Mitochondrial Respiration Analysis: BCH3021 Lab Report - Semester 1

This report details an experiment investigating mitochondrial respiration using both computer simulation (OxPhos) and an oxygen electrode. The experiment aimed to observe the effects of substrates (pyruvate/malate, succinate), ADP, and various inhibitors (rotenone, antimycin A, sodium azide) on oxygen consumption and the electron transport chain. The study involved the isolation of mitochondria from beef heart muscle and monitoring oxygen uptake to assess respiratory control ratio (RCR) and ADP/O ratios. The results, including calibration curves and traces of oxygen consumption, demonstrated the impact of different substances on mitochondrial function. The report discusses the roles of each inhibitor, the limitations of the oxygen electrode, and the importance of differential centrifugation for mitochondrial isolation. The findings align with established concepts of mitochondrial respiration, highlighting the significance of each component in the electron transport chain and the overall process of ATP synthesis.

Assessing the effects of substrates, ADP and inhibitors on mitochondrial
respiration in isolated mitochondria
Background
Mitochondria are double-layered cellular organelles found in most eukaryotes. Often, these are
referred to as the power house of the cell since they perform cellular respiration which is vital to
the survival and proper functioning of the cell. Errors in mitochondrial function often lead to the
development of diseases in humans. Several methods have been developed to study mitochondrial
respiration. Among these, software-based simulations provide an alternative and convenient
method to oxygen electrode and aid in visualising mitochondrial respiration. One of such
computer-based programs is OxPhos. It produces a graph that enables users to visualise the
oxygen uptake, ADP consumption and study the effects of different activators and inhibitors on
mitochondrial function.
For in vitro experiments, mitochondria are obtained by isolation from beef heart muscle.
Respiration occurs at the mitochondrial membrane and involves ATP synthesis through the
electron transport chain (ETC) (Liu et al., 2002). Respiration (oxygen consumption) can,
therefore, be monitored with an oxygen electrode. The method can be used to monitor the effects
of substrates and inhibitors like rotenone (Li et al., 2003), antimycin A (Rieske et al., 1967) and
sodium azide (Bogucka and Wojtczak, 1966), on the mitochondrial membrane complexes I, II, III
and IV of the ETC.
From the data obtained from the oxygen electrode, the rate of oxygen consumption can be
calculated. Also, respiratory control ratio (RCR), which represents the integrity of mitochondrial
membrane, can be calculated from the data obtained from oxygen electrode. An intact and
functioning mitochondria yield a value of more than 10 while damaged mitochondria have a
value of less than 2. Fully uncoupled mitochondria yield an RCR of 1. The amount of ATP
produced from the movement of two electrons through the ETC can be expressed as the ADP/O.
Hence, an accurate interpretation of the effects of different substrates and inhibitors on
mitochondrial respiration can be made by following the foregoing methods.
Aims
 To conduct a computer simulated mitochondrial respiration using OxPhos and analyse the
results.
 To measure oxygen consumption using an oxygen electrode and visualise mitochondrial
respiration using polarography.
 To observe the effects of inhibitors and change in respiration states using polarography.
 To explore the use of clear native electrophoresis for examination of mitochondrial protein
complexes.
Approach
The experiment was conducted in three sessions (week 5-7). Computer simulated OxPhos
program to explore respiration was completed in Week 5. While the calibration of an oxygen
electrode and measurements of oxygen consumption by mitochondria isolate with different
substrates and ADP additions was conducted in week 6. In week 7, testing of different inhibitors
were conducted in addition to the tasks of week 6.
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Methods
The method for week 5 was followed as outlined in the BCH3021 laboratory manual pages 85-
90. No amendments made.
The method for week 6 was followed as described in the BCH3021 laboratory manual pages 104-
112. The volume of ADP added was changed from 10 μL to 20 μL.
The method for week 7 was followed as described in the BCH3021 laboratory manual pages 115-
117. Changes were made to the second run with inhibitors as detailed below.
Table 1: Volume adjustment for substrate and inhibitors in run 2 (week 7).
Substrate/inhibitor Original volume (μL) Volume adjustment (μL)
Rotenone 40 40
Succinate 50 60
Antimycin A 40 50
Ascorbate + TMPD 50 50
Sodium azide (NaN3) 40 60
Results
During weeks 6 and 7, the data obtained could not be used due to equipment failure. Therefore,
all data for this report was obtained from another group with the appropriate volume adjustments
for traces detailed in the method above except for week 7 experiment 1 pyruvate + malate run.
Calculations and tables of result for rate of oxygen consumption, RCR and P/O ratio are included
in appendix 1 and 2.
