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Introduction
Aerobic respiration is the process by which mitochondria consume oxygen and organic
fuel to produce ATP for the organism to use. Fermentation is another source of ATP for
cells, but this method is by far, less effective in its yield of ATP. Glucose is the main
source of organic fuel for cellular respiration, which releases as many as 38ATP
molecules per glucose molecule in aerobic respiration. Unlike fermentation, which only
releases 2 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
Krebs cycle to release electrons in certain steps. These electrons then enter the electron
transport chain where a series of steps produce the majority of the ATP formed during
aerobic respiration.
Glycolysis 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. Using 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. Sucrose 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 enzymes in
the system that are involved with the Krebs cycle. In one step, succinate is converted to
fumarate. During 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, DPIP is a blue dye used to indicate the presence and level of electrons
in the solution of mitochondria before the electrons reach the electron transport chain.
When a DPIP molecule grabs an electron, its color changes from blue to colorless. The
degree of color change indicates the level of enzymatic activity occurring and using a
spectrophotometer, the color change can be monitored and recorded. The transmittance
level of the sample will indicate how much enzymatic activity is occurring.
Electrons are released gradually down the electron transport chain. This is the stage
where ADP is converted to ATP. DPIP acts as a substitute for ubiquinone, the only lipid
in the transport chain. Since DPIP has a greater affinity for electrons than ubiquinone, it
accepts the electrons and becomes reduced and colorless before the electrons reach the
bottom of the chain.
Using these methods, we are able to observe the level of aerobic respiration occurring
within the mitochondria of living cells.
Methods
A. Mitochondria Isolation.

1.Use a scalpel to cut soaked lima beans into pieces. (The lima beans must be soaked for
about 12 hours in water before cutting them up.) Grind the cut seeds with 25 mL of assay
buffer for two minutes in a blender.
2.Pour the mixture through 4 layers of cheesecloth and collect the filtrate.
3.Add 10 mL more of the buffer to the blender to rinse it out and filter this as well.
4.Chill the filtrate over ice for 10 minutes.
5.Fill a microtube with 1.5 mL of filtrate, close the lid, and centrifuge it in a
microcentrifuge for at least 5 minutes at 2000 rpm. (Make sure the tubes are balanced in
the centrifuge.)
6.Transfer the supernatant to another microtube, leaving the pellet behind.
7.Add 1.5 mL of cold sucrose/phosphate buffer to the pellet and use a pipet to resuspend
the pellet.
B. Procedure to Calibrate Spec 20.
1.Turn the Spec-20 and allow it to warm up for 20 minutes.
2.Set the wavelength to 600 nm and switch the filter lever to the right.
3.Set the mode to transmittance and turn the transmittance knob (the left one) to zero
without the blank in the chamber.
4.Set the mode to absorbance and place the blank cuvette into the chamber. Make sure
the white line on the cuvette is lined up with the arrow on the spectrophotometer.
5.Close the lid and turn the absorbance knob (the right one) to a reading of zero.
6.Each time you zero the Spec-20, you must use the black to reset the absorbance to zero
before performing another run.
C. Preparing Standard Curve Solutions.
1.Label 6 cuvettes # 1-6.
2.To each tube, add the DPIP and sucrose/phosphate buffer as labeled in the given table.
3.Using tube # 1 as the blank, zero the absorbance for Spec-20.
4.Take the absorbance at 600 nm for each concentration of DPIP.
5.Record the data in the data table given.
6.Graph the absorbance vs. concentration, which gives you the standard curve for DPIP.
Tube #Conc. Of DPIPAmount of DPIP (mL)Amount of Assay Buffer (mL)
10%05
22%0.14.9
34%0.24.8
48%0.44.6
512%0.64.4
616%0.84.2
D. Preparing Cellular Respiration Tubes.
1.Label 4 cuvettes # 1-4.
2.Add the solutions to each cuvette in order. Once the supernate is added readings must
be taken immediately. The time readings start at 0 minutes.
3.Tubes number 1 and 3 are controls. You will zero the instrument with tube 1 and then
read tube 2. Do the same with tubes 3 and 4. Three is used to zero, and four to read.

