VII] FATS AND ALLIED SUBSTANCES 95 



may be demonstrated by autolyzing the crushed seed with a little dilute 

 acetic acid; the increase of acidity will be found to be much greater 

 l-han in the case of a control, experiment in which acid has not been 

 added. 



It has not been found possible to extract the enzyme from the resting 

 seed. An active material can be obtained by digesting the residue, after 

 extraction of the fat, with dilute acetic acid and finally washing with 

 water. This material can then be used for testing the hydrolytic power 

 of the enzyme on various fats. 



There is little doubt that lipase catalyzes the synthesis of fats as 

 well as the hydrolysis; the reaction, in fact, has been carried out to a 

 certain extent in vitro. 



Expt. 95. Demonstration of the existence of lipase in ungerminated Ricinus seeds. 



A . Remove the testas from about two dozen Ricinus seeds and pound the kernels 

 up in a mortar. Into three small flasks («), {h) and (c), put the following: 



{a) 2 gms. of pounded seed + 10 c.c. of water. 



(6) 2 gms. of pounded seed + 10 c.c. of water + 2 c.c. of N/10 acetic acid. 



(c) 2 gms. of pounded seed + 10 c.c. of water + 2 c.c. of N/10 acetic acid, and 

 boil well. 



Add a few drops of chloroform to all three flasks, plug them with cotton-wool, 

 and allow them to incubate for 12 hours at 37° C. Then add 2 c.c. of N/10 acetic 

 acid to flask (a), and 25 c.c. of alcohol to all three flasks. Titrate the fatty acids 

 present with N/10 alkali, using phenolphthalein as an indicator. A greater amount 

 of fat should be hydrolyzed in (6) than in (a), and also slightly more in {a) than in 

 (c). The addition of alcohol checks the hydrolytic dissociation of the soap formed on 

 titration. 



B. Pound up about 15 gms. of Ricinus seeds which have been freed from their 

 testas, and let the pounded mass stand with ether for 12 hrs. Then filter, wash with 

 ether and dry the residue. Weigh out three lots, of 2 gms. each, of the fat-free meal 

 and treat as follows : 



(a) Grind up the 2 gms. of meal in a mortar with 16 c.c. of N/10 acetic acid 

 (i.e. 8 c.c. of acid to 1 gm. of meal), and let it stand for about 15 minutes. Then 

 wash well with water to free from acid, and transfer the residue to a small flask. 

 Add 5 c.c. of castor oil, 2 c.c. of water and a few drops of chloroform. 



{h) Treat the 2 gms. of meal as in (a), but, after washing, and before transferring 

 to the flask, boil well with a little distilled water. Add 5 c.c. of oil, 2 c.c. of water 

 and a few drops of chloroform. 



(c) Put the 2 gms. of meal into the flask without treatment and then add 5 c.c. of 

 oil, 2 c.c. of water and a few drops of chloroform. 



Incubate all three flasks for 12 hours, and then titrate with N/10 caustic soda, 

 after addition of alcohol as in ^. A certain amount of acetic acid is always retained 

 by the seed residue, and this is ascertained from the value for flask (6). Flask (c) will 

 act as the control. 



