RANELLE JANINE L.

ASI
2008-07554
BIO 120 S-5L
Exercise 7. Cellular Respiration
DISCUSSION
Cells require energy to fuel chemical processes that maintain its viability, the
source of which is from the hydrolysis of Adenosine Triphosphate (ATP), an exergonic
reaction that releases free energy that is reabsorbed into metabolic pathways. ATP
generation is achieved through the general process of Cellular Respiration wherein
large food molecules like polysaccharides and proteins are catabolised or broken
down to their simplest units to harvest energy.
In aerobic organisms, cellular respiration requires O 2 through aerobic
respiration that produces the highest yield of ATP among all of the cells energygenerating activities. One of the biochemical pathways involved in this activity is
the Tricarboxylic acid (TCA) cycle. This cycle is coupled with the Electron Transport
Chain (ETC), another pathway for the production of ATP and reducing powers like
Flavin Adenine Dinucleotide (FAD)
The TCA cycle breaks down pyruvate, a by-product of the breakdown of large
molecules, into CO2 while producing ATP and FAD.
Specifically, FADH2 is produced during the oxidation of succinate, a metabolic
intermediate of the TCA cycle, into fumarate. This is the reaction of interest in the
experiment as it is an established evidence of cellular respiration. This reaction is
catalyzed by the enzyme succinate dehydrogenase (SDH).
In the experiment, mouse liver homogenate was used as a source of
mitochondria as liver cells have relatively higher mitochondria compared to other
somatic cells due to its metabolic functions in detoxification. The mitochondria
contain the SDH enzyme to be used in the in vitro reaction of succinate.
To measure the activity if SDH, 2,6-dichlorophenolindophenol (DPIP) was used
as an indicator dye as it replaces FAD as an electron acceptor due to higher electron
affinity. DPIP is colored blue in its oxidized form and turns into colorless solution
upon reduction. The change in color indicates the progress of the oxidation of
succinate into fumarate and is measured using a spectrophotometer.
Also tackled in the experiment is the effect of inhibitors on the reaction of
interest. Two types of inhibitors are used. First is cyanide, an irreversible inhibitor,
which covalently binds to Cytochrome C oxidase, a component of the ETC, and
permanently deforms the component to stop the reaction pathway. Another is
Malonate, an analog of the substrate succinate, which acts as a competitive

inhibitor that consumes the oxidizing agent (DPIP in the set-up) that would react
with succinate.H2O is used as diluting agent.
To analyze the aforementioned reactions and each substances role, six test
tubes were filled with different contents enumerated in Table 1.
Table 1. Test tube contents for the measurement of respiration by dye reduction.
Tube no.
Volume of contents (mL)
Liver
Succinate
0.1 M
dH2O
Malonate
DPIP
homogen
solution
NaCN
solution
solution
ate
1
1
1
0
1
0
1
2
1
1
1
0
0
1
3
1
0
0
2
0
1
4
1
1
0
1
0
1
(heated)
5
1
1
0
0
1
1
6
1
1 (+ 1g
0
0
1
1
solid
succinate)
Blanks were prepared with identical contents but without the DPIP solution as
the spectrophotometer should only record the absorbance of the DPIP molecules.
Absorbance values were recorded in 1-minute intervals for 6 minutes. Partial
rates were computed for easier comparison of trends. (Table 2)
In test tube 1, the substrate (succinate), enzyme (SDH in liver homogenate)
and electron acceptor (DPIP) are present. Theoretically, the reaction will proceed
normally as no inhibitor is present. In the result of the experiment, the values did
not follow the decreasing trend exhibited by a normal respiration rate but, instead
stayed constant.
In test tube 2, the substrate, enzyme and electron acceptor are present, but
an inhibitor (cyanide, NaCN) was added. Theoretically, NaCn would stop ETC leading
to the accumulation of NADH which inhibits SDH. Since the enzyme would be
inhibited, there would be little to no respiration present. In the results, however, a
decreasing trend is observed, implying the normal progress of respiration.
In test tube 3, the enzyme and DPIP was added but no substrate was present,
hence no respiration occurred. The absorbance values were hence constant as there
is no reaction with DPIP. This confirms that succinate is needed as the substrate for
the reaction to proceed.

In test tube 4, the liver homogenate was heated, denaturing the SDH it
carries. Even though the substrate and DPIP is added, there is no evidence of
respiration as it proceeded too slowly to be recorded in the time period. This set-up
confirms that the reaction requires catalysis by the SDH enzyme.
In test tube 5, the substrate, enzyme and electron acceptor are present, but
an inhibitor (malonate) was added. Malonate is a competitive inhibitor that binded
with the enzymes active site but was not dehydrogenated by SDH due to its
difference in structure from succinate (it lacks a CH 2 group present in the original
substrate). No respiration happened as the absorbance values exhibited a
fluctuating trend.
In test tube 6, the enzyme, electron acceptor and a competitive inhibitor
(malonate) was present but the substrate concentration was increased.
Theoretically, competitive inhibition will be overcome by increasing the
concentration of the original substrate. In this set-up, moderate respiration should
have occurred as some of the succinate have overcome competition with malonate.

Figure 1. Absorbance values of six test tube setups measured for respiration by
dye reduction

Table 2. Computed partial rates of absorbance readings of six test tubes with
varying content for measurement of respiration by dye reduction.
Time
Partial rates
1
2
3
4
5
6
(min)

1
2
3
4
5
6

-0.002
0.001
-0.003
0.001
0.001
-0.001

-0.005
-0.005
-0.005
0.001
-0.002
0.001

-0.005
0.002
0.001
-0.001
0.001
-0.001

0.001
0
-0.007
0.011
-0.010
0.018

-0.010
0.008
-0.008
0.013
-0.009
-0.021

-0.002
0
0.002
-0.002
0
0

Figure 2. Computed partial rates of six test tube setups measured for respiration
by dye reduction
Errors in the experiment may have come from the wrong labelling of test
tubes 1 and 2, the results of which were opposite of the theoretical results of each
other. Contamination was also a factor considered for the increase of absorbance in
test tube 4. Improper handling of the device and ineffective wiping of the sample
holder may have caused fluctuating values in absorbance.
REFERENCES:
"The rate of reaction of Succinate dehydrogenase." 123HelpMe.com. 25 Mar
2015 Retrieved from <http://www.123HelpMe.com/view.asp?id=48176>.