BI2RT1 / MD1006P4 2015-16

OXIDATIVE PHOSPHORYLATION, ELECTRON

TRANSPORT AND PROTON TRANSLOCATION

Students must watch the audio visual demonstration How to Use an

Oxygen Electrode before you come to the laboratory

1. Learning Outcomes
Upon successful completion of this practical you will be able to:
Use an oxygen electrode to monitor oxygen uptake
Explain how ATP synthesis is coupled to electron transport
Reproduce and interpret the results of classic experiments involving inhibitors of
electron transport, membrane transporters and uncouplers.
Interpret the results of experiments designed to investigate Ca2+ transport into
mitochondria.

2. Safety Information
DO NOT MOUTH PIPETTE ANYTHING! All of the inhibitors (Table 2) are
toxic and there are no specific antidotes
WEAR GLOVES
REPORT SPILLAGES to a Demonstrator.
SYRINGE NEEDLES are used to modify the Gilson and Finn micropipettors.
Needle-stick injuries can be serious (eye damage etc.), so be careful when
handling and using them.
DO NOT RE-SHEATH NEEDLES, you may get a needlestick injury.
ELECTRICITY AND WATER Because of the range of electrical apparatus in
use, and the presence of water aspirators, care has to be taken to avoid electric
shock. Keep electric apparatus well away from the sink and taps and do not
allow splashing to occur.
AZIDE Do not expose azide solutions to an acidic environment (danger of toxic
gas evolution).

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3. Introduction
The background supplementary material for this practical includes a PDF
file and a video, both in Blackboard:
The PDF file Oxidative Phosphorylation, Essential Reading contains diagrams that you
will need to refer to during the practical. You should print it and bring it with you to
the practical. It is essential that you watch the video before you come to the
lab.

Relevance of the practical to medicine In mammals, the majority of

oxygen that we consume is as a result of the ATP synthesis. There are many disease
syndromes associated with mutations in mitochondrial proteins. A subset of these
diseases is associated with mutations in the proteins subunits of the electron transport
chain and ATP synthase. These diseases are sometimes called mitopathies and can
result in myopathies (muscle diseases) and neuropathies (neuronal diseases). The
majority of the electron transport chain and ATP synthase proteins, are encoded by
nuclear DNA. However, 13 oxphos proteins, 2 rRNAs and 22 tRNAs are encoded by
mitochondrial DNA (mtDNA). Some of the myopathies and encephalomyopathies that
can result from damage to mtDNA are Lebers Heredity Optic Neuropathy (LHON),
Myoclonic Epilepsy with Ragged Red Fibres (MERRF) and Mitochondrial
Encephalopathy, lactic acidosis and stroke-like episodes (MELAS).

The oxygen consumption assay performed in this practical is one of a battery of

assays used to detect defects in mitochondrial bioenergtic function from (muscle)
biopsies of patients predicted to have a mitopathy.

4. Practical Session

SUMMARY of the laboratory work

You will be supplied with a preparation of rat liver mitochondria. The
mitochondria will have been prepared using a protocol very similar to the one you
used in the Subcellular Fractionation Practical.
You will use the mitochondria to calibrate an oxygen electrode. You will then
investigate O2 consumption and ATP synthesis in the presence of glutamate plus
malate, in the presence of succinate and in the presence of the uncoupler 2,4-
dinitrophenol. The various solutions you will use are described Table 1.
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You will demonstrate how inhibitors of electron transport can be used to
elucidate the structure of the electron transport chain. The various inhibitors are
described in Table 2.
The mitochondrial inner membrane possesses a variety of transport systems
involved in the movement of nucleotides, substrates, metabolites and metal cations
into and out of the mitochondrial matrix. You will investigate two of these transport
systems, the adenine nucleotide and the calcium transporter (see Table 2).

ORGANIZATION

You will work in pairs, two students to an oxygen electrode.

