IntroductionAerobic Respiration is the Process by Which Mit Report High Dissertation 1071205796

respiration lab
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  Click to visit our student-to-student file sharing network. IntroductionAerobic respiration is the process by which mitochondria consume oxygen and organic fuel to produce ATP for the organism to use. ermentation is another source of ATP for cells! but this method is by far! less effective in its yield of ATP. lucose is the main source of organic fuel for cellular respiration! which releases as many as #$ATP molecules per glucose molecule in aerobic respiration. %nlike fermentation! which only releases & ATP molecules per glucose molecule.The first step in the process of aerobic respiration is glycolysis! which produces two molecules of pyruvate per cycle using one glucose molecule. Pyruvate is then used in the'rebs cycle to release electrons in certain steps. These electrons then enter the electron transport chain where a series of steps produce the ma(ority of the ATP formed during aerobic respiration. lycolysis takes place in the cytoplasm! while the other two steps occur within the mitochondrion organelle. In order to study the reaction of aerobic respiration! a source of mitochondria is needed. %sing a microcentrifuge! the mitochondria are removed from a solution of ground up lima beans. The mitochondria still carry out aerobic respiration even when they are removed from the rest of their cell contents. The source of glucose for this suspension is sucrose. )ucrose is processed readily by the mitochondria as it is a rich source of the sugar glucose.To measure the rate of cellular respiration! you need to monitor the activity of en*ymes inthe system that are involved with the 'rebs cycle. In one step! succinate is converted to fumarate. +uring this step! electrons are released from the reaction! which travel to the electron transport chain where they are used to make ATP.In this experiment! +PIP is a blue dye used to indicate the presence and level of electronsin the solution of mitochondria before the electrons reach the electron transport chain. ,hen a +PIP molecule grabs an electron! its color changes from blue to colorless. The degree of color change indicates the level of en*ymatic activity occurring and using a spectrophotometer! the color change can be monitored and recorded. The transmittance level of the sample will indicate how much en*ymatic activity is occurring.lectrons are released gradually down the electron transport chain. This is the stage where A+P is converted to ATP. +PIP acts as a substitute for ubiuinone! the only lipid in the transport chain. )ince +PIP has a greater affinity for electrons than ubiuinone! it accepts the electrons and becomes reduced and colorless before the electrons reach the  bottom of the chain.%sing these methods! we are able to observe the level of aerobic respiration occurring within the mitochondria of living cells./ethodsA. /itochondria Isolation.  0.%se a scalpel to cut soaked lima beans into pieces. 1The lima beans must be soaked for about 0& hours in water before cutting them up.2 rind the cut seeds with &3 m4 of assay  buffer for two minutes in a blender.&.Pour the mixture through 5 layers of cheesecloth and collect the filtrate.#.Add 06 m4 more of the buffer to the blender to rinse it out and filter this as well.5.Chill the filtrate over ice for 06 minutes.3.ill a microtube with 0.3 m4 of filtrate! close the lid! and centrifuge it in a microcentrifuge for at least 3 minutes at &666 rpm. 1/ake sure the tubes are balanced in the centrifuge.27.Transfer the supernatant to another microtube! leaving the pellet behind.8.Add 0.3 m4 of cold sucrose9phosphate buffer to the pellet and use a pipet to resuspend the pellet.:. Procedure to Calibrate )pec &6.0.Turn the )pec-&6 and allow it to warm up for &6 minutes.&.)et the wavelength to 766 nm and switch the filter lever to the right.#.)et the mode to transmittance and turn the transmittance knob 1the left one2 to *ero without the blank in the chamber.5.)et the mode to absorbance and place the blank cuvette into the chamber. /ake sure the white line on the cuvette is lined up with the arrow on the spectrophotometer.3.Close the lid and turn the absorbance knob 1the right one2 to a reading of *ero.7.ach time you *ero the )pec-&6! you must use the black to reset the absorbance to *ero  before performing another run.C. Preparing )tandard Curve )olutions.0.4abel 7 cuvettes ; 0-7.&.To each tube! add the +PIP and sucrose9phosphate buffer as labeled in the given table.#.%sing tube ; 0 as the blank! *ero the absorbance for )pec-&6.5.Take the absorbance at 766 nm for each concentration of +PIP.3.<ecord the data in the data table given.7. raph the absorbance vs. concentration! which gives you the standard curve for +PIP.Tube ;Conc. =f +PIPAmount of +PIP 1m42Amount of Assay :uffer 1m4206>63&&>6.05.?#5>6.&5.$5$>6.55.730&>6.75.5707>6.$5.&+. Preparing Cellular <espiration Tubes.0.4abel 5 cuvettes ; 0-5.&.Add the solutions to each cuvette in order. =nce the supernate is added readings must  be taken immediately. The time readings start at 6 minutes.#.Tubes number 0 and # are controls. @ou will *ero the instrument with tube 0 and then read tube &. +o the same with tubes # and 5. Three is used to *ero! and four to read.  @ou must do this every two minutes for a total of &6 minutes. Tube 0 is with supernate and without +PIP. Tube # is without supernate and without +PIP.5.<ecord your data in the data table.3./ake a graph of absorbance vs. time.Tube ;:ufferA*ide)uccinate+PIP)upernate0#.36.36.366.3&#.<esultsigure 0. )tandard curve plotting absorbance vs. concentration of +PIP at 766 nm.igure &. Aerobic respiration readings taken over time using +PIP dye to measure absorbance. A mitochondrial suspension was used as the source of electrons that +PIP would take up in order to change color.