Effect of temperature and SDS concentration on cell membranes of beet root cell
Five test tubes were labelled with the appropriate SDS concentrations to be tested. 6ml of 0, 0. 025, 0. 05, 0. 25, and 0. 5 %SDS concentration were added to each appropriately labelled tube. A beet cylinder was then placed in each tube for 20 minutes and gently shaken occasionally. The rest of the procedure was performed as outlined in the laboratory manual (Danyk, 2013/14) Data collection and measuring absorbance The tubes from both experiments were arranged from the palest to darkest and placed in a spectrophotometer to measure the absorbance.
Each absorbance was then converted to determine the concentration using the standard curve formula. The curve was prepared by plotting various known betacyanin concentrations with their respective absorbance. The concentrations were calculated using the formula x=y/0. 0084. Where x is the independent variable representing betacyanin concentration (µM) and y is the dependent variable representing betacyanin absorbance (460nm). Data was collected from the same studies done by other people and the mean and standard deviation calculated.
The mean data and standard deviation were obtained from the class data and then plotted on two graphs using excel. Results Figure 1. The Effect of SDS concentration on the membranes of the beetroot cell obtained from the Mean and Standard Deviation of Class Data. From Figure 1, the results show an increase in the betacyanin concentration as the % SDS concentration increased. The first three concentrations do not have a great effect on the betacyanin concentration, however, as the %SDS increased to 0. 025 there was an obvious rise in the betacyanin concentration. The standard deviations for the 0. 25 and 0.5 concentrations show a large deviation from the mean value, implying varying values for the betacyanin concentrations at that amount of SDS. The data however clearly reveals an effect on the membranes of the beetroot cells. Figure 2. The Effect of Temperature on the membranes of the beetroot cell obtained from the Mean and Standard Deviation of Class Data. Figure 2 shows increasing betacyanin concentrations at -5? C as well as 70? C. The other temperature treatments also resulted in varying betacyanin concentrations. The temperature treatment at 25? C resulted in the least amount of betacyanin concentration.
The standard deviation at -5? C showed least deviation from the mean data whereas the deviations for the other treatments from the mean value increased as the temperature increased. The results show a steady decrease in betacyanin concentrations from -5? C to 25? C, a gradual increase to 40? C and then a sharp increase to 70? C. Discussion The results supported the hypothesis that at extremely high concentrations of SDS concentrations and at extremely low and high temperatures, there will be a deeper red color in the water surrounding the beetroot, hence a greater betacyanin concentration.
The increased concentration at -5? C could be due to rigidity within the phospholipids. A membrane remains fluid as temperature decreases until finally the phospholipids settle into a closely packed arrangement and the membrane solidifies. (Reece et al. , 2011) The rigid nature of the membrane would make it easy for cracks to occur within the cell and the betacyanin would leak out. The solidified membrane makes it lose cell integrity, since membranes must be fluid to work properly (Reece et al, 2011).
When a membrane solidifies, its permeability changes (Reece et al. , 2011) this explains the leaking of the betacyanin from the vacuolar membrane, as in Figure 2 at -5? C. Also from Figure 2, the betacyanin concentration at room temperature is similar to that at 5 ? C. Expected results should have indicated a slightly larger difference in betacyanin concentrations between the temperatures, since the temperature at 5 ? C is colder than 25 ? C (room temperature) hence, there would be more rigidity in the membrane at 5 ? C than at 25 ? C.
Similar betacyanin concentration readings could also be due to delay in the addition of 6ml water to the beet after it was removed from the refrigerator. The delay could have caused a change in the temperature of the beetroot cylinder, hence changing the structure of the membrane from its previous structure at 5 ? C. Membranes that are too fluid cannot support protein function either and so do not function appropriately. (Reece et al, 2011) Rapidly moving phospholipids would be unable to support membrane proteins and the structure of the membrane would be compromised.
The change in the structure would cause an opening in the cell which affects membrane integrity. This explains why higher temperatures also have high amounts of betacyanin concentration, as seen in Figure 2 at 70 ? C. The extreme fluidity of the membrane caused the betacyanin to leak out because the cell was unable to maintain its structure, and therefore lost cell integrity. Detergents can be used to remove proteins from cell membranes, (Freeman, 2002) the absence of proteins destroys the cell membrane structure which makes it lose its integrity.
SDS is a detergent and increased concentrations would remove proteins from within the cell membrane. The cell integrity is lost because of the composition of the membrane which includes integral and peripheral proteins. These proteins maintain the membrane function (Reece et al). SDS also contains sodium which is usually part of the substances within the membrane. The cell membrane has a function which involves regulation of Na+ ions by driving them out, and allowing K+ cells in.(Reece et al, 2011) Increasing SDS concentration forces the cell to leak betacyanin since the concentration gradient is tampered with and the absence of the proteins within the cell membrane makes it lose its structure leading to increased betacyanin concentration as seen in Figure 1. The relatively large values for some of the betacyanin concentrations make the data obtained fairly reliable. The plotted data was from the means and standard deviations of a class data, this implied the experiment was carried out more than once to obtain the data and errors were included.
The objective of the experiment was to study membrane integrity under stresses of SDS concentration and temperature. The results show that membrane integrity is disrupted by extremely low and high temperatures as well as high SDS concentrations. This suggests that cell membrane integrity can be compromised by stresses that are applied to it. A further study would be to test the effect of potassium chloride concentrations on the cell membrane, to determine any changes in the cell membrane integrity.