190 INTRODUCTION TO GENERAL PHYSIOLOGY 



alcohol. The former combines with the sodium hydroxide, form- 

 ing a neutral salt, so that when sufficient has been produced 

 the red colour of the phenol-phthalein disappears. This dis- 

 appearance will be seen to take place slowly. 



The experiment may be made more accurately by taking 

 methyl acetate and water in molecular proportions, that is, 74.1 

 parts by weight of the former to 18 parts by weight of the latter. 

 Approximately, 10.5 c.c. of the ester to TO c.c. of water. Take 

 out a sample, say 2 c.c., by means of a pipette every day or two, 

 and determine, after dilution, the amount of acid present, by 

 titration with standard caustic soda. The rate of change will 

 ultimately become very slow and finally cease. By calculation 

 of the amount of acid formed, it will be found that there is 

 still a notable quantity of the ester left unhydrolysed, so that 

 the reaction has come to a balanced position. Take now a sample 

 of the mixture in equilibrium, dilute it with water to twenty times 

 its volume. Allow it to stand for a day or two and titrate again. 

 More acid will be present ; that is, further hydrolysis has taken 

 place. 



The experiment may be performed with ethyl acetate, but 

 the time taken will be much longer. 



Hydrolysis by Enzymes. The enzyme lipase may be used for 

 the first experiments on this question. This catalyst acts on 

 esters in general, and we will take the glycerol esters known as 

 fats Lipase is found in the pancreatic juice, in the liver, and in 

 various fatty seeds. The most convenient source is the seed of 

 the castor-oil plant ; but it is important that fresh seeds, capable 

 of germination, be used, otherwise there may be no enzyme 

 present. The seeds should, in fact, be obtained from a seedsman, 

 not from a druggist. 



It will suffice to rub some of the seeds with a little weak 

 acetic acid in a mortar, after removing the outer shells by a blow 

 with the pestle. The acid is required to form the active enzyme 

 from a preliminary stage in the resting seed. 



Add some of the paste thus obtained to an emulsion of a small 

 quantity of olive oil in water. After vigorous shaking for a 

 moment, take out a sample of 5 c.c. and titrate with sodium 

 hydroxide. It will contain a small amount of acid. It is best to 

 add the sample to 25 c.c. of methylated spirit to dissolve the oil, 

 and then to dilute with 25 c.c. of water and add phenol-phthalein 

 before titration. 



Allow the rest to stand in a warm place, shaking at intervals, 

 for a day or two, and then titrate again. The fat will have been 

 partially hydrolysed to oleic acid and glycerol. Under these condi- 

 tions, with excess of water, the ester is hydrolysed by the enzyme. 



