Exercise 7 – Cellular Respiration
1. What evidence did you obtain to prove that:
a. the reaction was catalyzed by the enzyme?
🡪 To be able to prove that the reaction was catalyzed by the enzyme, reduction of DPIP must be
observed. That reaction pertained to the action of the succinate dehydrogenase (SDH), the only
enzyme of the Krebs cycle bound to the inner mitochondrial membrane that facilitates the
chemical reaction
succinate + FAD ↔ fumarate + FADH2
In vivo, the prosthetic group, FAD, accepts a pair of electrons from the substrate succinate; thus,
becoming reduced to FADH2. SDH, in short, catalyzes the oxidation of succinate to fumarate. In
vitro, FAD is represented by DPIP. DPIP can be used as an artificial electron acceptor; hence,
functioning as FAD. There will be a change in color – DPIP is blue in its oxidized form and
becomes colorless as it is reduced. This can be, in turn, measured spectrophotometrically.
b. succinate was involved in the reaction?
🡪 To be able to prove that succinate was involved in the reaction, comparison of the absorbance
readings of tubes containing succinate and that of without succinate must be done. In tube 1, a
drastic decrease in the absorbance reading was observed wherein succinate was present
whereas in tube3 wherein no succinate was added, almost none or a little decrease in its
absorbance reading was observed. Liver homogenate was present in all tubes; thus, having
source for mitochondrion. Since succinate was present in tube 1, there would be a substrate for
SDH to catalyze; hence, the decrease in the color of DPIP and drastic lowered absorbance
reading. On the other hand, no succinate was present in tube3. There would be no substrate for
SDH to catalyze, no decrease in the color of DPIP, and no drastic lowered absorbance reading.
2. Discuss the role of these substances in the experiment:
a. liver homogenate
🡪 Liver is one of the most metabolically active organs in the body. It plays a major role in
metabolism; hence, containing a lot of mitochondria and of course, succinate dehydrogenase –
the subject of interest in the experiment. Being an enzyme that is tightly bound to the
mitochondria’s inner membrane, it can be easily isolated through disrupting the liver cells,
liberating the mitochondria. Afterwards, the homogenate would be subjected to 10 000 x g, to
isolate mitochondria from the other components of the cell.
b. cyanide
🡪 Cyanide was used in the experiment to inhibit the transfer of electrons from cytochrome a3 (cyt
a3) to the final electron acceptor, O2, in the electron transport chain (ETC). Cyanide combines
with the heme of cyt a3, preventing the re-oxidation of heme. Electron flow is now blocked,
leading to the accumulation of NADH. NADH donates electrons to the ETC and as a feedback
inhibition, accumulation of NADH will result to inhibition of TCA (tricarboxylic acid cycle) cycle. In
turn, SDH activity will also be inhibited since it is a part of the TCA cycle – an indirect inhibitor.
However, cyanide is also an irreversible inhibitor since it does not only inhibit TCA cycle but also
the respiration process as a whole.
c. malonate
🡪 Malonate is a structural analog of succinate and functions as a competitive inhibitor of SDH. Since
malonate has the same structure as that of succinate, malonate will compete with succinate in
This study source was downloaded by 100000899606070 from CourseHero.com on 03-04-2026 13:37:23 GMT -06:00
https://www.coursehero.com/file/108716308/Exercise-7-Post-Labdocxpdf/
1. What evidence did you obtain to prove that:
a. the reaction was catalyzed by the enzyme?
🡪 To be able to prove that the reaction was catalyzed by the enzyme, reduction of DPIP must be
observed. That reaction pertained to the action of the succinate dehydrogenase (SDH), the only
enzyme of the Krebs cycle bound to the inner mitochondrial membrane that facilitates the
chemical reaction
succinate + FAD ↔ fumarate + FADH2
In vivo, the prosthetic group, FAD, accepts a pair of electrons from the substrate succinate; thus,
becoming reduced to FADH2. SDH, in short, catalyzes the oxidation of succinate to fumarate. In
vitro, FAD is represented by DPIP. DPIP can be used as an artificial electron acceptor; hence,
functioning as FAD. There will be a change in color – DPIP is blue in its oxidized form and
becomes colorless as it is reduced. This can be, in turn, measured spectrophotometrically.
b. succinate was involved in the reaction?
🡪 To be able to prove that succinate was involved in the reaction, comparison of the absorbance
readings of tubes containing succinate and that of without succinate must be done. In tube 1, a
drastic decrease in the absorbance reading was observed wherein succinate was present
whereas in tube3 wherein no succinate was added, almost none or a little decrease in its
absorbance reading was observed. Liver homogenate was present in all tubes; thus, having
source for mitochondrion. Since succinate was present in tube 1, there would be a substrate for
SDH to catalyze; hence, the decrease in the color of DPIP and drastic lowered absorbance
reading. On the other hand, no succinate was present in tube3. There would be no substrate for
SDH to catalyze, no decrease in the color of DPIP, and no drastic lowered absorbance reading.
2. Discuss the role of these substances in the experiment:
a. liver homogenate
🡪 Liver is one of the most metabolically active organs in the body. It plays a major role in
metabolism; hence, containing a lot of mitochondria and of course, succinate dehydrogenase –
the subject of interest in the experiment. Being an enzyme that is tightly bound to the
mitochondria’s inner membrane, it can be easily isolated through disrupting the liver cells,
liberating the mitochondria. Afterwards, the homogenate would be subjected to 10 000 x g, to
isolate mitochondria from the other components of the cell.
b. cyanide
🡪 Cyanide was used in the experiment to inhibit the transfer of electrons from cytochrome a3 (cyt
a3) to the final electron acceptor, O2, in the electron transport chain (ETC). Cyanide combines
with the heme of cyt a3, preventing the re-oxidation of heme. Electron flow is now blocked,
leading to the accumulation of NADH. NADH donates electrons to the ETC and as a feedback
inhibition, accumulation of NADH will result to inhibition of TCA (tricarboxylic acid cycle) cycle. In
turn, SDH activity will also be inhibited since it is a part of the TCA cycle – an indirect inhibitor.
However, cyanide is also an irreversible inhibitor since it does not only inhibit TCA cycle but also
the respiration process as a whole.
c. malonate
🡪 Malonate is a structural analog of succinate and functions as a competitive inhibitor of SDH. Since
malonate has the same structure as that of succinate, malonate will compete with succinate in
This study source was downloaded by 100000899606070 from CourseHero.com on 03-04-2026 13:37:23 GMT -06:00
https://www.coursehero.com/file/108716308/Exercise-7-Post-Labdocxpdf/