You must do this every two minutes for a total of 20 minutes. Tube 1 is with supernate
and without DPIP. Tube 3 is without supernate and without DPIP.
4.Record your data in the data table.
5.Make a graph of absorbance vs. time.
Tube #BufferAzideSuccinateDPIPSupernate
13.50.50.500.5
23.00.50.50.50.5
34.00.50.500
43.50.50.50.50
Results
Figure 1. Standard curve plotting absorbance vs. concentration of DPIP at 600 nm.
Figure 2. Aerobic respiration readings taken over time using DPIP dye to measure
absorbance. A mitochondrial suspension was used as the source of electrons that DPIP
would take up in order to change color.
Discussion
In 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 enzyme activity. In order
to do this, we had to use a substance that could show us the change in the amount of
enzyme present in the test tubes. In order to accomplish this, a dye called DPIP 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
Krebs cycle. The DPIP 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. DPIP is used in place of ubiquinone, which is the traditional acceptor of the
electrons when they are moved to the transport chain. DPIP has a greater affinity for
electrons, so it attaches to them readily. Once a DPIP molecule attaches to an electron, it
turns from its original blue color, to a clear color. Measuring the absorbance of the DPIP
color indicates how much enzyme activity is occurring. The spectophotometer indicates
the level to which enzyme activity is occurring according to how much color is left in the
test tubes.
A standard curve of varying concentrations of DPIP in solution was measured in Figure 1
to show that as the concentration of DPIP goes up, the absorbance does also because
there are more molecules of blue DPIP to be absorbed by the light measuring instruments
in the spectrophotometer. This is useful information to have when measuring the levels
of enzyme activity in a test tube with mitochondrial suspension in it. For example, in this
experiment, you know the absorbance of DPIP is going down in the tube with supernate
in it, indicating that the concentration of DPIP is also going down, but in reality, it is just

changing to a clear color with the concentration of DPIP staying the same. The standard
curve is just a guide to explain how the concentration of blue DPIP relates to the
absorbance levels.
Sodium azide is added to all of the test tubes also whether they contain supernate or not.
The azides' function is to block FADH2 from passing electrons to ubiquinone. This
ensures that DPIP is the only substance that receives electrons so that the true level of
enzyme activity can be measured through absorbance readings. If FADH2 was allowed
to pass on electrons to ubiquinone, the electron transport chain would proceed as normal
and DPIP 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 Figure 2, indicates that adding
supernate increased the level of enzyme activity. The supernate was composed of a
mixture of mitochondria and sucrose/phosphate buffer. The buffer was the source of
energy for the mitochondria. According to Figure 2, the enzymatic activity was greater
over a period of 20 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 DPIP was greater in that tube. Therefore more
electrons were being taken up by the DPIP in the supernate test tube as opposed to the
DPIP 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 DPIP 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 enzymatic 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. Without 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. Without 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. For instance, if sodium azide were present in a living organism, it would
block NADH2 from releasing electrons to the electron transport chain and therefore the
major 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
enzymes as well as the organism. Also, once the reaction occurred, where would the cells
get enough energy for the next oxidation reaction if all the "fuel" were used up in one
step? 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 enzymes
which is used to break down energy sources such as glucose in a slow fashion, one step to
the 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
Kreb's cycle by which the transport of raw electrons can be interrupted by DPIP. The

DPIP could not take the place of ubiquinone and "steal" electrons away from NADH2.
Pyruvate is converted to lactate and ethanol via NADH, not NADH2 so there would be
no way to block the transport of electrons. Sodium azide might not be the correct
chemical to block the transport of electrons to NADH. In this case, DPIP would not be
able to measure the level of enzyme activity in the Kreb's cycle if there was no Kreb's
cycle and no NADH2 to block using the sodium azide. The absorbance levels of the
DPIP would not change considerably using the spectrophotometer, and perhaps they
would even remain the same. This procedure then, would not be a good way to measure
anaerobic respiration.
The process of cellular respiration is an important one and can be measured using simple
techniques utilizing the source of all cellular respiration in organisms: the mitochondria.
This is one of the single most important processes that occur within and organisms' cells.

