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Influence of Malonate and Sodium Fluoride on the Activity of Enzyme Succinate Dehydrogenase to Assess Inhibitors of Cellular Respiration

Thaovy Mai Tran

BI 151.5233 (Winter 2013)

Todd Tiano

03/25/2013

Introduction

Most organisms produce adenosine triphosphate (ATP) as a source of energy for cellular work, using cellular respiration. Cellular respiration is a process that involves glycolysis -breaking down glucose into two molecules of 3- carbon pyruvate, the Krebs cycle - oxidizing organic fuel derived from pyruvate-, and the electron transport chain - moving electrons through a series of protein complexes to generate ATP (Reece, et.al, 2011). The process of cellular respiration occurs in the Cytoplasm and the Mitochondria in plants and animals. In this process, many more ATP molecules are produced compared to alcoholic fermentation which is a process that releases energy in glucose to generate ATP in the absence of oxygen. Aerobic respiration produces 38 ATP compared to 2 ATP of alcoholic fermentation, which is respectively (Morgan, et. al, 2011). As part of cellular respiration, the Krebs cycle is a series of eight steps, each catalyzed by a specific enzyme. One enzyme used is succinate dehydrogenase which catalyzes the conversion of succinate to fumarate, thus loses hydrogen ions and electrons for use in generating ATP. It is important to know what inhibitors affect the activity of this enzyme which eventually will affect the whole process of cellular respiration. In this experiment, 1M Malonate( CH2(COO)22-) and 0.1M of Sodium Fluoride (NaF) ,were employed as inhibitors to the enzyme succinate dehydrogenase to assess their effect on cellular respiration. Malonate is a molecule that inhibits the oxidization of succinate. Besides that, Sodium Fluoride is the main ingredient that is commonly found in toothpaste. It also does not allow bacteria to grow on teeth. They are both expected to be good inhibitors that block the activity of the enzyme succinate dehydrogenase and hence would slow down the cellular respiration rate. The purpose of this experience is to determine how Malonate and Sodium Fluoride inhibit the cellular respiration. DPIP ( dichlorophenol -indophenol) is used to measure the rate of cell respiration occurring inside mitochondria. DPIP is blue in its oxidized (not binding H+) state and clear in its reduced state (BI 151 Lab, 2012). Therefore, the increasing of the colorless of DPIP, succinate is broken down fast. If the succinate is broken, then the DPIP will become colorless faster. The spectrometer (Spec-20 D) is also used to measure the rate of H+ capture by DPIP.

Materials and Methods

The spectrometer was set to 600nm and zero on transmittance. A blank cuvette B was prepared by mixing 3.8 mL buffer, 0.3 mL mitochondrial suspension, and 0.2 mL Succinate and 1 mL Reagent. Three other cuvettes labeled 1, 2, and 3 were filled with measured amount of the buffer, DPIP (di-chlorophenol-indophenol which changed from blue oxidized state to colorless reduced state when receiving hydrogen ions and electrons released from succinate), mitochondrial suspension, malonate, sodium fluoride, and succinate (Table 1). A blank and control tube were used to calibrate the Spec-20D and eliminate the independent variables. Succinate was added last to each cuvette and was quickly mixed. These tubes were then inserted into the sample holder of the spectrophotometer to record the transmittance level. Kim Wipe papers were used to clean each cuvette before placing into spectrometer to minimize errors from other variables. The transmittance level of tubes 1, 2, and 3 was recorded at 5-minute intervals for 30 minutes. This transmittance level revealed the amount of light transmitted by the DPIP. As the DPIP changed from blue to clear, more light would...
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