a dye. It is this
characteristic that can be exploited to test which conditions affect
the integrity of the cell membrane.
Because we are experimenting with the effects of temperature on the
membrane, we will place the samples of beetroot into a water baths of
varying temperatures and measure the colour change in the water.
Temperature is just one of the possible variables. Others include
effects of poisonous substances such as alcohol and/or various
solvents. So what happens when you heat this? When you heat something you give it energy. Molecules start to spin and vibrate faster. The water will 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 (think of what happens when you heat butter) so the membranes become more fragile. Proteins are remarkable machines: they're formed of coiled and folded strings of amino-acids, held together by hydrogen bonds and disulphide bridges. If you heat them too much, they will untangle and break apart (vibrations again). When this happens to the proteins spanning a lipid membrane, they will form holes that will destroy the delicate structure. Now, any pigments in the innermost compartment will spill out.
The variables kept constant
The same diameter corer is used so to keep the surface area of each beetroot the same size.
When the beetroot has been cut some of the cell membranes are broken, which means some betalain will leak out. This must be completely washed off in order to maintain the reliability of the results.
I will use distilled water to so that I have a reliable substance to test. Each piece of beetroot will be the same size (3 cm), by using a segregated knife to maintain the thickness of the surface though we could clearly see the concentration got higher as the temperature increased - Each person used a different beetroot –
The discs were cut from different parts of the beetroot - No instructions were given on how long to wash the beetroot before analysing - The water baths were not at the suggested temperatures. - Everyone used a different beetroot; there could have been variation between them.
Evaluation
As there is an anomalous result at 60oC, it is possible to assume that the experiment could have been done more accurately. There are several ways which this could have been achieved.
Firstly, the results could have been made more reliable by repeating the experiment (triplicates), and also by taking more measurements (10 rather than 5).
The results could also be made accurate in several ways. Firstly, the beetroot cylinders were not cut accurately, but only estimated. If the beetroot was cut more accurately, there would be a similar surface area on each cylinder and each cylinder would have the same potential to release betalain.
Half-life:
The half-life of beetroot pigment is 413 mins at 250C but only 83.5 mins at 600C. These values are doubled in 0.1% ascorbic acid. Metal ions speed up the breakdown – iron is particularly effective.
They are stable between pH 4.0 and 7.0 – indeed, at high temperatures they are most stable in a pH between 4.0 and 5.0 – and most fruits and vegetables are acidic!
- When the beetroot has been cut some of the cell membranes are broken, which means some betalain will leak out. This must be completely washed off in order to maintain the reliability of the results.
- I will use distilled water to so that I have a reliable substance to test with a colorimeter.
Each piece of beetroot will be the same size (3 cm), by using a segregated knife to maintain the thickness of the surface.
The reason why the amount of betalain pigment released (colorimeter reading) from the vacuole increased directly proportionally to the temperature of the water bath (from 20oC to 40oC) is because the amount of random movement of betalain molecules out through the cell membrane depends on the amount of heat energy the betalain molecules are given to convert into kinetic energy- hence the higher the temperature the more betalain lost from the vacuole.
This is because the betacyanin pigment of beet root cells is normally sequestered in the vacuole and by means of the cell membrane which maintains the integrity of the cell and the tonoplasts, it does not leak into the cytosol or the extra-cellular sap of the beet root. However when we increase the temperature the relatively weak forces holding the different parts of the polypeptide chains together (like hydrogen bonds, sulphur bridges and ionic bonds) can be disrupted very easily- this damages the vacuole and makes holes in the cell membrane, inducing leakage.
The cell membrane is also damaged and so diffusion of betalain occurs through the partially permeable membrane- the betalain molecules move from an area where they are more highly concentrated to an area where they are at a lower concentration, along a concentration gradient.
As long as the temperature does not go beyond what the membrane is
supposed to withstand, the permeability of the plasma membrane (PM) should
not be affected. There might be a higher controlled permeability for such
compounds that are supposed to transit the PM, but it will not break down,
spewing out red beet colour. So what happens when the temperature goes
beyond these limits? Water expands, putting pressure on the membranes from
within. The lipid part of the membrane liquefies, making it more prone to
leakage. The proteins that span the membrane fall apart, creating holes in
the fabric. All this combined will allow compounds to exit the cell. Why
does this happen? That is physics. Higher temperature makes all molecules
shake and vibrate more. The faster movement disrupts any ordered structure
there might have been, eventually destroying the structure altogether. So
much for the rephrasing recap
The reason why the curve starts to decrease slightly and flatten out (between 60oC and 80oC), is because although the denaturing of the protein causes a rapid rise in the amount of betalain released to start with, when the temperatures begin to get higher still, the protein's tertiary structure blocks some of the holes in the cell membrane and therefore slows down the release of betalain.
