Krebs Cycle Succinate to Fumarate Reaction by Soraya Pashaei Marandi 9 March 2016 Biology 111 Section 521 TA Wang Xiang Daisy Abstract An experiment was conducted to examine the conversion of succinate to fumarate through the process of the Krebs cycle Four samples containing phosphate buffer DPIP bean extract malonate and varying amounts of succinate were tested in a spectrophotometer to see the absorbance values of fumarate produced Tube one and two were used to test the different rates of cellular respiration between when the enzyme was kept at a normal temperature versus when the enzyme was denatured in boiling temperatures Tube three and four were used to test the affects of malonate concentration inhibitor versus succinate concentration substrate on the formation of fumarate Tube one had an overall increase in the absorbance and was comparatively higher than that of tube two due to the denaturation of the enzyme causing the reaction rate to decrease The absorbance rate of tube three increased with the presence of more malonate than succinate while tube four decreased in absorbance with equal amounts of both substances The hypothesis of this experiment was that if an increased amount of succinate was present then the rate of cellular respiration would be greater showing a decrease in the absorbance in DPIP Introduction Eukaryotic cells carry out aerobic respiration in order to produce the energy that is required for metabolic function Glycolysis produces pyruvic acid in the presence of oxygen which is then directed to the mitochondria Inside the mitochondria the process continues within the Krebs cycle Chemical energy oxidizes glucose through enzymatic reactions When a molecule is reduced it gains an electron and when it loses an electron it is oxidized The energy that is generated by the removal of both hydrogen and carbon dioxide electrons are stored in NAD and FAD Hydrogen ions move against the concentration gradient as they move down the electron transport chain Oxygen is the final electron acceptor in the electron transport chain For each glucose molecule that becomes oxidized there are thirty six to thirty eight ATP produced An important substrate for the mitochondria to function is succinic acid succinate When succinate is present in sufficient quantities succinate molecules bind the enzyme complex called succinate dehydrogenase The succinate dehydrogenase complex is also known as complex II of the electron transport system therefore when succinate is oxidized into fumarate it takes place in the inner membrane of the mitochondria Caprette 2005 The succinate dehydrogenase complex also contains the energy carrier FAD Due to the enzyme and FAD both being apart of the same complex the initiation of succinate oxidation is done by binding succinate to the enzyme During this experiment succinate was converted to fumarate catalyzed by the reaction of succinic dehydrogenase In this process two hydrogen atoms transferred from succinate to the coenzyme FAD which reduced to FADH2 For this experiment FAD was replaced by DPIP as an electron carrier As DPIP is reduced it changed from blue to clear Malonate is a competitive in hibitor that competes for the active site on succinate dehydrogenase If malonate is bound to the active site then no reaction will come of it Methods To conduct this experiment a spectrophotometer was set up at 600nm to test the absorbance of each reaction tube The Blank test tube which is the control contained 200 l bean extract 4 6ml of phosphate buffer and 66 l of succinate In order to test the other four tubes their absorbance levels were compared to this blank Each test tube was tested every two minutes for a total of sixteen minutes and results were recorded Test tube 1 contained 200 l bean extract 200 l DPIP 4 3ml phosphate buffer and 66 l succinate Test tube 2 contained 200 l boiled bean extract 200 l DPIP 4 3ml phosphate buffer and 66 l succinate Test tube 3 contained 200 l bean extract 200 l DPIP 4 0ml phosphate buffer 200 l malonate and 66 l succinate Test tube 4 contained 200 l bean extract 200 l DPIP 3 8ml phosphate buffer 200 l malonate and 200 l succinate Results Shown in Table 1 and Graph 1 are the results for the experiment Table 1 shows the absorption of light taken by the spectrophotometer at a wavelength of 600nm by DPIP This table lists the absorption of light at two minute intervals for sixteen minutes for each sample The table provides information that tube 2 and tube 3 increased in their absorbance levels of DPIP with some fluctuations Tube 2 started at 478A and ended with 500A while tube 3 started at 279A and ended with 285A Tube 1 had spikes of high absorbances but started at 316A and ended with 336A Lastly tube 4 had a steady decrease from 271A to 262A Graph 1 is a visual representation of the comparison between the absorbance of light at 600nm and the amount of time the succinate was in the mixture to form fumarate The absorbance of light was determined by testing the solution in a spectrometer with the electron acceptor DPIP The graph shows that tube 2 was the fastest at absorbing light and tube 4 is the slowest at absorbing light Discussion The hypothesis for this experiment was that if an increased amount of succinate was present then the absorbance levels would decrease due to the reducing of DPIP from blue to clear It was found that tube 2 had the highest increase in absorbance This is due to the fact that because the bean extract was boiled the enzyme was denatured causing it not to react which increases the levels of DPIP Since no cellular respiration occurred the DPIP stayed a blue color and did not reduce The same goes for tube 3 where there was more malonate inhibitor than succinate The malonate blocked the enzyme from binding to the substrate causing cellular respiration to fail Since DPIP couldn t reduce the absorbance levels increased However for tube 1 there should have been a steady decrease in the absorbance levels because succinate did reduce DPIP to form fumarate The lack of cellular respiration could have came from the test tubes being contaminated from other labs with the substances that cause the reaction to not occur Tube four contained equal amounts of malonate and succinate which means that they stabilized each other The substrate still had the same chance of binding to the enzyme The absorbance levels for tube 4 decreased suggesting that DPIP reduced meaning that succinate formed fumarate and cellular
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