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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.
4. Practical Session
ORGANIZATION
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CARE OF THE MITOCHONDRIA
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.
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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.
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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).
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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
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(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?
(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)
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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.
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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 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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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.
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