BIOL 2070H Lab 2

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1 Divine Orji BIOL 2070 H W13 January 31, 2024. Chaitali Shetty, Effects of Sodium Dodecyl sulphate (SDS) Detergent on the permeability of beetroot cell membrane.

2 Abstract I n this experiment, we aimed to explore how different solutions and their concentrations affect cell membranes' properties. We used beetroot to assess and measure pigment release in four solution treatments: 0% SDS detergent, 1% SDS detergent, 3% SDS detergent, and 5% SDS detergent. The method involved soaking beetroot sections in each solution and observing pigment leaching over time. After an hour, we removed the beetroot samples and analyzed the solutions using a spectrophotometer to measure pigment absorption. Interestingly, the solutions containing 1% and 3% SDS detergent had the most significant impact on beetroot pigment release. In conclusion, our results support the idea that cell permeability is affected by varying concentrations of SDS detergent, although our initial predictions were not confirmed. Introduction The cell membrane is a lipid structure that encloses the cell and serves as a protective barrier that selectively allows entry of nutrients when needed, and facilitates the removal of waste (Alberts et al., 2014). The distinctive red colour of Beetroot cells is derived from the betalain pigments that are kept within their cells. Through the examination of these pigments release in the event of membrane compromise, we can get knowledge regarding the stability and functionality of membranes. Sodium docedyl Sulphate (SDS) is an anionic surfactant that possesses both hydrophilic and hydrophobic sulphate groups (Debbie, 2024). Its amphipathic nature allows it to form micelles and break hydrophobic interactions, denaturing proteins by protein binding to hydrophobic areas (Debbie, 2024). The hypothesis proposed that cell permeability will be affected by different concentrations of SDS detergent. If this is the case,

3 then more pigment will be released at larger concentrations of the SDS detergent. This is because SDS detergent breaks down the cell membranes, so higher concentrations should cause it to break down and release its pigment quicker. To test the hypothesis, we conducted an experiment where beetroot chunks were immersed in different concentrations of SDS detergent. The levels pf pigment absorption and leaching were measured and observed. Methods We received a beetroot and cut it into twelve pieces, each measuring about 10mm by 7mm by 7mm. For our experiment, we prepared three different solutions along with a control, each replicated three times. The control consisted of three test tubes filled with 5 ml of water. The remaining nine test tubes contained a mixture of water and SDS detergent. Specifically, three tubes had a 5ml solution with 1% detergent, another three had 3% detergent, and the last three had 5% detergent. We achieved the desired concentrations by diluting the 5% detergent solution with the C1V1 = C2V2 calculation. After soaking the beetroot pieces in the solutions for an hour, they were removed, and the solutions were transferred to new tubes. We then measured the absorbance of the pigment in each solution using a Synergy HTX multi-mode plate reader). The optical density of each solution was determined by inserting the test tubes into the cuvette holder one by one for measurement. Results

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4 Water 1% SDS 3% SDS 5% SDS 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 Avg Absorbance of Leeching Pigment (abs) vs Concen tration of SDS Detergent with water (%) Concentration of SDS Detergent with water (%) Average Absorbance of Leeching Pigment (abs) Figure 1: The mean absorbance of beetroot pigment released per concentration of SDS detergent, with a water treatment included, and error bars showing standard deviation. Figure 1 reveals a clear trend in the data. As the concentration of SDS detergent rises, there is a decrease in pigment leaching. Notably, the average absorbance at 3% detergent concentration is lower than that at 1%. The most significant decrease in absorbance occurred with 5% SDS detergent and 0% (water), while the most notable increase was observed between 1% and 3% SDS detergent. The control treatment with water/0% SDS detergent had a mean absorbance of 0.0503 with a standard deviation of 0.0066. For the 1% SDS detergent treatment, the mean was 0.662 with a standard deviation of 0.5001, while the 3% SDS detergent treatment had a mean of 0.3646 and a standard deviation of 0.1625. The 5% SDS detergent treatment showed a mean absorbance of 0.197 with a standard deviation of 0.0719. The absorbance values for each test tube are provided in Appendix A. Additionally, Figure 1 suggests that solutions containing detergent have overlapping standard deviations. Qualitatively, during the initial thirty minutes, we observed pigment release, displaying a slight-red tint across all twelve test tubes. However, in the last thirty minutes, the pigment appeared to spread further into the tube. We observed more