In weeks 6 and 7, calibration of the oxygen electrode was confirmed by adding sodium sulphite to
the electrode. Rapid oxygen uptake was observed (figures 1 and 6) indicating that the electrode
was functioning properly. Oxygen consumption with substrate pyruvate + malate is shown in
Figure 2 (experiment 1) and Figure 3 (experiment 2). Oxygen consumption with succinate is
shown in Figures 4 and 5 (experiment 1 and 2 respectively). These traces indicate a steady
uptake of oxygen accompanied by an increase in ATP concentration due to conversion of ADP
which was added after the addition of substrate.
Figures 7 shows the effects of adding ADP instead of mitochondria on the second occasion.
Figure 8 shows the trace of oxygen consumption in the presence of mitochondria, substrate and
ADP only.
Figure 9 and 10 show the effects of addition of different types of inhibitors and substrates on
mitochondrial respiration. The effect is evident in each trace as significant decrease or saturation
in oxygen consumed when either inhibitors or substrates are added. This demonstrates respiratory
control in which the addition of inhibitors temporarily halts the electron transport between
complexes. However, further addition of substrate re-establishes electrochemical gradient
overcome inhibition.
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Figure 1: Calibration curve using sodium sulphite to deplete oxygen from chamber
The calibration curve was obtained from the experiment performed in week 6
Figure 2: Trace of oxygen consumption with pyruvate + malate substrate (experiment 1).
Figure 3: Trace of oxygen consumption with pyruvate + malate substrate (experiment 2).
In the above traces, M denotes addition of mitochondria into the Clarke oxygen electrode. S
denotes addition of substrate (pyruvate + malate) to the reaction system and A denotes the
addition of ADP.
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Figure 4: Trace of oxygen consumption with succinate (experiment 1)
Figure 5: Trace of oxygen consumption with succinate (experiment 2)
Figure 6: Calibration curve using sodium sulphite to deplete oxygen from chamber
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Figure 7: Trace of oxygen consumption when second m is replaced with ADP
Figure 8: Trace of oxygen consumption
Figure 9: Trace of oxygen consumption with substrates and inhibitors (run 1)
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Figure 10: Trace of oxygen consumption with substrates and inhibitors (run 2)
Order of addition: Mitochondria (M), Pyruvate + Malate substrate (S), ADP (A), Inhibitor (I)
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Discussion
Mitochondrial respiration refers to the series of metabolic reactions that require oxygen to
synthesize ATP. The process involves sequential transfer of electrons among the various
complexes participating in the process. The complexes are designated as complex I, complex II,
complex III, complex IV, and complex V. Electron flow among these complexes leads to the
transport of protons across the inner mitochondrial membrane to generate proton gradient that
ultimately leads to the conversion of ADP to ATP. In the current experiment, mitochondrial
respiration was investigated using computer simulated program, OxPhos, and the Clarke oxygen
electrode. Respiratory control was studied, which involved the initiation of state IV by an outside
source following state III respiration. Stimulation of state III respiration was achieved through the
increase of substrates like pyruvate/malate and succinate in the system. State III is further
stimulated by addition of ADP, as shown in figures 2, 3, 4 and 5. When inhibitors are added into
the system, they target and bind various complexes of the electron transport chain and hence
affect the electrochemical gradient formed during oxidative phosphorylation. ATP synthesis in
this case is temporarily disabled until inhibition is overcome or bypassed.
The traces (Figures 7 and 8) representing oxygen consumption for the experiment yielded
expected results. Oxygen consumption through all the traces was quite slow, meaning that state
III was maintained for a prolonged period before state IV (2) was achieved.
Figure 9 and 10 show the effects of introducing inhibitors into the oxygen electrode. The
inhibitors used in this experiment were rotenone, antimycin A and sodium azide, all of which
were added sequentially. These inhibitors generated the expected results, in which respiration
inhibition was achieved. Each addition of inhibitor was followed by the addition of a substrate
like succinate or an electron donor like ascorbate/TMPD which enabled the restart of respiration.
The roles of each inhibitor are quite specific as they selectively target and bind certain complexes
in the ETC. Rotenone targets and binds complex 1, stopping the transfer of electrons from
complex 1 to ubiquinone (Li et al., 2003), hence after rotenone was added, succinate substrate
followed, as it can feed electrons straight into complex II essentially bypassing complex 1
inhibition. Antimycin A is an inhibitor of complex III, where it binds to cytochrome c reductase,
inhibiting the oxidation of ubiquinone to ubiquinol and stopping reparation (Rieske et al.,1967).
