Summary 1
Succinate Dehydrogenase Enzyme Activity In Cellular Fractures (Lab 5) The nuclear, mitochondrial and cytoplasm fractions of cauliflower inflorescence tissue was isolated . The concentration of the proteins in the fractions was then measured, denatured and reduced by SDS-PAGE. The purpose of this lab was to measure the activity of the succinate dehydrogenase (SDH) enzyme. I predict that the presence of heat will denature the enzyme. I also predict that out of the different cellular fractions, the mitochondrial fractions will have the highest activity. In order to measure the activity of the SDH enzyme, 12 tubes were with varying combinations of sodium azide, which inhibits the 4th complex of the electron transport chain, DCPIP (dichlorophenolindophenol), an artificial electron acceptor, malonate, a competitive inhibitor, and SDH, which deprotonizes FADH2 into FAD+. This results in a reduction of DCPIP, once DCPIP is reduced, it becomes colorless. The spectrophotometer was utilized to measure the color of the reaction at an absorbance of 600nm, which was determined to be the absorbance of the enzyme activity. The absorbance of the 12 tubes were taken and recorded a intervals of 0,5,10 and 20 min, then the change from each of the intervals from 0 min was recorded. The absorbencies were then graphed in the form of a Michaelis Menten Plot. Prior to have an absorbance read, Tube 4 was the only tube heated in a hot water bath. All of the tubes were put in a chilled bath to prevent enzyme activity. When the absorbance levels were read, the mitochondrial fraction showed the highest absorbance, which indicates that the mitochondrial fraction had the greatest activity. This supports my first hypothesis. Another observation that was made was tube 4, which was heated had one of the lowest absorbance, and therefore had the lowest activity which indicates that the heat denatured the proteins.