KE0026 Biochemistry Labs

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Wet Lab: Oxidative phosphorylation

The purpose of this practical is to investigate the respiratory chain by using electrochemical techniques and to increase the understanding of the respiratory chain and the flow of electrons through the major enzyme complexes.

The following experiments will be done:

1. Determination of ADP:O quotient and respiratory control
2. Effect of diffferent inhibitors on the respiratory chain

Respiratory investigation on liver mitochondria with oxygen electrode technique
Mitochondria are the energy producing centre in the cell and are responsible for the conversion of the main part of chemically bound energy to high energetic bonds in the form of ATP. About 50 to 2500 mitochondria can be found in one single cell. Each of them is equipped with a complete collection of enzymes to carry out the oxidative reactions in the citric acid cycle. The energy producing system is called the respiratory chain (or the electron transport system). The respiratory chain couples the oxidation of the citric acid substrates to the formation of ATP by transforming the oxidative energy to energy conserved in phosphate bonds. The respiratory chain can be blocked at several different places by agents such as amytal, rotenone, antimycin, KCN etc. Oxidative phosphorylation can be uncoupled from phosphorylation by for example free fatty acids and di-nitrophenol (DNP).

The oxygen electrode is very often used in studies of different mitochondrial functions. By using the oxygen electrode technique, several experiments can be made very fast in a very instructive way. Registration of rapid changes in the rate of oxygen utilisation by cellular and subcellular systems makes it possible to determine the ADP:O quotient as well as the mitochondria quality, effects of different inhibitors etc.

Experiment 1.
Determination of ADP:O quotient and respiratory control
Oxygen is consumed when suitable substrates are oxidised by the mitochondrial electron transport system. This decrease in oxygen concentration in a system closed from the atmosphere can be measured with an oxygen electrode, which is in effect an oxygen polarograph. In "coupled mitochondria", electron transport and the synthesis of ATP from ADP and Pi are mutually dependent processes, i.e., in addition to an oxidisable substrate, the presence of both ADP and inorganic phosphate is required for oxygen uptake to occur. Each group of students will measure the amount of consumed oxygen linked to substrate oxidation.

Materials
Equipment
Clarkes oxygen electrode
Reaction vessel
Amplifier
Magnet
Magnetic stirrer
Water bath (30 °C)
Recorder
Water ejector
Glass and Gilson pipettes
Hamilton syringes 10 and 20 ul
   Solutions
Air saturated incubation buffer (KCL 80 mM, MgCl2 5 mM, KH2PO4 5 mM, Tris-HCL 20 mM, pH 7.4)
Rat liver mitochondria
ADP 90 mM
DNP (dinitrophenol) 15 mM

Substrates
Na-glutamate 0.75 M
Na-succinate 0.75 M
Na-ascorbate/TMPD 150 mM/50 mM (TMPD = N,N,N,N-tetramethyl-p-phenylidene-diamine)
Performance
Check the adjustments on the apparatus:

Recorder:   Chart speed 20 mm/min
Range 10
Zero supress 0
Stirrer:   ON (no heat)

Adjust the recorder so that a total deviation, over the whole recorder paper, corresponds to the total amount of O2 in the reaction vessel by following the instructions below.
First empty the reaction vessel, thereafter fill the reaction vessel with new degassed buffer.
Press the red zero button on the amplifier and adjust to zero on the recorder by using the zero "wheel" on the recorder.
Adjust the oxygen content in the reaction vessel to 100% by turning the knob "calibrate" on the amplifier.

The initial O2-content in the reaction vessel is 0.25 mmol O2 /ml. The recorder should be adjusted so that a total deviation, over the whole recorder paper, corresponds to the total amount of O2 in the reaction vessel before any additions are made. When the recorder pen is at the right side no oxygen has been consumed, and when the pen reaches the left edge all oxygen has been consumed.

Experimental apparatus
  1. Determine the volume of the reaction vessel (the reaction vessels have different volumes). Calculate how much of the incubation media that has to be added to fill the reaction vessel with the other substances added afterwards. NB! The incubation media should be kept in an Erlenmeyer flask placed in a thermostated water bath (30 °C) and it should be gassed with a continuous air flow.

  2. Add the calculated amount of incubation media to the reaction vessel.

  3. Add 100 ml mitochondria suspension after about 2-4 cm registration. Mark on the record paper which additions are made in consecutive order.

  4. Follow the graph for 2-4 cm and add 20 ml substrate (start with glutamate) and observe what happens after the addition.

  5. After about 2-4 cm registration add 10 ml ADP via a Hamilton syringe and record the O2-concentration until all ADP has been consumed.

  6. Finally, when all ADP is consumed, add 20 ml of DNP. The DNP addition shall be done only with one of the substrates e.g. with glutamate.

  7. Repeat the experiment by using succinate and ascorbate/TMPD as substrate. When ascorbate/TMPD is used add only 5 ml ADP (instead of 10 ml).