Hence, the addition of ascorbate/TMPD was added as TMPD acts as an artificial electron carrier
which is readily reduced by ascorbate and oxidised by cytochrome c. Ascorbate/TMPD is used to
elucidate the process of oxidative phosphorylation, enabling transfer of electrons straight into
complex IV. Addition of sodium azide is known to inhibit cytochrome oxidase and hence stops
the transfer of electrons to the final reaction in ETC causing a rapid depletion of ATP (Bogucka
and Wojtczak, 1966).
In the current experiment, the Clarke oxygen electrode posed as a significant limitation.
Introduction of air bubbles into the electrode can have detrimental effects on the results. Also,
human error during experimentation and data collection is another source of variation in the
results.
The mitochondrial isolate was prepared using differential centrifugation of beef heart muscle as
they are dense in mitochondria and provide a high yield. Differential centrifugation is a process in
which a sample is subjected to multiple centrifugations with increasing centrifugal force each
time. Separation occurs based on particle mass and size meaning that the mitochondrial isolate is
most likely relatively pure. However, to further assess the purity of the preparation, and SDS-
PAGE, western blot or immune-detection could be carried out.
Conclusion
The experiment involved visualising mitochondrial respiration using a Clarke oxygen electrode.
The effects of different substrates, ADP and inhibitors were explored the respiratory control was
investigated. It was observed that the results obtained were in concurrence with the prevailing
concepts.
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References
BCH3021 laboratory manual
Bogucka, K. and Wojtczak, L., 1966. Effect of sodium azide on oxidation and phosphorylation
processes in rat-liver mitochondria. Biochimica et Biophysica Acta (BBA)-Enzymology and
Biological Oxidation, 122(3), pp.381-392.
Frazier, A.E. and Thorburn, D.R., 2012. Biochemical analyses of the electron transport chain
complexes by spectrophotometry. In Mitochondrial Disorders (pp. 49-62). Humana Press.
Li, N., Ragheb, K., Lawler, G., Sturgis, J., Rajwa, B., Melendez, J.A. and Robinson, J.P., 2003.
Mitochondrial complex I inhibitor rotenone induces apoptosis through enhancing
mitochondrial reactive oxygen species production. Journal of Biological Chemistry, 278(10),
pp.8516-8525.
Liu, Y., Fiskum, G. and Schubert, D., 2002. Generation of reactive oxygen species by the
mitochondrial electron transport chain. Journal of neurochemistry, 80(5), pp.780-787.
Rieske, J.S., Lipton, S.H., Baum, H. and Silman, H.I., 1967. Factors affecting the binding of
antimycin A to complex III of the mitochondrial respiratory chain. Journal of Biological
Chemistry, 242(21), pp.4888-4896.
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Appendix I
Worksheet 1
Results Table 1
Parameter
0.1 M Pyruvate + 0.1 M Malate 0.1 M Succinate
Expt 1 Expt 2 Mean Expt 1 Expt 2 Mean
ΔY/ΔX (% O sat. min-1)
State 4(1) -4.5 -2.1 -3.3 2.1 -1.5 0.7
State 3 -15.3 -13.8 -14.55 -8.9 -7.5 -8.2
State 4(2) -1.8 -2.1 -1.95 -1.9 -1.8 -1.85
Respiratory
Control Ratio
(RCR)
8.5 6.6 7.55 4.68 4.17 4.425
Respiration
Rate
μmol atom O.min-1..ml-1. Mitochondria
State 4(1) 0.00046073 0.00021501 0.0003377 0.00021998 0.0005713 0.00018856
State 3 0.00156648 0.00141290 0.0019897 0.00093231 0.0007856 0.00085899
State 4(2) 0.0018429 0.00021501 0.0001997 0.00019703
2
0.0001885 0.00019380
ADP:O ratio
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Calculations for respiration rate, respiration control ratio (RCR) and ADP:O ratio
Respiration rate
State 4(1) – Pyruvate + malate
-[-ΔY/ΔX]/100 x 2.37 x 2.16 x 1/50
-[-3.3]/100 x 2.37 x 2.16 x 1/50
= 0.0003377 μmol atom O. min-1.ml
State 4(1) – Succinate
-[-ΔY/ΔX]/100 x 2.37 x 2.16 x 1/50
-[0.7]/100 x 2.37 x 2.16 x 1/50
= 0.00018856 μmol atom O. min-1.ml
Same calculations were carried out for state 3 and state 4(2) of pyruvate + malate and succinate,
summarized in results table 1 above.