Error Evaluation
There was a source of error that may have affected the accuracy of my results. My method should have stated that I must wash the beetroot thoroughly and then dry them with a paper towel to remove excess dye. As washing each of the beetroot pieces may not have removed all of the red pigment on the outside, so this would have affected my results very slightly. Again this would only have had a small effect on my results, as a very slight increase in betalain molecules would not have changed the reading on the colorimeter.
Another source of error was the time the tubes were left in the water baths. It is impossible to take out every tube at exactly 30 minutes- you can not pick them all out at the same time with only two
hands. This would have had little affect on my results, as I would have only released slightly more betalain molecules.
Another potential source of error was that there may be different amounts of dye originally in the different pieces of beetroot, thus affecting the diffusion gradient between beetroot and water and therefore rate of diffusion.
The method should have stated to push the cylinder out with a pencil carefully- otherwise the membrane of beetroot is damaged. This again would have had little affect on my results, as I would have only released slightly more betalain molecules.
There were also limitations of my experiment. Firstly I only had an hour in which to conduct my experiment- a more accurate method would have included a larger number of results taken at different temperatures between 20oC and 60oC would have allowed me to find out more accurately where the point of denaturising occurred.
It would also have been better to have had the time to repeat each temperature more times to make the results more reliable and so I could be sure the results were not gained through chance. This may have eliminated my anomaly, but I did repeat the experiment twice and the two results on each temperature were almost identical, so this would have had very little effect on the accuracy of my results.
Another limitation was cutting the beetroot into pieces. The pieces cut had roughly the same surface area to volume ratio- but not exactly the same. My method would have been more accurate had I used a template to control the size of the beetroot pieces. This would have had a slight effect on my results because the rate of diffusion of betalain particles across the plasma membrane is increased, as the surface area of the beetroot increases. So the slightly thinner and smaller pieces of beetroot I cut would have released more betalain from their vacuole.
Another limitation was that using a thermometer is less accurate than using a biosensor. Using a stopwatch is also less accurate than using an electronic timer. However these limitations would have had very little affect on my results, as only slightly more betalain molecules may have been recorded and it is very difficult to get the exact number of betalain molecules released from the vacuole, using even the most advanced equipment.
The method only looked at one type of plant cell, so I cannot be sure that every plant cell and indeed eykaryote- which have different plasma membranes, that may be adapted to cope with heat better or worse than beetroot cells will have the same results.
I can firmly conclude that there are no apparent anomalies in my results and none of my sources of error or limitations of my experiment are enough to deem my results unreliable. However the sources of error and limitations in my results may have made my results slightly less accurate, but other students in my class found the same patterns occurring and roughly the same results- which would vary slightly between each beetroot anyway.
Cell surface membranes have a number of different functions within the cell, but their major role is to act as a selectively responsive barrier to control the entry and exit of things. According to the fluid mosaic theory, the cell surface membrane consists of a double layer of phospholipids molecules the lipid bilayer, which is a fluid like structure and contains a ‘mosaic’ of protein molecules where movements of molecules can take place.
The plasma membrane also provides a boundary to cell cytoplasm separating it from other cells. Phospholipids have a polar head, which is hydrophilic and a non polar tail which is hydrophobic. Proteins have a hydrophobic part which is buried in the lipid bilayer and a hydrophilic part which can be involved in a variety of activities.
Some proteins move freely some are fixed in one place. Some proteins only span the top bilayer which is the extrinsic or the bottom which is the intrinsic or they may span the entire membrane. Some proteins on the outer edge of the membrane have carbohydrates molecules attached, usually short sugar chains called glycoprotein or lipids with carbohydrates attached to them called glycolipids. These are important in cell recognition and the immune system. Membranes are therefore vital to cellular function.
As long as the temperature does not go beyond what the membrane is
supposed to withstand, the permeability of the plasma membrane (PM) should
not be affected. There might be a higher controlled permeability for such
compounds that are supposed to transit the PM, but it will not break down,
spewing out red beet colour. So what happens when the temperature goes
beyond these limits? Water expands, putting pressure on the membranes from
within.
The lipid part of the membrane liquefies, making it more prone to
leakage. The proteins that span the membrane fall apart, creating holes in
the fabric. All this combined will allow compounds to exit the cell. Why
does this happen? That is physics. Higher temperature makes all molecules
shake and vibrate more. The faster movement disrupts any ordered structure
there might have been, eventually destroying the structure altogether.
References
Modern Biology (Revised Edition 1995) CD ROM: Albert Towe
Wikipedia:
Science And Plants for Schools: http://www-saps.plantsci.cam.ac.uk