5 pigment leaching in the 1% concentration, followed by the 3% SDS concentration, with 5% showing the least pigment release. Discussion Our hypothesis that cell permeability is affected by different concentrations of SDS detergent was supported based on the data shown in Figure 1. However, our prediction was not supported as the most significant decrease in absorbance occurred with 5% SDS detergent and 0% (water) while the most notable increase was observed between 1% and 3% SDS detergent, which was contrary to our prediction that higher concentrations would result in more pigment release. These inconsistent findings may have been caused due to several miscalculations while undergoing the experiment, like the difference in beet size. The beet size in one of the 1% SDS detergent test tubes were observed to be a lot bigger than the rest of the beets in the remaining 11 tubes, which most likely caused it to have a lot higher absorbance. Other factors like the plate reader could have also interrupted with reading the results more accurately. Another possible explanation could be that the concentrations of the SDS being very high (5%) might have damaged the cell membranes and inhibited pigment release. Journals publishing results from comparable trials have reported findings like ours. Two studies examining the interaction of detergents with proteins or cell membranes were conducted. Reynolds and Tanford (1970) discovered that the SDS detergent damaged the protein membrane, while Sagawa et al. (1993) demonstrated that membrane breakdown accelerates with greater concentrations of solution. This happens because of hydrophobic damage, which intensifies with increasing detergent solution concentration (Reynolds and Tanford, 1970; Sagawa et al., 1993). According to Schchuck et al. (2003), detergents have an impact on the cell membrane by

6 damaging and upsetting the hydrophobic lipids in the lipid bilayer interface, which causes the membrane to rupture. Moreover, our most alarming and significant “error” was the Treatment A1 (1% SDS) with the most absorbance and most pigment released, and we figured it was because of the big difference in size of beetroot. In conclusion, our hypothesis was supported and while our results do not support the prediction by showing a decrease in pigment leaching with increasing detergent concentration, unexpected findings suggest complex interactions. Further research is needed to fully understand the relationship between detergent concentration, cell membrane integrity, and pigment release. Reference Alberts, B., D. Bray, K. Hopkin, A. Johnson, J. Lewis, M. Raff, K. Roberts, and P. Walter. 2014. Essential Cell Biology, 4th ed. Garland Science, New

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7 York, NY. p. 360-369 and 385- 389 Alberts, B., D. Bray, K. Hopkin, A. Johnson, J. Lewis, M. Raff, K. Roberts, and P. Walter. 2014. Essential Cell Biology, 4th ed. Garland Science, New York, NY. p. 360-369 and 385- 389 Alberts, B., D. Bray, K. Hopkin, A. Johnson, J. Lewis, M. Raff, K. Roberts, and P. Walter. 2014. Essential Cell Biology, 4th ed. Garland Science, New York, NY. p. 360-369 and 385-389 Sagawa, H., S. Tazuma, and G. Kajiyama. (1993). Protection against hydrophobic bile salt

8 induced cell membrane damage by liposomes and hydrophilic bile salts. Am J Physiol 264:835–839 Table 1: Absorbance of beetroot pigment raw data from the Spectronic 20 readings in Part II. Percent Concentration (%) Repetition Spectrophotometer Absorbance Reading (abs) 2% A 0.995 2% B 1.47 2% C 1.38 1% A 1.29

9 1% B 1.30 1% C 1.39 0.5% A 1.27 0.5% B 0.84 0.5% C 1.13 0% (Control) A 0.15 0% (Control) B 0.13 0% (Control) C 0.32 Solution Dilution Calculation Example (Concentration): C1V1 = C2V2 (0.2) * (5ml) = C2 *(10ml) C2 =0.1

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10 Table 1: Absorbance of beetroot pigment raw data from the Spectronic 20 readings in Part II. Percent Concentration (%) Repetition Spectrophotometer Absorbance Reading (abs) 2% A 0.995 2% B 1.47 2% C 1.38 1% A 1.29 1% B 1.30 1% C 1.39

11 0.5% A 1.27 0.5% B 0.84 0.5% C 1.13 0% (Control) A 0.15 0% (Control) B 0.13 0% (Control) C 0.32 Solution Dilution Calculation Example (Concentration): C1V1 = C2V2 (0.2) * (5ml) = C2 *(10ml) C2 =0.1

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