Respiration control ratio (RCR)
Pyruvate + malate experiment
RCR = State 3/State 4(2)
= -14.55 (% O sat.min-1) / -1.95 (% O sat.min-1)
= 7.55
Succinate experiment
RCR = State 3/State 4(1)
= -8.2 (% O sat.min-1) / -1.85 (% O sat.min-1)
= 4.43
P/O ratio
P/O ratio = μmol ADP consumed/ μmol atomic oxygen consumed when mitochondria is in state 3.
Calculating P:
n = cv
n = 0.05 mol/L x 0.00001 L = 0.05 μmol
Calculating O:
Oxygen consumed during state 3 = [(%O saturation at the start state 3 - %O saturation at the end state
3)/100] x 0.95 μmol/ml x 2ml (volume of added buffer)
From the graph of pyruvate + malate
100 %O saturation – 10 %O saturation = 90
(90/100) x 0.95 x 2
= 1.71 μmol
P/O ratio = 0.05 μmol /1.71 μmol = 0.029
The same calculations were carried out for P/O ratio of succinate, summarized in results table 1
above.
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QUESTIONS
1. Comment on the reproducibility of the measured rates for each of the substrates used.
Reproducibility cannot be measured for each substrate used due limitations placed on the experiment
by factors beyond our control including; time constraints, mitochondria availability and electrode
malfunction.
2. Comment on any differences in the mean rates of respiration comparing pyruvate-malate or
succinate as substrates.
The rates of respiration between pyruvate-malate and succinate seem to indicate that the respiration of
succinate occurred much faster than pyruvate-malate. This could be due to the rate at which succinate
and pyruvate are feeding electrons into the ETC. Pyruvate is slower in feeding electrons as it needs to
be oxidized to acetyl-CoA before entering the citric acid cycle where it can then start feeding
electrons. On the other hand, succinate is already an intermediate of the citric acid cycle and of
formed in step five by substrate level phosphorylation of succinyl-CoA. Hence succinate can provide
electrons to complex II faster producing a quicker respiration rate.
3. Comment on the RCR for your preparation of mitochondria, using pyruvate-malate or
succinate as substrates. Do you consider yours to be a well coupled preparation?
The RCR calculated for pyruvate-malate and succinate were 7.55 and 4.43 respectively. RCR value of
greater than 10 indicates that the mitochondria have been well prepared. Despite the RCR of succinate
to be just under 10, using succinate substrate can also be classified as well prepared although to a
lower quality than pyruvate-malate. Both values did not fall into the range of damage mitochondria
(RCR < 2) or fully uncoupled mitochondria (RCR = 1).
4. Comment on the P:O ratio measured for each of pyruvate-malate and succinate as substrates.
Are the P:O ratios of the anticipated order of magnitude? Are there systematic differences
between the P:O ratios measured for the two types of substrates used? Explain why you would
expect there to be a difference between pyruvate-malate and succinate.
For the pyruvate-malate substrate, P/O ratios can be generated in the range of 2.5-2.7, whereas for
succinate substrate can generate values in the range of 1.5-1.7. The values calculated for the
experiment indicate that both pyruvate-malate has a P/O ratio of 0.029. This indicates that the values
obtained from the experiment are incorrect in comparison as they are well below the expected range
of both pyruvate-malate. The P/O ratio should be greater for pyruvate-malate compared to succinate
because the theoretical values are obtained based on the number of protons pumped from the
movement of two electrons donated by reduction of oxygen atom divided by the number of protons
needed for ATP synthesis. Hence, oxidation of pyruvate/malate will pump more protons compared to
succinate leading to a greater P/O ratio.
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APPENDIX 2
Additions to chamber ΔY/ΔX (% oxygen saturation.min-1)
Expt 1 Expt 2 Mean
Pyruvate/malate + ADP -39 -10.1 -24.55
Rotenone -1 -0.53 -0.765
Succinate -31 -45 -38
Antimycin -0.63 -6 -3.315
Ascorbate + TMPD -8.7 -12.2 -10.45
Sodium azide (NaN3) -1.9 -0.79 -1.345
4. How would you set up experimental assays, using intact mitochondria with various substrates
and inhibitors, to measure the activity of the following enzymes spectrophotometrically?
[Note: that the inter-conversions of NADH/NAD+ and cyt cox/cyt cred can each be measured
spectrophotometrically]
(a) Complex I
Rotenone can be used to measure the activity of complex I as it selectively inhibits this complex.
Measure the absorbance of complex 1 with and without rotenone.
Performing a complex 1 assay.
• Prepare two cuvettes containing cell sample and Cl assay buffer. o First cuvette contains Cl buffer
and rotenone.
o Second cuvette contains Cl buffer and ethanol (rotenone replacement).
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