404 PRACTICAL ORGANIC AND BIO-CHEMISTRY 



VI. Lipase. 



Preparations of lipase are most conveniently obtained from castor-oil seeds 

 and from pigs' pancreas : 



(a) Lipase from castor-oil seeds. 



The seeds are shelled, freed from oil by pressure or by treatment with 

 ether or petroleum ether and finely ground up with -iN acetic acid. 



The lipase is liberated by the treatment with acid. The insoluble matter 

 is filtered off and washed free from acid. A suspension of it is made in water. 



(b) Lipase from pancreas. 



The pancreas is freed from fat, weighed, finely minced and ground up 

 with sand. It is then extracted for 24 hours with a mixture consisting of 

 90 parts of pure glycerol and 10 parts of i per cent, sodium carbonate solu- 

 tion, 10 c.c. of this mixture being used for every gram of pancreas. The 

 fluid is strained through muslin and is kept at o. The lipase is destroyed as 

 soon as the fluid becomes acid ; this happens generally in about three days. 



An active extract may also be prepared by treating the fresh and finely 

 minced pancreas with twice its weight of '5 per cent, sodium carbonate solu- 

 tion for 1 2 hours and straining through muslin. 



As substrate for the action of lipase neutral olive oil (see p. 381), an 

 emulsion of egg-yolk in water, milk and esters, such as ethyl butyrate, may 

 be used. Hydrolysis occurs with the formation of fatty acids which are 

 recognised by the acidity of the solution. 



The activity of the enzyme is shown as follows : 



(1) 5 c.c. of oil, or ester, are mixed with 5 c.c. of suspension, or 

 extract 



(2) 5 c.c. of oil, or ester, are mixed with 5 c.c. of boiled sus- 

 pension, or extract 



The two mixtures are placed in a water-bath at 40 for at least 

 half an hour, preferably 2 or 3 hours, and the contents are occasion- 

 ally mixed by shaking. At the end of this time a few drops of phenol- 

 phthalein are added to each and they are titrated with -iN alkali. 

 More acid will be required to neutralise the contents of (i). 



The presence of lipase in castor-oil seeds may be demonstrated 

 as follows : I gm. of seed, freed from shell, is ground up with 25 c.c. 

 of water saturated with chloroform ; two equal parts of the suspension 

 (10 c.c.) are placed in two test tubes and to each is added i c.c. of 

 dilute acetic acid to liberate the enzyme. One portion is immediately 

 boiled. The substrate is the oil of the seed. Both test tubes are kept 

 at 40 for half to one hour. A few drops of phenolphthalein are 

 added and they are titrated with 'iN alkali. More alkali will be re- 

 quired to neutralise the acid in the tube containing unboiled enzyme 

 showing that fatty acids have been formed. 



