LABORATORY WORK 191 



To observe the opposite process of synthesis, add the paste 

 containing lipase to a mixture of oleic acid and glycerol, to which 

 a very small quantity of water has been added. Titrate as above 

 at once, and again at intervals of a few days, in a warm place. The 

 sample is best weighed in a flask, taking 2 or 3 gm. and working 

 out the result for 10 gm. in each case. 



The difficulty in this experiment is that the constituents of 

 the mixture separate from each other, so that the action is very 

 slow, unless continual shaking is practised. But if the mixture 

 be well shaken at intervals, an obvious synthesis, shown by decrease 

 in the oleic acid present, should be detected. 



If a polarimeter is available, an experiment showing the same 

 facts may be made with the enzyme emulsin made from almonds. 

 An active preparation can be bought. This enzyme acts on 

 glucosides, in particular that kind called the /3-glucosides, which are 

 laevo-rotatory. The synthesis of the /3-glucoside of glycerol can 

 be shown thus : Take 9 gm. of glucose, dissolve by aid of heat 

 in 6 gm. of water. Cool. Add 20 gm. of glycerol, and rub in 

 a mortar with I or 2 gm. of emulsin. The exact quantities are 

 not essential ; the proportions given ensure the most rapid result. 

 Take a sample of the mixture at once, say 2 gm. weighed as in 

 the olein experiment. Add 2 or 3 c.c. of a solution of mercuric 

 nitrate to precipitate the proteins introduced with the enzyme, 

 make up to 50 c.c., and filter. Determine the degree of rotation. 

 It is well to allow the solution to stand for some time after addition 

 of the mercuric nitrate, to allow aggregation of the particles. 

 Place the remainder in a warm place, preferably at 45 C. Take 

 samples every day for the first four days, then every second day. 

 The dextro-rotation due to the glucose will become less and less, 

 owing to the formation of the glucoside with the opposite rota- 

 tion. The rotation ultimately passes to the opposite side of zero. 

 Finally, take a sample, dilute about twenty times with water, add 

 a little fresh emulsin, and warm for a day. Treat as before, and 

 it will be found that the original dextro-rotation returns. Thus 

 the same enzyme hydrolyses in dilute solution. 



Ethyl-glucoside (/?) in crystals can be obtained by the aid of 

 emulsin thus (Bourquelot) : Add powdered glucose in excess to 

 100 c.c. of 90 per cent, alcohol, so that part remains undissolved. 

 Then add about a gram of emulsin and leave in a warm place, 

 shaking at intervals, for two or three weeks. Filter. Evaporate 

 to dryness on a water bath. Extract with a small quantity of 

 pure, cold, dry acetone. This dissolves the glucoside, leaving 

 glucose. Crystals separate on standing. Filter off and dry over 

 sulphuric acid in a desiccator. The crystals will be found to 

 form a laevo-rotatory solution and not to reduce alkaline copper 



