52 PHOTOSYNTHESIS 



Exercise X 



with the concomitant evolution of oxygen. 

 You will be able to follow the course of the 

 reaction by observing the loss of blue color as 

 the dye is reduced: 



Dye (blue) + H2O —T^, — * 



■' ^ ' chloroplasts 



Dye — H2 (colorless) + JO2. 



Spinach chloroplasts have been prepared as 

 follows: Leaves were homogenized with 0.5- M 

 sucrose solution at 0°C for 30 sec in a Waring 

 blendor. The suspension was then filtered 

 through two layers of cheese cloth. The filtrate 

 was centrifuged at 50 times the force of gravity 

 (50 G) for 10 minutes. The supernatant was 

 then decanted and recentrifuged for 10 minutes 

 at 600 G. The supernatant was decanted and 

 discarded. The pellet at the bottom, containing 

 the chloroplasts, was resuspended in 0.5-M 

 sucrose. It is important to keep the chloroplasts 

 at 0°C; they deteriorate rapidly at higher tem- 

 peratures. 



In each of two test tubes, mix: 



2 ml of phosphate buffer, 0.1 M, pH 6.5; 

 2 ml of dye solution 



(2,6 dichlorophenolindophenol, 



2.5 X 10-4 M). 

 0.1 ml of chloroplast suspension (2 drops); 

 6 ml of distilled water. 



Swirl to stir, wrap one tube immediately in 

 aluminum foil to protect it from light, and 

 expose the other to bright light for 10 minutes. 

 Compare. (Protect the chloroplasts from heat 

 radiation by placing a glass tumbler filled with 

 water between the light source and the reaction 

 tubes.) 



Devise experiments to show (a) that the 

 chloroplasts and dye must be illuminated 

 together to obtain this result; (b) that the reac- 

 tion depends upon catalysis by enzymes. In- 

 clude the results of these experiments in your 

 notes. 



Oxygen evolution in photosynthesis 



Place three leafy sprays of Elodea in one test 

 tube of the volumeter described in Exercise VI 



(pp. 35-36), and fill this tube with 1% sodium 

 bicarbonate solution. Add sufficient solution so 

 that when the rubber stopper is inserted, an air 

 space of about 3 to 6 mm is left between the 

 liquid surface and the stopper. The bicarbonate 

 solution will provide the carbon dioxide used 

 in photosynthesis. Fill the second tube, which 

 as before will serve as thermobarometer, 

 with the same volume of sodium bicarbonate 

 solution. 



A 60-100 watt lamp mounted upright in a 

 standard receptacle will be used as light source. 

 Between this lamp and the volumeter place ajar 

 or glass filled with water, to serve as a heat filter, 

 which, by absorbing the infrared (heat) radia- 

 tion from the lamp, will prevent large tempera- 

 ture changes in the volumeter from distorting the 

 readings. Place the lamp as close to the volu- 

 meter as it will go with the heat filter in between. 

 Draw a drop of kerosene into the proximal end 

 of each side-arm, as described in Exercise VI, 

 and close the pinch clamps. Allow the system 

 to equilibrate for 5 to 10 minutes. This equilibra- 

 tion time is needed for the oxygen evolved to 

 saturate the water; thereafter all the gas pro- 

 duced is given off". 



Take readings in both side-arms at 2-minute 

 intervals, each time subtracting the reading in 

 the thermobarometer from that in the experi- 

 mental tube. Go on with the readings until the 

 rate of change remains constant through three 

 consecutive readings. 



After the rate has stabilized, move the light 

 source to twice the distance from the plant, 

 and after taking readings at this distance double 

 the light distance once again. Assuming that 

 the light intensity is inversely proportional to 

 the distance, plot the rate of oxygen evolution 

 (change of volume in units/min) vs. light inten- 

 sity (relative). {Note: The intensity of light 

 coming from a point source falls off" as the square 

 of the distance; i.e., at twice the distance the 

 intensity has fallen to one-quarter. Your lamp, 

 however, is not a point source, particularly if 

 frosted, or equipped with a reffector; and the 

 light intensity declines more nearly in propor- 

 tion to the distance.) 