Reagent Function

Reaction Medium Buffer and osmotic protectant

20 mM Tris-HCl, 80 mM KCl, 5 mM MgCl2, pH 7.4
Glutamate plus Malate mix Fuel substrate, supplies electrons to NADH
160 mM sodium glutamate plus 40 mM sodium malate
Succinate Fuel substrate, supplies electrons to FADH2
0.5 M sodium succinate
ADP Substrate for ATP synthase
40 mM ADP
0.5 M Phosphate Source of inorganic phosphate
0.5 M potassium phosphate, pH 7.4
Calcium
10 mM CaCl2
Table 1 Substrates and Reagents Used to Study Electron Transport in Mitochondria

Rotenone Inhibitor of electron transport Obtained from the roots of plant

-1 at Complex I. species belonging to the genus
100 g ml in ethanol
Derris. Used by Amazonian
Indians to poison fish and by
European gardeners as an
insecticide (Liquid Derris).
Antimycin A Inhibitor of electron transport An antibiotic secreted by
1 at Complex III Streptomyces.
100 g ml in ethanol
Sodium azide Inhibitor of electron transport A substitute for cyanide or carbon
0.4 M sodium azide in H2O at complex IV monoxide.
Oligomycin Inhibitor of ATP synthase An antibiotic secreted by
-1 Streptomyces.
100 g ml in ethanol
Atractyloside Inhibitor of the adenine A highly toxic glycoside isolated
nucleotide transporter from Atractylis gummifera and
other thistles.
Ruthenium red 2+ Oxychloride ammoniated complex
Inhibitor of the Ca / H+
1.0 mM ruthenium red in H2O of the lanthanide metal
antiporter
ruthenium
DNP Chemical uncoupler
2,4-dinitrophenol, 3 mM in
H2O
Table 2 Inhibitors and Uncouplers Used in the Study of Electron Transport in Mitochondria

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CARE OF THE MITOCHONDRIA

Mitochondria are very fragile and it is important that the mitochondrial

suspensions must be kept cold and must not be contaminated by pipette tips,
which might have been used for inhibitors or which have come into contact with
perspiration from your hands or contamination from dirty benches. Keep your glass
pipettes clean by placing them upright inside clean test tubes held in a
suitable rack. Never leave them horizontally on the bench.
Mitochondria will sediment with time and thus all suspensions must be mixed by
gently inverting the tube (covered with parafilm) prior to withdrawing a sample.
Before you start: Use a scissors to cut about 4.0 mm off the tip of a yellow tip.
Use this cut-off tip to pipette the mitochondria. A narrow orifice could produce
shearing forces that may damage the mitochondria. Keep this tip for pipetting
mitochondrial suspension only. Keep it clean. This tip is not to be confused with the
more severely truncated type used to fit the needles to the micropipettes.

4.1 ORGANISE YOUR PIPETTES AND TIPS

Organise your pipettes and tips as demonstrated in the video. The 5 ml pipette
in the test tube rack is for pipetting reaction medium. Prepare a paper concertina and
label it Suc (for succinate), ADP, Glu (for glutamate / malate), Rot (rotenone), antim
(antimycin) and Azide. Assemble the syringe needles (in a beaker) and the cut-off
yellow tips (in another beaker) by fitting the green end of a needle snugly to a
truncated tip, with a slight twist and push. The fit must be air-tight. Lay the
assembled needles on the concertina. Use the same tip each time you pipette a
particular solution.

4.2 CHECK THAT THE ELECTRICS WORK CORRECTLY

1 Your oxygen electrode chamber will have been left containing water.
2 Switch on the electric stirrer, the control box and the recorder (chart speed 10
-1
mm min ). Check with demonstrator See Fig1 A & B for chart recorder
details
3 The voltage on the recorder will be set at 1 mV. Check that it is. See Figure 1
C.
4 Zero the recorder (left side of paper). See Fig. 1C.

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5 Switch the recorder to electrode output as described in bold in note to Fig
1C. Check that the pen is moving across the paper towards 100% (right side of
paper).
6 Switch off the stirrer, the pen should move back towards zero.

Figure 1 Chart recorder

details.
Fig 1A: Note the oxygen
content is measured on the left-
right axis as indicated. The
electronic zero point is set on
the extreme left hand side. The
oxygen content will be set by
you using the oxygen electrode
control box at a position just short of the edge of the right hand side of the chart
paper.

Fig 1B: First, check the left handside of the chart

recorder. Is the chart recorder on? Check that the
chart speed is set for 10 mm/min. As indicated the
dial should be set at 10 and the indicated button
should be up.
You do not need to press any other buttons. If necessary you can change the dial to
vary the speed the paper moves towards you. Always indicate chart speed on the
chart.

