Effect of Solvent Exposure and Temperature on Beet Cell Membrane

The Effect of Solvent Exposure and Temperature on Beet Cell Membrane Integrity Introduction: Cell membranes are vital to proper cellular function • Cell membranes consist of a lipid bilayer made up of phospholipids • Phospholipids are amphipathic molecules meaning they have a dual nature in that they show both hydrophilic (water-loving) and hydrophobic (water-fearing) properties • The amphipathic nature of phospholipids causes them to spontaneously form bilayers in water based solutions o The inner and outer surfaces of the bilayers are hydrophilic and can interact with the aqueous environment, while the core of the bilayer is hydrophobic and able to exclude water and other polar molecules o Because of these properties, phospholipids bilayers are able to serve multiple functions including: ? act as barriers to protect the cell from the outside environment ? house proteins and carbohydrates necessary for cell to cell and extracellular communication ? rganize and provide surface areas for metabolic reactions • While the phospholipids bilayer is a highly organized system with multiple functions, it is also highly dynamic and can be functionally described using the fluid mosaic model (FMM) o FMM characterizes the phospholipids bilayer of the cell membrane as highly dynamic and fluid meaning that the position (location) of any given phospholipids in the membrane is not fixed but fluid; thus, individual phospholipids can change positions are needed based on the current conditions. o Similarly proteins and carbohydrates are generally mobile in the membrane as well. • This experiment was aimed at investigating the conditions that affect cell membrane integrity. o Because cell membranes are part of a living system they are sensitive to environmental conditions o Like most biological systems, cell membranes show a relatively narrow range of function with respect to pH, temperature, and exposure to organic solvents • The experimental model employed was red beets Red beet cells contain large, membrane bound central vacuoles that house a pigment, betacyanin o When cell and vacuole membranes are in tact, betacyanin in contained within the cell o If the cell and vacuole membranes are disrupted, betacyanin can leak in the surrounding solution turning it pink o Higher amounts of membrane damage will lead to more leakage of betacyanin pigment from the cells. The more pigment present the higher the absorbance reading. o Therefore, betacyanin release, as determined by measuring absorbance, serves as a proxy for quantifying membrane damage. • In this experiment, we tested the effect of changing two environmental conditions on cell membrane integrity.

First, we tested the effect of temperature on cell membranes o To accomplish this we exposed beet cells to temperatures ranging from -5(C to 70(C o Because cell membranes maintain integrity under a narrow range of conditions, we hypothesize that exposure to higher temperatures (50-70(C) and extreme low temperatures (-5(C) will cause severe damage to the cell membranes which will result in higher amounts of betacyanin being released from the cells. • We also tested the effect of exposing beet cells to two different solvents o Methanol and acetone are organic solvents capable of dissolving hydrophobic species such as phospholipids o Acetone is generally considered a stronger organic solvent compared with methanol o Thus, we hypothesize that exposure to acetone will cause more damage to beet cell membranes compared with exposure to methanol.

Additionally, we hypothesize that higher concentrations of acetone will cause more damage than lower concentrations. Similarly, higher concentrations of methanol will also cause more damage. Results (the data numbers are for example only and meant to reflect the general trend in the class; data from individual groups will vary): • Experiment 1: The effect of temperature on cell membrane integrity o We tested the effect of exposing beet cells to various temperatures ranging from -5(C to 70(C. (Question) o To do this, uniform pieces of beet were placed in empty test tubes then incubated at a specific temperature. (How the question was addressed) ?

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Following this water was added to the test tube and the cells were incubated for 20 minutes. ? After incubation, the water was transferred to a clean tube and absorbance at 425 nm was determined. o Absorbace readings were similar at temperatures of 5? C, 25? C, 40? C, 55? C, and 70? C (A425 0. 25-0. 30) (Figure 1) (Experimental data) o Incubation at -5? C showed a dramatic increase in absorbance (A460 = 0. 95) (Figure 1) (Experimental data) o We conclude from this data that temperatures ranging from 5-70? C have only minor effects on cell membrane integrity. In contrast; exposure of cells to a temperature below freezing causes much more membrane damage. (Conclusion) Experiment 2: The effect of solvent on cell membrane integrity o We tested whether exposure to two different organic solvents, methanol and acetone, disrupted cell membrane integrity (Question) o Beet cells were incubated in 10%, 25%, and 50% methanol or 10%, 25%, and 50% acetone. Additionally, additionally cells were also incubated in saline, a solvent that mimics physiological conditions. (How the question was addressed) ? Following exposure to solvent, membrane damage was determined by measuring absorbance. o Incubation in 10% or 25% methanol yielded absorbance readings of 0. 2 and 0. 25 respectively; however, incubation in 50% methanol showed an increase in absorbance to 0. 5. Figure 2) (Experimental data) o Incubation in 10% or 25% acetone produced higher absorbance readings compared to the same concentrations of methanol (0. 4 and 0. 42 respectively). Similar to 50% methanol, 50% acetone yielded the highest absorbance reading of 0. 8 (Figure 2). (Experimental data) o Taken together, these data show that acetone causes more membrane damage than methanol and that concentrations of 50% solvent are more damaging than lower concentrations. (Conclusion) Overall Conclusion: • We hypothesized that temperatures outside of the normal physiological range would disrupt membrane integrity causing betacyanin to leak out of the cells. Our data shows that our hypothesis was partly correct, low temperature, -5? C, inflicts the most damage to cell membranes while higher temperatures, ranging from 55-70? C induce little damage when compared with physiological temperatures. • We also hypothesized that exposure to high concentration organic solvents would disrupt membrane integrity and that acetone, being a stronger solvent, would show more damage than methanol. o In this case our hypothesis was support. Acetone showed more damage as measured by betacyanin release than methanol. Additionally, the highest concentration of each solvent showed the most damage. Data: [pic] Figure 1: The effect of temperature on cell membrane integrity.

Cells were exposed to temperatures ranging from -5C to 70C. Following incubation at the indicated temperature, water was added and the cells were allowed to incubate at room temperature for 20 minutes. Damage to cell membrane integrity was assessed by quantifying leakage of the pigment betacyanin via measuring light absorbance at 460 nm. [pic] Figure 2: The effect of solvent on cell membrane integrity. Cells were incubated in the presence of methanol, acetone, or saline 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.

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