+iscussionIn this experiment! the process of aerobic respiration was being studied. The procedure that was used presented some specific problems that needed to be addressed so that we might be able to observe respiration taking place.The first problem presented was how to measure the levels of en*yme activity. In order to do this! we had to use a substance that could show us the change in the amount of en*yme present in the test tubes. In order to accomplish this! a dye called +PIP was added which turns clear when it attaches to electrons. As the mitochondria go through aerobic respiration! they process the glucose into pyruvate! which is then sent to the 'rebs cycle. The +PIP intercepts the electrons at the point where succinate is converted to fumarate! and in the process! is releasing electrons that assist in the electron transport chain. +PIP is used in place of ubiuinone! which is the traditional acceptor of the electrons when they are moved to the transport chain. +PIP has a greater affinity for electrons! so it attaches to them readily. =nce a +PIP molecule attaches to an electron! it turns from its srcinal blue color! to a clear color. /easuring the absorbance of the +PIP color indicates how much en*yme activity is occurring. The spectophotometer indicates the level to which en*yme activity is occurring according to how much color is left in the test tubes.A standard curve of varying concentrations of +PIP in solution was measured in igure 0 to show that as the concentration of +PIP goes up! the absorbance does also because there are more molecules of blue +PIP to be absorbed by the light measuring instruments in the spectrophotometer. This is useful information to have when measuring the levels of en*yme activity in a test tube with mitochondrial suspension in it. or example! in thisexperiment! you know the absorbance of +PIP is going down in the tube with supernate in it! indicating that the concentration of +PIP is also going down! but in reality! it is (ust  changing to a clear color with the concentration of +PIP staying the same. The standard curve is (ust a guide to explain how the concentration of blue +PIP relates to the absorbance levels.)odium a*ide is added to all of the test tubes also whether they contain supernate or not. The a*ides function is to block A+B& from passing electrons to ubiuinone. This ensures that +PIP is the only substance that receives electrons so that the true level of en*yme activity can be measured through absorbance readings. If A+B& was allowed to pass on electrons to ubiuinone! the electron transport chain would proceed as normal and +PIP would have no electrons to bind to because they would be used in the transport chain.Plotting the absorbance over time graph! as shown in igure &! indicates that adding supernate increased the level of en*yme activity. The supernate was composed of a mixture of mitochondria and sucrose9phosphate buffer. The buffer was the source of energy for the mitochondria. According to igure &! the en*ymatic activity was greater over a period of &6 minutes for the test tube with the supernate as opposed to the one without it. The absorbance levels go down at a greater rate using the supernate test tube! indicating the color change of the +PIP was greater in that tube. Therefore more electrons were being taken up by the +PIP in the supernate test tube as opposed to the +PIP only test tube.These results indicate that mitochondria are the source of cellular respiration in cells. If the test tube with mitochondria had lower and lower absorbance readings over time! that indicates the +PIP was attaching to electrons and changing to its clear color. The test tube without supernate had little change in absorbance levels indicating minimal changes in the en*ymatic activity of the solution. Therefore! the mitochondria are the source for electron movement and also of cellular respiration.The electron transport chain is an important process to all organisms. ,ithout it! the  production of ATP would not occur to a great extent. ATP is needed for all almost all  processes that occur within an organism. ,ithout it! there would be few processes that could occur as usual within cells. An organism would not be able to handle all of its life  processes without the use of ATP. It would be severely restricted in its movement and or metabolisms. or instance! if sodium a*ide were present in a living organism! it would  block A+B& from releasing electrons to the electron transport chain and therefore the ma(or source of ATP during cellular respiration would be lost. This would have the same effect on an organism as the electron transport chain not functioning at all.Also during cellular respiration! ATP is released in a series of small steps instead of one large step. This is a more effective method of releasing energy within a cell. There would be great heat released if the reaction occurred in one step and this could kill en*ymes as well as the organism. Also! once the reaction occurred! where would the cellsget enough energy for the next oxidation reaction if all the DfuelD were used up in one stepE It is difficult to harness the energy present in glucose. The only thing keeping the slow rate going is the high activation energy barrier of electrons retained by the en*ymes which is used to break down energy sources such as glucose in a slow fashion! one step tothe next.The procedure used in this experiment could not be used to determine anaerobic respiration either. In anaerobic respiration! there is no electron transport chain and no 'rebs cycle by which the transport of raw electrons can be interrupted by +PIP. The
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