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Fig. 1C: Check the right handside of the chart recorder. The voltage on the recorder
will be set at 1 mV. To set electronic zero press indicated button and adjust the pen to
the extreme left of the chart paper (electronic
zero). When this button is down the recorder
is isolated from the oxygen electrode. Now
press it again the button will rise up and
now the chart recorder is responding to the
electrode. The pen should move somewhere
to the right when you do this. The exact
position of the pen can be set by you by
adjusting the controls (fine control) on the
oxygen electrode control box.
NOTE: Make sure that the coarse control is set at
the lowest setting first and then pen position with
the fine control. (if you have difficulty ask the
demonstrator).

4.3 CALIBRATION OF YOUR OXYGEN ELECTRODE

One member of each pair should act as an event marker by annotating the
chart every time an addition is made to the reaction chamber.
The aim of this experiment is to convert the distance across the chart paper into an
amount of oxygen, i.e. to calculate a value for nmole O/small division to know how
much each small division represents in terms of the amount of oxygen in the electrode
chamber. To do this you will add a volume of reaction buffer and phosphate that is
representative of the total volume of the chamber in subsequent experiments (~ 4.3
ml would be such a value). Once you have set the 100% position using the control on
the oxygen electrode box ask a demonstrator to add a small amount of sodium
dithionite which will react with the dissolved oxygen in the medium and remove it all.
Sodium dithionite (also known as sodium hydrosulfite) is a white crystalline powder
which reacts very rapidly with oxygen to generate sodium bisulfate and sodium
bisulfite.
Na2S2O4 + O2 + H2O NaHSO4 + NaHSO3

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STEPS:
1 Shake your bottle of Reaction Medium vigorously to ensure air-saturation and
record its temperature by placing a clean thermometer carefully into it.
You will need this temperature measurement later to calculate the amount of oxygen
dissolved in the reaction medium.
2 Aspirate the water out of the oxygen electrode chamber.
3 Pipette 4.3 ml of Reaction Medium into the oxygen electrode chamber
and turn on the stirrer.
4 When a steady-state response is obtained adjust the control box so that the
recorder pen reads 90 scale divisions i.e. the pen is off-set ten small divisions
from the right-hand side of the recorder paper. This is the 100% oxygen you
should use later in your calculations.
5 Make sure your recorder is switched on. At this point ask a demonstrator to
add some sodium dithionite to your electrode chamber. The pen should
move rapidly to the left until all of the oxygen has been removed. Your trace
should resemble that shown in Figure 4 of Oxidative Phosphorylation Background
Reading. Note that in Figure 4 the zero point is obtained using mitochondria and
succinate plus ADP rather than dithionite but the result is the same).
6 Record the position of anaerobiosis.- this position is your zero oxygen
position for use in calculations.
7 Immediately remove the solution and thoroughly wash out your chamber with
water, at least three times to remove all traces of dithionite. Add 3.9 ml of
Reaction Medium into the oxygen electrode chamber and turn on the stirrer.
8 Before moving on to the next experiment use the values in Table 4 to
calculate how many nmoles of oxygen each small division across the chart
paper represents. See Section 5 for details. Make a note of your
calibration: you should now have a value of X nmoles oxygen/small
division. Confirm this with your demonstrator. You must have this
calibration value before you proceed to next experiments.

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State Definition
State 1 The rate of oxygen consumption observed when
mitochondria and reaction medium are added to an
oxygen electrode.
State 2 Rate of oxygen consumption observed when a
substrate (e.g. succinate) is added to mitochondria
in State 1.
State 3 The burst of oxygen uptake following the addition
of ADP to mitochondria in State 2.
State 4 The slow rate of oxygen consumption observed
after all added ADP has been phosphorylated to
form ATP.
Table 3 Oxygen Consumption in States 1 to States 4

