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:

   	1	2	3	4
   Phosphate Buffer	2.0 ml	2.0 ml	2.0 ml	1.0 ml
   BSA	0.1 ml	0.1 ml	0.1 ml	0.1 ml
   KCN	1.0 ml	1.0 ml	1.0 ml	1.0 ml
   DCPIP	1.0 ml	1.0 ml	1.0 ml	1.0 ml
   Succinate	None	1.0 ml	None	1.0 ml
   Malonate	None	None	1.0 ml	1.0 ml

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.