Exercise 8.8 - Photosynthesis by Respirometry

LEVEL III

Table 8.1 Characteristics of isolated chloroplasts

Materials

• Gilson respirometer equipped with photoflood lamps
  
• Chloroplast suspension 11
  
• Chloroplast suspension buffer (suspension solution from Exercise 8.1 )
  
• 0.1 M NaHCO
  
• Inhibitor 1 (1 x 10 DCMU)
  
• Inhibitor 2 (5 x 10 DCMU)

Procedure

1. In general, use the procedure listed in Exercise 8.7 for mitochondria respiration, except that the reactions are run at 25° C and a bank of photoflood lights must be used to ensure photosynthesis.

2. Set up the reactions vessels in the following manner:

   Tube	Side arm	Main Vessel
   1-2		2.0 ml Chloroplast buffer
   3-4	0.5 ml NaHCO	0.5 ml buffer 1.0 ml Chloroplast Suspension
   5-6	0.8 ml NaHCO	0.2 ml buffer 1.0 ml Chloroplast Suspension
   7-8	0.5 ml NaHCO 0.3 ml Sodium Azide	0.2 ml buffer 1.0 ml Chloroplast Suspension
   9-10	0.5 ml NaHCO 0.3 ml Inhibitor 1	0.2 ml buffer 1.0 Chloroplast Suspension
   11-12	0.5 ml NaHCO 0.3 ml Inhibitor 2	0.2 ml buffer 1.0 ml Chloroplast Suspension
   13-14	0.5 ml NaHCO	0.5 ml buffer 1.0 ml Boiled Chloroplasts

3. Measure the amount of oxygen evolved every 10 minutes until a steady rate is reached. Plot results and compare the effects of the selected inhibitors.