4.4 MEASUREMENT OF RESPIRATORY CONTROL RATIO AND

P:O RATIO FOR VARIOUS SUBSTRATES AND THE EFFECT OF
INHIBITORS OF ELECTRON TRANSPORT ON THE RATE OF
OXYGEN CONSUMPTION
The P:O ratio is a measure of the amount of ATP produced per mole of oxygen
consumed. How to calculate P:O ratios will be explained in Section 5.
All of the additions must be made before the system becomes anaerobic (State
5). Speed is important. Do not sit about between additions, be ready with the next
addition.
Make the following additions to the oxygen electrode vessel:
1 3.90 ml of reaction medium
2 100 l of potassium phosphate buffer
3 100 l of mitochondria
4 Put the lid on, remove air bubbles, bring pen to the 95th division and observe a
stable blank rate (State 1) for 1-2 minutes.
5 Add 100 l of glutamate/malate, observe a stable State 2 for two minutes.
6 Add 20 l of ADP observe State 3 and wait for State 4 should take no longer
than 3 minutes.
7 Add 10 l of rotenone observe for 30 seconds.
8 Add 100 l of succinate, can you restart oxygen consumption with this
alternative substrate? Wait two minutes.
9 Add 20 l of ADP observe state 3 and wait for state 4 as before
10 Add 10 l of Antimycin A observe for 60 seconds.
Clean out the oxygen electrode with copious amounts of water

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4.5 EFFECTS OF OLIGOMYCIN, UNCOUPLER AND THE ACTIVE

TRANSPORT OF CALCIUM IONS ON THE RATE OF OXYGEN
CONSUMPTION
Make the following additions to the oxygen electrode vessel:
1 3.90 ml of reaction medium
2 100 l of potassium phosphate buffer
3 100 l of mitochondria
4 Put the lid on, remove air bubbles, bring pen to the 95th division and observe a
stable blank rate (State 1) for 1-2 minute.
5 Add 100 l of sodium succinate, observe State 2 for two minutes.
6 Add 10 l rotenone, wait one minute to confirm that rotenone has no effect (why
no effect?)
7 Add 20 l of ADP wait for State 4
8 Add 10 l of oligomycin observe the steady rate for 1 minute
9 Add 20 l of ADP observe for 1 minute
10 Add 50 l of CaCl2 observe rapid rate, wait until a slower rate (i.e. State 4) is
achieved
11 Add 20 l of ruthenium red wait 30seconds
12 Add another 50 l dose of CaCl2 observe for 2 minutes
13 Add 40 l of 2,4 DNP, what happens?
Clean out the oxygen electrode with copious amounts of water

4.6 THE EFFECT OF ATRACTYLOSIDE ON THE ADENINE

NUCLEOTIDE TRANSLOCATOR

Figure 1 The Adenine Nucleotide Translocator

Make the following additions to the CLEAN oxygen electrode vessel:

1 3.90 ml of reaction medium
2 100 l of potassium phosphate buffer
3 100 l of mitochondria
4 Put the lid on, remove air bubbles, bring pen to the 95th division and observe a
stable blank rate (State 1) for 1 minute.
5 Add 100 l of succinate, observe State 2 (2 minutes).
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6 Add 10 l of rotenone
7 Add 20 l of ADP. When State 4 has been achieved add 10 l of atractyloside
and a few minutes later add 20 l of ADP.
8 What do you observe, where does atracyloside act?
9 Add 40 l of 2,4 -DNP.
10 Why does the addition of 2,4 DNP cause oxygen uptake?

QUESTIONS to be resolved during the practical

(i) What are the respiratory control ratios measured for:

(a) glutamate/malate
(b) succinate

(ii) What are the P:0 ratios measured for:

(a) glutamate/malate ?
(b) succinate ?

(iii) What is the effect of rotenone, antimycin A and atractyloside on mitochondrial

oxygen consumption rate and phosphorylation and where do they act?

(iv) What is the effect of oligomycin on mitochondrial oxygen consumption rate and
phosphorylation and how does it act?

(v) Can you explain your observations with calcium ion addition. How does
ruthenium red act?

