OXPHOS COMPLEXES ACTIVITIES
1.
Harvest 5 x107 Dictyostelium cells growing to exponential phase in HL5 medium by
centrifugation at 1000g 5 min.
2.
Wash the pellet with PBS 1X and centrifugate 1000g 5min.
3.
Resuspend in 2ml cold buffer SETH and sonicate 3 times 10s on 30s off on ice.
4.
Centrifugate 8.000g 10 min 4ºC to eliminate cell debris and non broken cells.
5.
Use de supernatant from step 4 as a sample for spectrophotometric analyses.refer. Use
duplicates
6.
Measure the sample protein using Bradford. Normalize all the measures by protein and
citrate synthase specific activity.
COMPLEX I
 = 6.81 mM-1 cm-1 = 340nm
Temp 30ºC
1. Prepare the following mix in 1 cm plastic cuvette (duplicates)
Buffer KP 40mM pH 8
NADH 1mM (Roche)
NaN3 (Sigma) 50 mM
BSA (Sigma)-EDTA 1%
Sample
H2O
500 l
20 l
200 l
100 l
50 l
120 l
2. Mix and incubate at 30ºC (into the spectrophotomer) 8 min. At this point the Abs must be at
least 0.6-1.2
3. Add CoQ1 (Sigma) 10 mM
10ml
4. Mix and incubate 30s at 30 ºC.
Read 2 min
5. Add rotenone (Sigma) 0.25 mM
20ml
6. Mix and incubate 30s at 30 ºC
Read 2 min
7. Calculate activity sensitive to rotenone (CI) with:
(Abs/min)= Abs/min – Abs/min with rotenone
NOTE 1: The NADH must be new. Keep the solid compound at 4º C. the way to see if in not
valid is seeing a straight line instead of a curve during the 2 min. It indicates that it´s oxidized.
NOTE 2: Keep the buffer KP at –20ºC . It can be used for 1 month (see buffers for more info).
NOTE 3: How to prepare the rotenone 0.25 mM solution? First prepare a solution 30X 7.5 mM
(2,96 mg/ml) in EtOH 100%. Heat a bit in the microwave to dissolve and keep it at 4ºC. Take
50 l of this solution and add 325 l EtOH 100% + 1125 l H2O. Keep it at –20ºC.
NOTE 4: How to prepare the CoQ 10 mM solution? First prepare a 100mM stock in EtOH and
keep it at –20ºC. Then dilute 1:10 in EtOH and keep it at 4ºC (no light please) for 2 weeks. You
can use brown eppendorfs.
NOTE 5: Avoid bubles while mixing.
COMPLEX II
 = 19 mM-1 cm-1 = 600 nm
Temp 30 ºC
1. Prepare the following mix in 1 cm plastic cuvette (duplicates)
Buffer KP 100 mM pH 7
500 l
KCN(Sigma) 30 mM
50 l
DCPIP (Sigma) 1mM
100 l
Sample
20 l
H2O
230 l
2. Mix and incubate 2min 30ºC
3. Add succinate (Sigma) 320 mM
100 l
4. Mix and incubate 30s
Read for 2 min
5. Add CoQ1 10 mM
5
l
6. Mix and incubate 30s
Read 2 min
NOTE 1: See Complex I for preparing CoQ.
NOTE 2: Be careful preparing KCN. Use mask .
NOTE 3: To calculate the CII activity you can use both reactions from steps 4 or 6.
COMPLEX III
 = 21 mM-1 cm-1 = 550 nm
Temp 30 ºC
1. Prepare the following mix in 1 cm plastic cuvette (with and without antimicin A and
duplicates)
Buffer KP 100 mM pH 7.5.
NaN3 (Sigma) 50mM
BSA-EDTA 1%
Cytocrome C (Sigma) 1mM
Antimicin A (Sigma) 1mg/ml
H2O
DBH2 10mM
500 l
40 l
100 l
50 l
10 l
265 l
5 l
2. Mix and incubate for 2 min (30ºC)
3. Add Sample
30 l
4. Mix and read for 2 min
5. Calculate the activity sensitive to Antimicin (CIII) with:
Abs/min = Abs/min without Antimicin – Abs/min with antimicin
NOTE 1: The citocrome should be used completely. Use the stock compound (oxidized) but
before buy it ask first the customer service from Sigma. Not all batchs are good! You can re
NOTE 2: How to prepare the antimicin solution? Prepare to a final conc 1mg/ml in EtOH 50%.
