Laboratory 6: Aerobic respiration

Introduction:

    Mitochondria are key sites for oxidative metabolism in the cell.  The primary mitochondrial pathway for oxidation of carbon and production of high-energy intermediates is the tricarboxylic acid (TCA) cycle.  This pathway consists of a series of reactions that progressively oxidize acetate to CO2.  Enzymes of the TCA cycle are only found in mitochondria.  Likewise, other metabolic pathways are often localized to specific cellular organelles. 
    Succinate dehydrogenase catalyzes the oxidation of succinate to malate during the TCA cycle.  In this exercise you will measure the level of succinate dehydrogenase activity in your cell fractions.  If the tissue were intact, then several catabolic pathways would generate acetyl-CoA and intermediates that feed into the TCA cycle.  Since these sources are not present in the isolated mitochondria, you can add succinate to fuel the succinate dehydrogenase reaction.  The electrons from this reaction are normally transferred to FAD⁺ and then to the electron transport chain.  It turns out that FAD⁺ tends to be lost during mitochondrial isolation so electron transfer does not occur in vitro.  However, the artificial electron acceptor, dichlorophenol indophenol (DCPIP), can substitute for FAD⁺ in this reaction.  Oxidized DCPIP absorbs light at 600 nm but reduced DCPIP does not.  Therefore, you can measure the amount of succinate dehydrogenase in the mitochondria by adding succinate and DCPIP and following the change in absorbance at 600 nm. 




Materials:

Three cellular fractions (from last week)
0.2 M     Phosphate buffer pH 7.5
0.6 M     Sodium succinate with 1% BSA (Bovine serum albumin)
0.25 mM     DCPIP
0.39% Sodium azide (toxic)


Methods:

Succinate dehydrogenase assay
   
You should work in the same pairs you had last week.  Make sure your three fractions are thawed out, but kept cold on ice. 

In order to speed up the process of making appropriate blanks, four groups (e.g., the two benches on east side) can agree to have one group prepare one blank for the other four groups.  For example, one group will boil 375 µL of their 2K pellet and use that to make a blank cuvette.  Three other groups will use this one blank in one run.  Another group will boil 375 µL of their 15K pellet and a third 375 µL of the 15K supernatant.  This means that one group will not have to make up a blank (unless something goes wrong).  You can draw cards to see who's lucky. 

Coordinate with the four groups to have their first cuvettes ready to go (except for the cellular fractions). 

Reagent	Add to cuvette (µL)
Phosphate buffer	750
Succinate	375
DCPIP	375
Na azide (toxic)	375
ADD THIS JUST BEFORE YOU ARE READY TO LOAD THE SAMPLES INTO THE SPECTROPHOTOMETER Make sure the fraction is resuspended well.
Cellular fraction	375

 
When all four groups are ready, take the cuvettes to the spectrophotometer.  Then add the cellular fraction; quickly mix by inversion and place the cuvettes in the spectrophotometer (wear gloves).  Press the run key on the spectrophotometer and monitor the reactions for 5 minutes (#7 Advanced kinetics; DCPIP program).  Print out your results. 

Complete reactions with each of the three cellular fractions (2,000 x g pellet, 15,000 x g pellet & 15,000 x g supernatant).


Data analysis

Calculate the activity for each reaction and report it on a table such as the following. 

Fraction	Rate (millimoles/minute)
2K pellet
15K pellet
15K supernatant

In order to calculate the rate in millimoles of DCPIP reduced per minute, use the following formula:


 
    ∆ Abs  =  change in absorbance
    e_c^DCIP = Extinction coefficient: 19.1 L/millimoles
    ∆T  =  Time in minutes
    Vmit  =  Volume of cell fraction added: 0.000375 L (i.e., 375 µL)

The discussion should include your observations as to which fraction you believe was enriched for mitochondria. 

Questions:

1.  What's missing from the extinction coefficient?  Why doesn't it matter? 

2.  Did you get negative readings for absorbance?  Explain why.

3.  Is it possible that DCPIP was reduced by non-mitochondrial processes?