Keywords:
introduction aerobic respiration process which mitochondria consume oxygen organic
fuel produce organism fermentation another source cells this method less effective yield
glucose main source organic fuel cellular respiration which releases many molecules
glucose molecule aerobic respiration unlike fermentation which only releases molecules
glucose molecule first step process aerobic glycolysis produces molecules pyruvate cycle
using molecule pyruvate then used krebs cycle release electrons certain steps these
electrons then enter electron transport chain where series steps produce majority formed
during glycolysis takes place cytoplasm while other steps occur within mitochondrion
organelle order study reaction source mitochondria needed using microcentrifuge
mitochondria removed from solution ground lima beans still carry even when they
removed from rest their cell contents this suspension sucrose sucrose processed readily
rich sugar measure rate cellular need monitor activity enzymes system that involved with
krebs cycle step succinate converted fumarate during this step electrons released from
reaction travel electron transport chain where they used make experiment dpip blue used
indicate presence level solution before reach electron transport chain when dpip grabs
color changes blue colorless degree color change indicates level enzymatic activity
occurring using spectrophotometer color change monitored recorded transmittance level
sample will indicate much enzymatic activity occurring released gradually down stage
where converted dpip acts substitute ubiquinone only lipid since greater affinity than
ubiquinone accepts becomes reduced colorless before reach bottom these methods able
observe occurring within living cells methods isolation scalpel soaked lima beans into
pieces lima beans must soaked about hours water before cutting them grind seeds with
assay buffer minutes blender pour mixture through layers cheesecloth collect filtrate more
buffer blender rinse filter well chill filtrate over minutes fill microtube with filtrate close
centrifuge microcentrifuge least minutes make sure tubes balanced centrifuge transfer
supernatant another microtube leaving pellet behind cold sucrose phosphate buffer pellet
pipet resuspend pellet procedure calibrate spec turn spec allow warm wavelength switch
filter lever right mode transmittance turn transmittance knob left zero without blank

chamber mode absorbance place blank cuvette into chamber make sure white line cuvette
lined arrow spectrophotometer close turn absorbance knob right reading zero each time
zero spec must black reset absorbance performing another preparing standard curve
solutions label cuvettes each tube phosphate labeled given table tube blank take each
concentration record data data table given graph concentration gives standard curve tube
conc dpipamount amount assay preparing cellular tubes label cuvettes solutions cuvette
order once supernate added readings must taken immediately time readings start tubes
number controls will instrument then read same three four read every total supernate
without without supernate record your data table graph time
bufferazidesuccinatedpipsupernate results figure standard curve plotting concentration
figure readings taken over measure mitochondrial suspension that would take order
change discussion experiment process being studied procedure that presented some
specific problems needed addressed might able observe taking place first problem
presented measure levels enzyme substance could show amount enzyme present test
accomplish called added turns clear when attaches through they into pyruvate sent krebs
intercepts point succinate converted fumarate releasing assist ubiquinone traditional
acceptor moved greater affinity attaches them readily once attaches turns original blue
clear measuring indicates much enzyme spectophotometer indicates according much left
test varying concentrations solution measured figure show goes does also because there
more absorbed light measuring instruments spectrophotometer useful information have
measuring levels test mitochondrial suspension example experiment know going down
indicating also going down reality just changing clear staying same just guide explain
relates levels sodium azide added also whether contain azides function block fadh passing
ensures only substance receives true measured through fadh allowed pass would proceed
normal would have bind because plotting over graph shown adding increased composed
mixture phosphate energy according enzymatic greater period opposed rate indicating
therefore more were being taken opposed these results indicate cells lower lower
attaching changing little indicating minimal changes therefore movement important
organisms production occur great extent needed almost processes occur within organism
there processes could usual organism able handle life processes severely restricted
movement metabolisms instance sodium azide were present living block nadh releasing
therefore major during lost have same effect functioning released series small instead
large effective method releasing energy cell there great heat reaction occurred could kill
enzymes well once occurred enough energy next oxidation fuel were difficult harness
present thing keeping slow rate going high activation barrier retained enzymes break
sources such slow fashion next procedure determine anaerobic either anaerobic kreb
interrupted take steal away nadh lactate ethanol nadh block sodium azide might correct
chemical case kreb kreb considerably perhaps even remain good anaerobic important
measured simple techniques utilizing organisms single most important organisms
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