Exercise X 



PHOTOSYNTHESIS 51 



energy absorbed as light by chloroplasts gen- 

 erates not only hydrogen, but also ATP. Indeed, 

 isolated chloroplasts can carry out the whole 

 process of photosynthesis. 



Carbohydrate, having been prepared by 

 photosynthesis, is in turn degraded to provide 

 all the cell's energetic needs. The two principal 

 processes for deriving energy by the degradation 

 of sugars are fermentation and respiration. We 

 shall examine both processes in the next labora- 

 tory session. Fermentation is the process by 

 which cells derive energy anaerobically, by rear- 

 ranging the atoms of sugar to yield products of 

 lower energy. Respiration is a combustion, in 

 which sugar is burned with molecular oxygen to 

 yield carbon dioxide, water, and energy in the 

 form of ATP. 



Photosynthesis and respiration are opposed 

 reactions. The overall equation of the former 

 is just the reverse that of the latter. Green 

 plants respire in the dark; they simultaneously 

 respire and photosynthesize in the light. The 

 consumption of oxygen is a measure of their 

 respiration; the evolution of oxygen measures 

 their photosynthesis. In the light, with both 

 processes going on simultaneously, the oxygen 

 exchange represents a balance between these 

 opposed reactions. If the light is sufficiently 

 bright, however, photosynthesis may go so much 

 faster than respiration as to dominate the 

 oxygen exchange. 



With a fine capillary, apply this mixture to 

 the longer side of a 4.5 x 4.5-inch filter paper in a 

 narrow line, 3 inches long, 1 cm from the bot- 

 tom. Develop the chromatogram with a mix- 

 ture of 9 petroleum ether : 1 acetone. 



In this solvent the carotenes (C4nH,5fi) move 

 the fastest, followed by the xanthophylls 

 (C4oH54(OH)2) and then the chlorophylls a 

 and b. Outline the visible pigment spots lightly 

 with a pencil. Then examine the paper under 

 ultraviolet light, noting the fluorescence of the 

 various pigments and the presence of any addi- 

 tional spots which were not apparent in visible 

 light. (Caution: Recall our earlier warnings not 

 to look into the light.) 



Determine where the petroleum ether extract 

 of plant pigments has absorption maxima by 

 looking at this solution through the hand spec- 

 troscope. Chlorophyll a has a major absorption 

 band at about 680 m// and chlorophyll b at 

 about 665 myu. Those of you who have time 

 may cut out the two chlorophyll bands on your 

 chromatogram and elute the pigments by leach- 

 ing out the paper strips in a small test tube with 

 a few ml of acetone. Remove the filter paper 

 with forceps and observe the absorption of these 

 two solutions at the specified wavelengths in the 

 hand spectroscope, or measure it in a spectro- 

 photometer. Which pigment migrated faster on 

 your chromatogram? What is the chemical dif- 

 ference between chlorophylls a and A? Why is 

 chlorophyll green? 



EXPERIMENTS 



Analysis of chloroplast pigments 



The chlorophylls and carotenoids (xantho- 

 phylls and carotenes) are the major pigments of 

 the chloroplasts. These pigments can be ex- 

 tracted from green plant tissues with lipid sol- 

 vents, and separated by chromatographic ad- 

 sorption. 



Such an extract has been prepared before the 

 laboratory session by homogenizing spinach 

 leaves with 95*^^, ethanol in a Waring blendor. 

 The extract has been filtered, evaporated to 

 dryness, and redissolved in petroleum ether. 



The Hill reaction 



The photolytic cleavage of water in the pres- 

 ence of chloroplasts is known as the Hill reac- 

 tion. It can be represented by the following 

 equation : 



A + H2O 



light 



chloroplasts 



H2A + i02. 



In this reaction "A" represents an electron (or 

 hydrogen) acceptor. In plants this is usually 

 the coenzyme TPN. In our experiment we shall 

 use an artificial electron acceptor, the dye 2,6 

 dichlorophenolindophenol, which is reduced 



