Cytochrome Oxidase Manometric Analysis

Materials

 

• 0.01 M Potassium Phosphate Buffer, pH 7.4
  
• Sat. K Fe(CN)
  
• 0.6 M Sodium Malonate (Malonic acid, sodium salt)
  
• 5 mM KCN
  
• 0.02% (w/v) p-phenylenediamine oxalate (PPDO)
  
• Mitochondria suspension from
  
• Spectrophotometer and cuvettes

Procedure

 

1. Prepare a series of 5 tubes as follows:

    

   <center> <table> <tbody> <tr> <td> Substance</td> <td> 1</td> <td> 2</td> <td> 3</td> <td> 4</td> <td> 5</td> </tr> <tr> <td> Buffer</td> <td> 3.0 ml</td> <td> 3.0 ml</td> <td> 2.0 ml</td> <td> 2.0 ml</td> <td> 3.0 ml</td> </tr> <tr> <td> K Fe(CN)</td> <td> 0.1 ml</td> <td> 0.1 ml</td> <td> 0.1 ml</td> <td> 0.1 ml</td> <td> 0.1 ml</td> </tr> <tr> <td> Malonic Acid</td> <td> None</td> <td> None</td> <td> None</td> <td> 1.0 ml</td> <td> None</td> </tr> <tr> <td> KCN</td> <td> None</td> <td> None</td> <td> 1.0 ml</td> <td> None</td> <td> None</td> </tr> <tr> <td> Water</td> <td> 1.0 ml</td> <td> None</td> <td> None</td> <td> None</td> <td> None</td> </tr> </tbody> </table> </center>

    

2. Turn on a spectrophotometer and adjust the wavelength to 630 nm. Use tube #1 to blank the spectrophotometer.

    

3. Add 1.0 ml of the mitochondria suspension to tube #2 and mix by gentle inversion. Add 100 µ l of PPDO to the tube and immediately measure its absorbance at 630 nm.

    

4. Continue to measure the absorbance every 30 seconds for 4-5 minutes or until there is no further change in absorbance.

   Note that oxidation of p-phenylenediamine oxalate (PPDO) changes the dye from its colorless form (reduced) to purple (oxidized). Consequently, there should be a gradual increase in absorbance as the dye is oxidized, corresponding to the activity of cytochrome oxidase.

    

5. Add 1.0 ml of the mitochondria suspension and 100 µl of PPDO to tube #3. Measure the absorbance immediately.

    

6. Continue to measure the absorbance every 30 seconds for 4-5 minutes or until there is no further change in absorbance.

    

7. Repeat Steps 5 and 6 with tube #4.

    

8. Place 1.0 ml of mitochondria suspension into a tube, and place in boiling water for 5 minutes. Cool and add to tube #5. Add 100 µl of PPDO to tube #5, invert and measure the absorbance as with tubes #2-4.

    

9. Record the changes in absorbance for tubes 2-5 at 30 second intervals. Plot the changes in absorbance for each tube.

    

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Manometric Analysis

Each of the preceding exercises utilized the reducing power produced by the reactions of photosynthesis or respiration to monitor the color change in a dye. Another approach to the process would be to monitor the production or utilization of oxygen in the process. Since oxygen is a gas, it can be monitored through direct measurements of change in volumes of a gaseous phase. This is known as respirometry, and can be monitored simply (with testtubes and capillary pipettes) or by more complex and more accurate means (with the use of a Gilson Respirometer or a Warburg Apparatus). The respiration of single cells has been measured with Cartesian divers.

These systems lend themselves primarily to fluid systems with a gas intermediate, an ideal arrangement for isolated organelles in suspension. It also works well for cells in suspension (algae, bacteria, yeast, tissue culture) and for moist tissue slices (leaf disks, liver slices).

Because of the nature of gas volume measurements, the primary difficulty with these techniques is control over temperature and atmospheric pressure. For simple procedures (such as the measure of gas evolved from an elodea leaf in a test tube) these are usually ignored or some type of control is attempted (water baths and heat shields for the lights). For accurate measurement on the cell level more extensive control must be had.

The Warburg Manometer was developed as a system to maintain constant temperature and volume, thus measuring changes in gas exchange by changes in pressure. It is extremely accurate, but it also is tedious and requires a good deal of mathematical corrections (made easier, of course, by using a computer). The Gilson Respirometer keeps constant temperature and pressure and monitors the change in gas exchange by changes in volume. To its advantage, the volume can be read directly and thus much of the mathematical corrections needed for the Warburg Manometer are eliminated. The constant pressure respirometer suffers in accuracy when compared to the constant volume manometer of Warburg, but it is sufficient for nearly all cell physiology purposes. Its inherent ability for easy automation has made it a standard for most cell labs. By contrast, the manometer remains the tool of choice for physical chemists.

The Gilson Constant Pressure Respirometer

The reaction flask consists of a main reaction chamber, a side arm, a center well and connections to a micrometer and a second chamber. The reaction is started by pouring the contents of the sidearm (usually a substrate) into the main vessel (containing the main reactants). The center well contains any materials which are accessory to the reaction (such as KOH to absorb CO ). Changes in volume within the main vessel are altered through the use of a micrometer/piston system, and the micrometer is usually designed to read directly in microliters of gas exchanged. Interpretation of what gas has been exchanged is a function of the experiment design.

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