Kevin Lopez
03/18/13 (008945285)
Succinate Dehydrogenase Enzyme Activity in Mitochondrial Fraction
Succinate dehydrogenase (SDH) is an enzyme that catalysis the oxidation of succinate to fumarate in the citric cycle. During this step, electrons removed from succinate directly reduce an electron acceptor, known as FADH2; however, for this laboratory experiment an artificial electron acceptor, dichlororphenolindophenol (DCPIP), was utilized. By inhibiting complex IV of the electron transport chain with sodium azide, the electrons from succinate reduce DCPIP and alter its color from blue to colorless. Therefore the color of the reaction can be measured using a spectrophotometer, and a decrease in A600 indicates enzyme activity that corresponds with the concentration of succinate found in the tube samples (i.e. higher succinate concentration = higher enzyme activity; vice-versa). In addition, by plotting a Michaelis-Menten graph, the maximum velocity of the reaction (Vmax) and the concentration of the substrate that results in ½ Vmax (Km). The first step in this experiment was the creation of twelve tube samples that contained a mixture of reaction buffer (3.2 mL), DCPIP (0.5 mL), and succinate (varying from 0-0.5 mL). Of the twelve, tube 3 included the addition of 0.2 mL Malonate, which served as a competitive inhibitor and tube 4 was denatured affecting enzyme activity. These will be discussed later. Upon completion of all twelve mixtures, their absorbance values were taken at 600 nm in five minute intervals starting with 0 minutes and ending with 20 minutes. From the observed absorptions, the reaction velocity for SDH was determined for tubes 5, 6, 7, 8, 9, and 2 since these tubes included the mitochondrial fraction where SDH is mainly located (in the inner membrane and matrix of mitochondria), and higher SDH activity is expected. These tubes had 0, 0.1, 0.2, 0.3, 0.4, and 0.5 mL of succinate respectively. Based on calculated reaction velocities of DCPIP reduction for each tube, these values tend to increase in accordance with the amount of succinate present since its concentration will directly affect how much DCPIP is reduced, and thus a higher succinate concentration yields a higher reaction velocity as seen in tube 2 that had the most succinate available. In contrast tube 5 had 0 succinate that exhibited none, if hardly any, reduced DCPIP and thus no color change indicating relatively low SDH activity. Consequently, the Km value found at ½ Vmax (tube 7) yielded a relatively large Km which indicates that lots of substrate is needed for activity; as is observed through the plot of a Michaelis-Menten graph based on the collected data. Furthermore, the plot yielded a Vmax of 7.94*10-6 DCPIP mmoles/min and Km of 20 mM succinate. As for tube 3, malonate was added to serve as a competitive inhibitor that in turn will increase the Km requiring more amounts of succinate to out-compete the malonate at the SDH active site. While tube 4 was heated up to 100 °C to denature SDH and thus reduce its activity by “turning off” the enzyme into an inactive state showing a decrease in absorbance since none of the DCPIP is reduced. Ultimately, the observed results slightly differed from the expected results in that the reaction velocity of DCPIP for the observed increased, then decreased, and increased once again rather than consistently increasing. Nevertheless, the tube (2) with the most succinate yielded the highest SDH activity as hypothesized.