iv] CARBON ASSIMILATION 33 



that the chlorophyll in plants is accompanied by an enzyme, chlorophyl- 

 lase, which, in alcoholic media, brings about alcoholysis of the chlorophyll, 

 and replaces the phytyl by the ethyl radicle. The products, formerly 

 known as crystalline chlorophyll, are now termed cnlorophyllides : 



(C S2 H 3() ON 4 Mg)(COOCH 3 )(COOC 20 H3 9 ) + C 2 H 5 OH 



= C2oH3 9 OH + (C3,H3 ON 4 Mg)(COOCH 3 )(COOC 2 H 5 ). 



Phytol Ethyl chlorophyllide 



Similar chlorophy Hides are produced by other alcohols. In aqueous 

 solutions chlorophyllase brings about hydrolysis and the free acid 

 chlorophyllide is formed (see Scheme 2, p. 34): 



(C 32 H 3 oON 4 Mg) (COOCH 3 ) (COOC^) + H 2 O 



= C2oH3 9 OH +(C 32 H3oON 4 Mg) (COOCH 3 ) (COOH). 



Chlorophyllide 



Chlorophyllase is a very stable enzyme ; it is not even destroyed by 

 boiling in alcohol for a short time, but if leaves are boiled in water, the 

 enzyme is destroyed. 



Expt. 26. Microscopic examination of ethyl chlorophyllide. Prepare sections of 

 fresh Heracleum leaves and mount them in a drop of 90 % alcohol. Leave the slide 

 under a bell-jar containing a dish of alcohol. The section slowly dries in the course 

 of half a day or a day. It is then examined under the microscope when there svill be 

 observed the characteristic triangular and hexagonal crystals of ethyl chlorophyllide 

 (crystalline chlorophyll). 



Expt. 27. Production of methyl chlorophyllide in the leaf. Sections may be used 

 as in the preceding experiment, or a piece of a leaf may be employed. In the latter 

 case a test-tube with 4 c.c. of 75 / methyl alcohol is taken and 1 gm. of fresh leaf 

 is added to it. The leaf first becomes a darker green and then during the course of 

 a few hours becomes yellowish. On holding the leaf to the light there can be 

 observed with the naked eye a number of black points. If sections of the leaf be cut 

 and examined under the microscope, these spots appear as aggregates composed of 

 rhombohedral crystals, occurring in certain cells. 



Expt. 28. Extraction of ethyl chlorophyllide. Two grams of dry Heracleum leaf- 

 powder are left for a day in a test-tube containing 6 c.c. of 90 % alcohol. The extract 

 is then filtered through a small porcelain funnel and the powder on the filter washed 

 with a little acetone. The filtrate is mixed with an equal quantity of ether, and then 

 with some water. The ether solution is transferred to a separating funnel and 

 thoroughly washed with water, and then concentrated on a water-bath to or 1 c.c., 

 and 3 c.c. of petrol ether are added. On standing, the ethyl chlorophyllide is pre- 

 cipitated in the form of crystalline aggregates. It is freed from yellow pigments by 

 shaking with a little ether, and can be further purified by redissolving in ether and 

 precipitating again with petrol ether. 



Expt. 29. The action of chlorophyllase. Fresh leaves of a species rich in chloro- 

 phyllase, e.g. Heracleum or Galeopsis, are finely divided and put in a 70 % acetone 

 solution, 3 c.c. of solution being used for every gram of leaf-powder. The chlorophyll, 



o. 3 



