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Unformatted text preview: BI/CH 368 Laboratory Experiment 3 Death on the Farm! Inhibition of Citric Acid Cycle Enzymes Case Background Two agricultural workers were instructed to spray around the farm where they worked with a Mexican rodenticide for pest control (Figure 1). Over the course of the day, they both experienced increasing nausea, vomiting, and abdominal pain, and one of them died while returning to the farm for help. The other man was admitted to the hospital. His temperature was 39.3C, and he was sweating profusely. He was extremely anxious and confused, and his pupils were dilated. Blood tests revealed that he was experiencing severe lactic acidosis and ketosis. Despite strenuous attempts to treat him, he also died in a coma two days after admission. In view of the clinical findings, a sample of the rodenticide was obtained for testing. Your goal is to determine the mode of action of this potent toxin using a preparation of rat liver mitochondria. The symptoms suggest that you should focus on reactions relating to the citric acid cycle. Experimental Background In this project, you will use isolated liver mitochondria to assay the activity of several key reactions of the citric acid cycle. If any of these reactions are inhibited by the rodenticide, then this provides good evidence for the compounds mechanism of action. First, you will monitor the activity of citric synthase by using 5,5'-dithiobis-(2-nitrobenzoic acid (DTNB) , commonly used to quantify thiol groups. Thiols, such as that found in free coenzyme A, react with DTNB, cleaving the disulfide bond to yield the yellow species 2-nitro-5- thiobenzoate (NTB- ; Figure 2). Recall that citrate synthase catalyzes the following reaction: acetyl-CoA + oxaloacetate citrate + CoA-SH The velocity of this reaction will be monitored via the production of CoA-SH in the presence of DTNB as the change in absorbance at 412 nm ( A 412 ) over time. You will determine the initial velocity of the citrate synthase reaction in the presence of its substrates and then measure the rate in the presence of the rodenticide to see if the compound inhibits this enzyme. Figure 1. The last photograph taken of the two agricultural workers who met a tragic end. BI/CH 368 Laboratory Experiment 3 Spring 2012 2 Next, you will measure the activity of several dehydrogenases by monitoring the reduction of the artificial electron acceptor 2,6-dichlorophenolindolphenol (DCPIP) . To use an artificial electron acceptor, the normal path of electrons in the electron transport chain must be blocked....
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- Spring '12