Effect of Different Concentrations of Detergent on Nature of Cell Membrane

How protective our house is because of the door. Similarly, the cell has a cell membrane that provides the barrier separating it from the external world. It allows only some components to pass through while rejecting others. It is made up of Lipid bilayer which is hydrophobic on the outside and hydrophilic molecules on the inner side(Alberts, 2014). The membrane can be solubilized by certain molecules known as detergents. They form micelles with the lipid bilayer and helps in separating Detergent-lipid and protein-mixed micelles (Lichtenberg. D., et al. 2013). They also found that it requires energy to break these cellular membranes which is fastened by detergents. Detergents such as Sodium Dodecyl sulfate are widely used in Biology these days to lyse the cell membrane.

The current study aims to find the permeability of cell membranes to different concentrations of detergent which in fact is the nature of the cell membranes. Evenly cut beetroot was soaked in different concentrations of detergent and one test tube of water for 30 minutes and the resulting solutions were analyzed by a spectroscopic method to check the absorbance. A spectroscope is a device used to measure the absorbance or transmittance of compounds by the emission of wavelength. Based on previous work, it was hypothesized that different concentrations of detergent will affect the permeability of the cell. Permeability here refers to the lysis of cell to allow interchange of components. Detergents interaction with lipid bilayer was assessed at different concentrations. So it was predicted that a higher concentration of detergent will cause more pigmentation and more permeability.

Methods: For part 1, there were three unlabeled solutions and one labelled solution. Solution from each bottle was mixed with cow‘s blood to examine under the microscope. Cells were identified as Hypotonic, isotonic or hypertonic on the basis of swelling or shrinking in them. For part 2, there were two groups, the Treatment group and Control group. The treatment group was SDS which was used in different concentrations for testing on beetroot as 2%, 1% and 0.5%. The Control group was room temperature water. These solutions were mixed with the beetroot and allowed to stand for 30 minutes. These aliquots were diluted to 10% observed under the Spectron 20 spectrometer to check the absorbance of chemicals inside the cell(Leitz. D., 2018) The results from spectrometer was compared to find the mean and Standard deviations. Results: Line graph showing absorbance by beetroot cells as a measure of concentration of detergent. The graph shows the amount of pigmentation beetroot has due to the addition of detergent in it. It is evident from the graph that the pattern follows an increasing trend of absorbance with a slight bend between 0.5-1 %. Visual observation from the test tube was also made. The darkest color was obtained in 2% solution with the color going to brighter side as moving down to 0%. 2% was the maximum concentration in this experiment while the control group has a percentage of 0 i.e. it is water. It shows that water alone cannot help in determining the permeability of cells to chemicals.

As the detergent concentration increases, cells allows more pigment to go out and shine their color. Discussion: The result section shows that absorbance increases with the increase in the concentration of Sodium Dodecyl Sulfate. The mean shows an absorbance at 1.58 which might be due to dilution at the end of experiment or some errors. The mean is actually the mean of different measurement taken during the experiment. The graph and in turn results confirms the prediction that higher concentration will cause more pigmentation and more permeability. More pigmentation means darker color was seen in the test tubes. Our hypothesis was also supported by the fact that higher concentration of detergent affected the permeability of cell to pigmentation.With each increasing concentration of detergent there was a slight increase of pigment expelled from the cell. This is supported by the fact that cell membrane is made up of hydrophobic part as lipid on both sides with hydrophilic part protruding inside the lipid bilayer.

Detergents also have both hydrophilic and hydrophobic parts (Alberts 2014). When they are mixed into each other, the hydrophobic part of the detergent attach itself to lipids of cell membrane and hydrophilic part attach to water-soluble part, pulling them apart. This way the cell membrane is dissolved and pigments of the cell comes out. As concentration of detergent got higher it showed more and more pigmentation in the tube(Womack D.M., et al.,1983). The red color in the test tubes was due to this pigment. The graph in the result section might be bent between 0.5-1% due to the errors in performing experiments. Findings from one similar study have shown that detergents should be used at a concentration, micellar concentration where they work precisely (Womack D.M., et al.,1983). Any concentration above that would denature the protein and other products can also catabolize which could lead to huge difference in readings. It might be possible that the concentration for this detergent would have been around 0.5-1% where it works maximally. Another possible cause of error could be defect in spectrometer deflection or maybe the improper use of spectrometer. Also, dilutions during the spectrometry could have gone wrong and made a sudden curve in the graph. Spectroscopy misalignment can cause a shift in results if it is not corrected before the use (Stutz J., Platt U.,1996)

As Biology is a growing filed, continuing research on the topic could be done to find more about the cell membrane like Why the cell is lysed by only certain compounds not by other? Absorbance as a measure of detergent concentration with different sets. The mean was taken for each of them to make inferences from the statistical data. Mean came out to be 1.73, 1.83, 2.4, 0.34 in the order as the table is made. The observations made during the lab for solutions with different concentrations when mixed with cow‘s RBC. All these solutions explains a phenomenon known as Osmosis which states that water moves from lower solute concentration to a higher concentration. A solution is said to be isotonic if the solute concentration of the cell is same as the concentration of the cell. That’s what solution C and labelled solution have in common. They are of the same size so no water molecule leaves or enters the cell. Solution A is hypertonic because the solute concentration on the outside of the cell is more than the concentration inside. So, as a result of Osmosis, water moves outside the cell to compensate for the increase in solute concentration. Cell shrinks to about many times with its cellular components compressed giving it a spiky appearance. The solution labelled as “B” is hypotonic because the cell has more solute concentration on its inner side than the outside. So there is the net movement of water molecules inside, making the cell to swell. That is why the cell looks exploded.

References:

1. Bruce, A. et. al. 2014. Membrane Structure. In: Essential Cell Biology. Garland Science, United States of America. p. 359-377.
2. Huber, R., D. Leitz. 2018. OSMOSIS AND THE NATURE OF CELL MEMBRANES Trent University, Peterborough ON.
3. Stutz, J. and U. Platt. 1996. Numerical analysis and estimation of the statistical error of differential optical absorption spectroscopy measurements with least-squares methods. Applied Optics. 35: 6041-6053.
4. Womack, M.D., D.A. Kendall., R.C. MacDonald. 1983. Detergent effects on enzyme activity and solubilization of lipid bilayer membranes. Biochimica et Biophysica Acta (BBA) - Biomembranes, 733: 210-215. https://doi.org/10.1016/0005-2736(83)90524-2
5. Lichtenberg, D.,1 H. Ahyayauch2., A. Alonso., 2F., M.Goñi2 .2013.Detergent solubilization of lipid bilayers: a balance of driving forces.Trends in biochemical sciences, 38: 85-93