You can prepare firstly a stock of 10 mg/ml and keep it at –20ºC.
NOTE 3: How to prepare the DBH2 (decylubiquinone)? It´s a bit complicated!be patient!
a) Prepare a DB solution 40mM: Add 1938 l EtOH 100% to the stock (Sigma. 25 mg). Keep it
in the shade and at –20 ºC.
b) In a glass tube mix 250 l of the previous solution, 1ml EtOH 100% , 1 ml of H2O and test
the pH till achieve pH 2 with HCl. Add with a palette BH4Na (Sigma) till the color fades away.
c) Add then 1ml of diethyl ether, vortex, add 1 more ml, vortex, add 1ml hexane and vortex.
Wait till two phases are splitted away. Extract the upper phase (organic) to a glass tube.
d) Add 1ml of NaCl 2M, vortex, wait till two phases are splitted away and recover the upper
phase (organic) in another glass tube.
e) Evaporate all with N2 (be patient).
c) Add 500 l EtOH and add drops of HCl 6M to dissolve the decylubiquinol and go to pH 2.
Keep it at –70 ºC and in the shade.
COMPLEX IV
 = 21 mM-1 cm-1 = 550 nm
Temp 38 ºC
1. Prepare the following mix in 1 cm plastic cuvette (duplicates)
Buffer KP 100 mM pH 7
100 l
Citocrome C (Sigma) 800mM reduced
100 l
H2O
770 l
2. Mix and incubate 2min (38ºC)
3. Add sample
10 l
4. Mix and read 2min
NOTE 1:
How to prepare the cytochrome c 10mg/ml (800mM) solution?
a) Weigh at least 15 mg of cytochrome c (Sigma. Oxidized). Add 1 ml of buffer KP 10 mM pH
7. Mix
b) Add an edge of pallete of BH4Na. Mix
c) Put on ice 30min
d) Adjust pH to 7-7.3 with HCl 1N and 0.25 N annotating the volume added. Complete with
H2O to the required volume (1500 l if you have 15 mg)
Prepare this solution everytime you perform a new experiment.
NOTE 2: The BH4Na solid lasts only some weeks .
CITRATE SYNTHASE
 = 13.6 mM-1 cm-1 = 412 nm
Tris- HCl 0.75M pH 8
DTNB (Sigma) 1mM
Triton X-100 1%
Acetyl- coA (Roche) (7 mg/ml)
Sample
H2O
Temp 30 ºC
100 l
100 l
100 l
50 l
10 l
590 l
Mix and incubate 2min at 30ºC
Add oxalacetatic acid (Sigma) 10mM
50 l
Mix and incubate 30s (30ºC)
Read 2 min
NOTE 1: DTNB and oxalacetate are dissolved in Tris-HCl buffer 0.75M pH 8, not in water.
NOTE 2: How to prepare Acetyl- coA? Add 1428 l to the commercial tube with 10 mg.
Alicuote and conserve at – 20ºC.
NOTE 3: It is a good idea to start the experiments measuring this enzyme activity. If the values
are very different is not a good idea to go on and you should stop, measure the protein once
again, and try to equilibrate the protein load between the samples.
SOLUTIONS
Buffer A 100mM: 17.4 g K2HPO4 in 1 l H2O
Buffer B 100mM: 13.6 g KH2PO4 in 1l H2O
Buffer C KP 100mM pH 8: 94 ml A + 6 ml B
Buffer D KP 100mM pH 7.5: 83.4ml A + 16.6 ml B
Buffer KP 100mM ph 7: 61.5 ml A + 38.5 ml B
Buffer KP 40 mM pH 8 : 40 ml C+ 60 ml H2O
Buffer KP 10 mM pH 7.4 : 1ml D+ 9ml H2O
Keep them at –20ºC.
BSA-EDTA 1%: 100 mg BSA + 10 ml EDTA 10mM pH 7.4. Keep at 4ºC