An Investigation of the Effects of Temperature and Sodium Dodecyl Sulfate (SDS) Treatments on the Integrity and Function of the Phospholipid Bilayer Membrane of Beetroot (Beta vulgaris) Cells.
Introduction
Cellular membranes function as a selective barrier that allow the passage of select nutrients, chemicals and waste across their boundaries. The membrane is an extremely thin layer (8 to 10 nm) thick, which is partially permeable (Reece et al. 2011). It is a phospholipid bilayer that consists mostly of lipids and proteins.
Phospholipids are essential for cells because they make up cell membranes. The hydrocarbon tails point toward the interior bilayer and is hydrophobic, which means they are excluded from water. However the hydrophilic head has an affinity for water and makes up the exterior of the membrane (Danyk, 2013-2014). The plasma membrane and its proteins not only act as an external boundary but also permit the cell to carry out its functions (Reece et al. 2011).
The integrity of a cell membrane as a barrier, and to carry out its functions is dependent on its external environment. An environmental stressor such as temperature can damage the cell. Low temperature can result in ice formation within cell (Tan et al. 2002), and higher temperatures can denature proteins embedded in a cell membrane (Reece et al. 2011). Organic compounds, like sodium dodecyl sulfate (SDS); a commonly used detergent used to solubilize biological membranes and to isolate and purify membrane proteins and membrane lipids (Tan et al. 2002) also will damaged cells.
This study uses beetroots, which contain an abundant red pigment called betacyanin that is localized almost entirely in the large central vacuole of beet cells. The vacuole is further surrounded by the tonoplast membrane, and it, by the plasma membrane of the entire cell. If the membranes are stressed or damaged, the betacyanin will leak (Danyk, 2013-2014).
The objective of this experiment is to determine the effects of two treatments; temperature and SDS concentration, on the beetroot cell membrane. The resulting betacyanin concentrations of each treatment are an indirect measure of cell damage.
Methods
Beetroot cylinder preparation
A core borer with an inside diameter of 5-mm was used to cut ten uniform beet cylinders from cooked beetroots. A razor blade was used to trim each cylinder to 15mm in length. All cylinders were placed in a wash beaker and allowed to run under cool tap water for two minutes to wash the betacyanin from the injured cells on the surface (Danyk, 2013-2014).
SDS treatment sample preparation
6ml of appropriate concentration of SDS was added to tubes labelled 0, 0.025, 0.05, 0.25, and 0.5; a cylinder from the wash beaker was added to each tube. The tubes were then left to sit at room temperature for a total of 20min, and shaken gently periodically. The cylinders were removed from each tube using a dissecting needle; tubes were then arranged from darkest solutions to lightest (Danyk, 2013-2014).
Temperature treatment sample preparation
Two cylinders were removed from the wash beaker; most adhering water was removed by briefly touching each to paper towel. A cylinder each was placed in two tubes labelled -5ºC and 5ºC and was placed in a freezer and refrigerator with respective temperatures for 15 minutes. The third, fourth and fifth cylinders were each placed in beakers with water temperatures of 70º, 45ºC and 25ºC respectively and allowed to stand for exactly 15 minutes each. Each cylinder was then removed from tubes using a dissecting needle and solutions were arranged from darkest to lightest (Danyk, 2013-2014).
Data analysis Absorbance’s (A460) of each sample were read using a Spectronic 20™ spectrophotometer set at 460nm. The concentrations of betacyanin were then calculated using a standard curve (y=0.0084x). Finally the mean and standard deviations were calculate and graphed in Microsoft Excel 2007 to identify potential
