Respiration: Succinic Dehydrogenase

Materials

 

• Mitochondrial suspension from Exercise 8.4
  
• Sodium dithionite
  
• 0.2 M Sorenson Phosphate Buffer, pH 7.5
  
• 1% (w/v) Bovine Serum Albumin
  
• 0.005 M Potassium Cyanide
  
• 0.00025 M Dichlorophenolindophenol
  
• 0.6 M Sodium succinate, pH 7.5
  
• 0.6 M Sodium malonate, pH 7.5
  
• 0.033% (v/v) Phenazine Methosulfate in Phosphate Buffer
  
• Spectrophotometer and cuvettes

Procedure

 

1. Place 1.0 ml of mitochondrial suspension in a test tube and place in a boiling water bath for 5 minutes. Cool before using. This boiled preparation will allow measurement of background absorption due to the turbidity of the mitochondria when used in a spectrophotometer.

    

2. Prepare a series of tubes as follows:

    

   <center> <table> <tbody> <tr> <td>  </td> <td> 1</td> <td> 2</td> <td> 3</td> <td> 4</td> </tr> <tr> <td> Phosphate Buffer</td> <td> 2.0 ml</td> <td> 2.0 ml</td> <td> 2.0 ml</td> <td> 1.0 ml</td> </tr> <tr> <td> BSA</td> <td> 0.1 ml</td> <td> 0.1 ml</td> <td> 0.1 ml</td> <td> 0.1 ml</td> </tr> <tr> <td> KCN</td> <td> 1.0 ml</td> <td> 1.0 ml</td> <td> 1.0 ml</td> <td> 1.0 ml</td> </tr> <tr> <td> DCPIP</td> <td> 1.0 ml</td> <td> 1.0 ml</td> <td> 1.0 ml</td> <td> 1.0 ml</td> </tr> <tr> <td> Succinate</td> <td> None</td> <td> 1.0 ml</td> <td> None</td> <td> 1.0 ml</td> </tr> <tr> <td> Malonate</td> <td> None</td> <td> None</td> <td> 1.0 ml</td> <td> 1.0 ml</td> </tr> </tbody> </table> </center>

    

3. Mix 2 ml of 0.00025 M DCPIP with an amount of sodium dithionite sufficient to fully reduce it. The amount of sodium dithionite is not crucial. Add a small "pinch," shake to dissolve and continue until the color of the DCPIP becomes clear.

    

4. Use the reduced DCPIP from step 3 to blank a spectrophotometer at 600 nm.

    

5. Add 1.0 ml of the boiled mitochondrial suspension and 1.0 ml of PMS to tube #1, mix by inversion and immediately place in the previously blanked spectrophotometer. Record the absorbance and continue to record at 30 second intervals for 3 minutes.

    

6. Add 1.0 ml of the mitochondria suspension to tube #2 and measure the absorbance immediately. Read and record the absorbance every 30 seconds for 6-7 minutes or until no further changes occur.

    

7. Repeat Step 6 for tube #3.

    

8. Repeat Step 6 for tube #4.

    

9. Use a hemacytometer to count the number of mitochondria per ml of suspension used.

    

10. Calculate the reaction rate for each tube as millimoles of dye reduced/minute/100 mitochondria.
    Convert the absorbance readings to millimoles of dye by using E = 19.1 millimoles for DCPIP @600 nm. Refer to Appendix G for details of the Beer-Lambert law.

     

11. Record the amount of dye reduced at 30 second intervals. Use linear regression to plot the dye reduction over time for each tube.

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