Membrane Lab Report

The cell membrane consists of mostly phospholipids and proteins which gives the cell its selectively permeable nature. The function and permeability of the cell membrane depends on its whole structure. When destroyed, the permeability of the cell membrane is disrupted causing cellular contents to leak out. When cells are cut, the cell membranes are mechanically ruptured. High temperature disrupts the structure of proteins and certain chemicals such as acetone and methanol dissolve the phospholipids, leading to damage of the membrane and therefore leads to increased permeability. Beetroot is the material used in this experiment to demonstrate the effects of high temperature and chemicals on the permeability of the cell membranes. Beetroot contains a red pigment called betacyanin, which is located in the large central vacuole of the beetroot cells. The vacuole is enclosed by a single membrane called tonoplast and the whole cell is enclosed by cell membrane made up of phospholipids. As long as the cells and their membranes are intact, the pigment will remain inside the vacuoles. However, if the membranes are damaged, betacyanin will leak out and produce a red colour in the surrounding water. More cells being damaged will cause a higher red colour intensity in the solution. The beetroot cylinders were repeatedly washed following cutting until not more colour appeared in the wash water. The aim of this experiment is to observe the effect of physical and chemical stress on the membrane permeability of beetroot.

METHOD
A large beetroot was washed with water. 14 cylinders were cut, all to the exact length (approx. 1cm with all the skin removed, including each ends) using a cork borer (approx. 10mm diameter). All of the beetroot cylinders were washed under running tap water for a duration of 5 minutes until no more colour appeared in the wash water or on the surface.

The Effect of High Temperature

Each cylinder was gently held using forceps and placed in a water bath to incubate at 45℃, 50℃, 55℃, 60℃, 65℃, and 70℃ for 60 seconds. As a control, an unheated cylinder was placed in a boiling tube containing distilled water at room temperature (approx. 20℃). After heating, all the cylinders were placed into a boiling tube containing 15ml of distilled water and were then allowed to stand for 45 minutes. After the 45 minute period, the beetroot cylinders were removed from the distilled water and pigment leakage was recorded using a spectrophotometer set at 460nm. Distilled water was used as the blank. The data is shown in the graph below. The absorbance is a measure of the amount of light absorbed by the sample. The darker the sample, the greater the absorbance.

The Effect of Freezing

A single cylinder was cut from a beetroot that was frozen (-17℃) and was then allowed to defrost. Again, the cylinder was rinsed under running tap water and was then put into a boiling tube containing 15 ml of distilled water and left at room temperature for 45 minutes. After the 45 minute period, pigment leakage was recorded using a spectrophotometer set at 460nm. The Effect of Organic Chemicals

6 washed beetroot cylinders are placed into a bottle, each of 10ml of the following solvents (1% acetone, 25% acetone, 50% acetone, 1% methanol, 25% methanol and 50% methanol). The bottles were left to incubate for 20 minutes and then the beetroot cylinders were removed. The membrane permeability/damage was quantified visually depending on the amount of pigment, betacyanin.

The higher the temperature, the more betacyanin is lost from the vacuole. This is because the betacyanin pigment of beet root cells is normally seized in the vacuole and by means of the cell membrane which maintains the integrity of the cell and the tonoplast, it does not leak into the cytosol 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) can be disrupted very easily- this damages the vacuole and makes holes in the cell membrane, inducing leakage. The reason why the line starts to flattens out (between 45(℃) and 50(℃)), is because although the denaturing of the protein causes a rapid rise in the amount of betacyanin released to start with, when the temperatures begin to get higher still, the protein’s structure blocks some of the holes in the cell membrane and therefore slows down the release of betacyanin.

The data above shows that acetone showed more damage as measured by betacyanin release than methanol. Additionally, the highest concentration of each solvent showed the most damage. Cells were incubated in the presence of methanol and acetone at the indicated concentrations. Following incubation, damage to cell membrane integrity was assessed by quantifying leakage of the pigment betacyanin via measuring light absorbance at 460 nm. This data supported the portion of our hypothesis that states, “If a higher percentage of solution is added to a cellular membrane, then the colour of solution will be stronger”. One example of how this data supports our hypothesis is found in the relationship between the acetone 25% solution and the acetone 50% solution.

DISCUSSION
There were a number of sources of error that may have affected the accuracy of my results. Firstly I kept a note of the time and timed all the water baths for 45 minutes, so there was a slight delay however between the placing of the tubes in each water bath, I had to walk to each water bath so it would have affected my results slightly. This would only have had a small effect on my results, because the vacuole of the beetroot would have only released slightly more betacyanin, as the molecules would have only had a very small amount more heat energy to convert into kinetic energy. Secondly, when I washed each of the beetroot pieces I may not have removed all of the red pigment on the outside, so this would have affected my results very slightly. 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.

An increase in temperature will affect the permeability of the cell membrane. Low surrounding temperature such as 30℃ and 40℃ does not have a significant effect on permeability. At 50℃ and 60℃, permeability starts to increase while at over 70℃ the cell membrane becomes freely permeable due to protein structure destruction. Meanwhile, according to the result of this experiment, the temperature to denature the function of protein is approximately at 70℃. Another limitation was cutting the beetroot into pieces. The pieces cut had roughly the same surface area – but not exactly the same. This would have had a slight effect on my results because the rate of diffusion of betacyanin 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 betacyanin from their vacuole. 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.

REFERENCES
Reed,R; Holmes, D; Weyes, J and Jones, A. Practical Skills in Biomolecular Sciences, 4th Ed., 2013, Longman

Willis, J. (2004) Data Analysis and Presentation Skills: An Introduction for the Life and Medical Sciences. Wiley, Chichester.

APPENDIX- Safety Assessment

1. General Aspects
Gloves must be worn at all times to protect from pigment staining. Safety glasses must also be worn throughout the lab to protect the eyes from chemicals. Potential injury may arise from razor blades and cork borers. Lab coats must be fully fastened to protect clothes from staining. 2. Chemical Hazards

The solvents (acetone and methanol) should not be inhaled as it can cause damage to mucous membranes. They can also cause damage to skin permeability barrier. 3. Disposal of Material and Equipment

All gloves and pipette sleeves must be disposed of in the blue waste bins. The used pasteurs and cuvettes go in the pots on the bench. All of the other items go into separate white buckets. Bottles containing solvents must never be emptied.