Question

Stability of a biological membrane
[Protocol] [HS-level]
Keywords: Structural biology, organic chemistry, biochemistry

Answer 1

\(\Large \sf Introduction\)
The majority of molecular biological processes are directly connected to membranes or surrounding areas. Cells are therefore divided into multiple subareas using membranes. Most plant cells contain a central vacuole rich on water, which fills out 95% of the cells cytoplasm. In the roots of Beta vulgaris, also known as beetroots, the vacuole is filled with the water-soluble red colorant, which is characteristic for beetroots. The colorant is known as betanin and belongs to a group of pigments called betacyanins. The membrane surrounding the vacuole reassembles very much the structure of the cell membrane, but is a little thinner, with the vacuole being ~7.5 nm and the cell membrane ~10 nm.
Studies of alcohol’s (ethanol) intoxicating mechanisms, reveals that ethanol dissolves membranes lipid layer, thus destabilizing the membrane making it more fluent. It is therefore speculated, if the concentration of alcohol became sufficiently high, the membrane would be destroyed which would resolve in the cell’s death. If the vacuole-membrane of beetroots was destroyed, the content of the vacuole would be emptied into the cell’s surroundings. This would have the consequence that the color intensity would be proportional with the number of destroyed cell membranes.

In this project we will investigate alcohols destabilizing effect on biological membranes, using roots of B. vulgaris as model organism in the presences of the alcohols; methanol, ethanol and 1-propanol in the concentration range of 0-30%. By monitoring the change in color intensity, using spectrophotometry, we get an estimate of the amount of destroyed membranes as a function of the alcohol centration. From the results we can then determine the apparent maximum alcohol centration which does not result in membrane destruction and the concentration in which it is assumed 50% of the membranes are destroyed.

\(\Large \sf Protocol\)
A) Extraction of betanin:
1. Microtiter plates are filled with alcohols and water according to the wished concentration using a disposable syringe on 1 mL. The total volume in each well is recommended to be 4.0 ml.
2. The beetroot dice are created by using a pommes frites cutter by cutting ones and a second time perpendicular to their longitudinal direction.
3. The beetroot dice are placed in water in a 250 mL beaker and carefully stir around with a spatula. Change the water (as minimum 3 times) until the dice no longer gives any color to the water (use a spectrophotometer for the most accurate controls).
4. Remove the water from the dice and very carefully dry the dice using some paper towels. With a tweezer carefully transfer the dried beetroot dice to the microtiter plates.
5. With a match (do not let the sulfur get in contact with the solution!) stir carefully in the wells on shift. The total amount of time for the dice’s exposure to alcohol should be approximately 10 min.
6. After the approximately 10 min, remove the beetroot dice from the wells carefully with a tweezer. Be very carefully not to damage the dice by any means when doing this. The beetroot dice can next be discarded.

B) Determination of betanin concentration:
Using a spectrophotometer calibrate with 100% water at the wavelength 470 nm. Next measure all the samples from the microtiter plates. Data should be visualized and should be figured out by the one performing the experiment, possibly under guidance.

Considerations for additional experiments and report writing:
1. Give a schematic of a cell membrane along with a description
2. The membrane surrounding the vacuole was thinner than cell membranes (7.5 nm ≠ 10 nm). What determines the thickness of a membrane?
3. We washed the dice before we started experiment to remove any excess betanin. But why did this betanin come?
4. We are using very high concentrations of alcohol:
a. What is the limit of alcohol for driving?
b. What is the percentage of alcohol required to die from alcohol poisoning and what is the mechanism of death?
5. We are using 3 different alcohols in this experiment:
a. What similarities do the 3 alcohols have?
b. What differences are there in the alcohols structure?
c. What part of the molecule is hydrophobic?
d. What part of the molecule is lipophobic?
6. How would alcohol molecules place them self relative to the cell membrane and why would it cause the membranes to become more fluent?
7. Which of the alcohols are the most harmful for the membranes? Include the concentration in which 50% of the membranes are destroyed along with the highest concentration no observable damaged could be detected. (Include statistics if the experiment has been performed multiple times).
8. Are there any correlation between alcohol’s chemical structure and their harmful effects on cell membranes based on your experimental data?
9. Make a prediction outside your data range if any correlation is found in 8).

Answer 2

Any sort of guidance or questions regarding actual performance of the experiment can be addressed to me.

The reason why I put this up, is among others due to the increasing number of replica experiments I'm correcting in the high school where I used to go and today teach.

Answer 3

This is awesome. Thanks for sharing!!!

Answer 4

great work

Answer 5

Wow!
Thanks! :)