(vi) Calculate the Ca2+/O ratio

(vii) Calculate the final concentration of the following in the reaction system
assuming a volume of 4ml and ignoring the increase in volume caused by any
addition :
glutamate
malate
succinate
ADP
calcium

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5 CALCULATIONS
Calibrating a recorder trace and calculating the rate of oxygen consumption
You need the temperature of the reaction medium. From that the concentration of O
in the reaction medium can be derived from Table 4.
Temperature 0C nanomol O per ml
16 562
17 551
18 540
19 530
20 521
21 512
22 503
23 495
24 486
25 479
26 471
Table 4 Concentration of O in Air-Saturated
0.15M KCl as a function of Temperature
[From: Reynafarje, B., et al (1985). Anal. Biochem. 145,406-418]

The full-scale span in the trace in Figure 2 = 91.5 divisions i.e. 100% oxygen = 91.5
divisions. Based on Table 3, 1 ml of KCl based reaction medium at 220C = 503
nmoles O. In the experiment described in Section 2.3, the total reaction volume was
4.3ml.

Therefore using the information above, a sample calculation can be worked out as
follows:
1ml medium = 503 nmoles O
4.3 mls medium = 2162.9 nmoles O (or 2.2moles O)

Total Full Scale Span = 91.5 divisions

1 division = 2162.9/91.5 = 23.6 nmoles O per division (or 0.024moles O).

The chart speed was 1 cm min-1.

For a State 3 rate of 34 divisions per minute:
34 x 23.6 nmoles O per min = 802.4 nmoles O per min (or 0.80moles O per min).

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Figure 2 A Typical Trace Obtained During an Oxygen Electrode Calibration

The full-scale span = 91.5 divisions i.e. 100% oxygen = 91.5 divisions.

P:O ratio
The P:O ratio is a measure of the amount of ATP produced per mole of oxygen
consumed. The amount of ATP produced is equivalent to the amount of ADP used up
and so the P:O ratio can be calculated as the ratio of a known amount of ADP added
vs the amount of O taken up during the phosphorylation of that ADP.

To calculate the amount of ADP used:

Use the State 3 Rate obtained with 20 l of ADP in Experiment 4.4.
You added 20 l of 40 mM ADP to the reaction mixture one litre of 40 mmol of ADP
contains 40 mmoles. It follows that 1 ml of 40 mM ADP contains 40 moles of ADP
and 1 l of 40 mM ADP contain 40 nmoles of ATP. How many nmoles of ADP in 20 l?

To calculate the amount of O used:

Work out the State 3 rate of O consumption form the trace of Experiment 4.4 as
described above. That will give you a value in moles of oxygen per min. Then you

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just need to work out how many minutes the State 3 rate lasted for and you can
calculate the amount of O consumed as 20 l of ADP is used up.
To calculate the Respiratory Control Ratio:
The respiratory control ratio is State 3 rate / State 4 rate and is a measure of the
tightness of the { HYPERLINK
"http://www.bmb.leeds.ac.uk/illingworth/oxphos/history.htm" \l "coupling" } between
respiration and phosphorylation.

APPENDIX

THE CLARK-TYPE OXYGEN ELECTRODE

The type of electrode (Rank Bros., Bottisham, Cambridge, U.K.) shown above consists
essentially of a Ag/AgCl reference half-cell, joined to a Pt/02 cathode by a saturated
KCl bridge. Its purpose is to measure the rate of transfer of O2 to the platinum
cathode, this rate being proportional to the activity of O2 in the reaction chamber.

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At the plateau region, the current flowing is proportional to the concentration of O 2 in

the reaction chamber. Therefore, a polarising voltage of 0.6 volts is placed across the
cell. Since many reactions involve redox components which would interfere with this
system, a teflon or polyethylene membrane theoretically permeable only to O2, is
interposed between the platinum electrode and the reaction mixture. The rate and
extent of uptake or evolution of O2 during a reaction can thus be measured by the
rate of change of the current passing through the cell. Although N2 and CO2 are also
present in air-saturated aqueous solutions and can diffuse across the membrane they
undergo no electrode reaction.
The polarising voltage is provided by the "Black Box", which also includes a
sensitivity control to enable the current change to be recorded conveniently on a strip-
chart recorder.
The reaction at the cathode is :
2H+ + 2e- + 1
/ 20 2 = H 20

When a membrane is present to prevent other oxidizing species reacting with the
cathode, electrons may only enter solution when O2 is present. The current flowing is
proportional to the activity of O2 provided the solution is adequately stirred to
minimize the formation of an unstirred layer next to the membrane.

The black box converts the current into a potential difference down the potential
divider, which allows the voltage across the recorder to be varied.

6/2/2015 Porter / Nolan / Nic a' Bhird / Robinson

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