Calculate the ADP:O quotient and the respiratory control ratios for all three substrates used.
Indicate the theoretical ADP:O values for the three substrates.
Calculate the respiratory control ratio (the slope with ADP/without ADP) for each substrate.
Explain why KH2PO4 and MgCl2 are included in the buffer.
What is DNP, and how is the respiration affected by addition of DNP?
Make a drawing of the respiratory chain and show where the substrates enter the respiratory chain.

Litterature:
Methods in Enzymology, vol X, pp 41
Stryer 5/e chapter 18; Horton, chapter 15

Experiment 2.
Effect of different inhibitors on the respiratory chain
Different inhibitors block the electron flow through the respiratory chain. The students receives three different inhibitors which should be added to a solution of mitochondrias and substrates in definite order. From this experiment conclusions can be drawn about which enzyme complex is blocked by which respective inhibitor.In these experiments, the following reagents are used instead of ADP.

Materials
Solutions:   Incubation buffer (same as in Exp. 1)
Glucose 0.6 M
ATP 120 mM
Hexokinase 20 mg/ml
   
Substrates:   Na-glutamate 0.75 M
Na-succinate 0.75 M
Na-ascorbate/TMPD 150 mM/50 mM
   
Inhibitors:   Rotenone 0.5 mM in 50% methanol (dissolve in 100% methanol first and add water afterwards)
Antimycin-A 100 mg/ml in 100% methanol (keep at -20 °C)
KCN 150 mM in KH2PO4 62 mM pH 7.5 (NB! Keep KCN in a closed tube!)
Performance
  1. Calculate how many ml of buffer solution will be needed to fill up the reaction vessel.
  2. Add x ml incubation buffer, 100 ml glucose, 100 ml ATP, 100 ml mitochondria and 100 ml hexokinase.
  3. Add reagent nr 1 and register the O2-consumption. Two to four cm registration is usually enough.
  4. Add reagent nr 2 and register; continue adding substances and registering until all substances have been added.
Nr ReagentVolume
1.Glutamate20 ml
2.Rotenone5 ml
3. Succinate20 ml
4. Antimycin A20 ml
5.TMPD/ascorbate20 ml
6.KCN20 ml

Explain why glucose, ATP and hexokinase are added. Make a drawing of the respiratory chain and mark where the different inhibitors attack the respiratory chain.

Litterature:
Methods in Enzymology, vol X, pp 48.
Stryer 5/e chapter 18; Horton, chapter 15.

Lab report
Prepare your report according to the general guidelines in "How to write a lab report". In addition to the results of your measurments, graphs and calculations, you shall include answers to all the questions above and a schematic drawing of the respiratory chain where the entry points for substrates are shown, as well as where the different inhibitors attack. Discuss how well your results correspond to what would be expected, possible sources of errors and limitations of the method.

...AND DONT FORGET UNITS : g/mol, mM, mg/ml, nm, M-1*cm-1 etc.

APPENDIX

Preparation of mitochondria
(The preparation of the mitochondria for this laboratory experiment will be made by the supervisors.)
Buffer: Sucrose 0.25 M, Tris 5mM, EDTA 0.5 mM, pH =7.2 (adjusted with 6 M HCl). The pH must be checked the same day as the experiment is performed since pH for sucrose buffers (even fresh buffers) decrease during storage.

  1. Sacrifice a rat and transfer the liver to a beaker containing ice chilled sucrose buffer.
  2. Cut 6 g of the liver in small pieces in a beaker containing 10 ml of sucrose buffer. Decant the sucrose solution and rinse again. Transfer the liver pieces with 25 ml sucrose buffer to a Potter-Elvenhjem homogenizer.
  3. Homogenize the liver sample by moving the pistil up and down until the pistil reach the bottom of the homogenizer. After that, move the pistil up and down 3 more times.
  4. Dilute the homogenate to 10% (w/v) with sucrose buffer.
  5. Centrifuge at 600 g (2400 rpm) during 10 min at 4 °C.
  6. Transfer the supernatant very carefully to new ice chilled centrifuge tubes.
  7. Centrifuge the supernatant at 11,000 g (9000 rpm) for 10 min at 4 °C.
  8. Remove the supernatant and the "fluffy" layer and make a suspension of the mitochondria (the pellet) by carefully pipetting the sucrose buffer up and down in the pipette (50% of the homogenate volume).
  9. Centrifuge the mitochondria suspension at 11,000 g (9000 rpm) during 10 min at 4 °C. Remove the supernatant and suspend the mitochondria as described according to point 8.
  10. Repeat as point 9. Suspend the mitochondria in about 5 ml sucrose buffer.


Risks and protection:

Chemicals:
  • 2,4-dinitrophenol.
  • Rotenone.
  • Antimycin A
  • KCN
    All of the listed compounds are highly toxic (affect cellular respiration). Avoid inhalation and contact with skin and eyes. Use gloves!

    Organisms:


  • Radioactivity:


  • Other:


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    Lab by Torbjörn Lundh© 1994 - 2003
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