ix] GLUCOSIDE-SPLITTING ENZYMES 147 



the Columbine (Aquilegia vulgaris\ the Arum (Arum ma-culatum) and plants of the 

 Bird's-foot Trefoil (Lotus comiculatus) : also with bitter almonds and apple pips, and 

 young shoots of Flax (Linum perenne). In the case of the seeds, these may be used 

 crushed, both with and without chloroform, the uninjured seed being used as a 

 control. 



Expt. 141. Preparation of amygdalin. Weigh out 100 gms. of bitter almonds. 

 Remove the testas by immersing them for a short time in boiling water. Then 

 pound up the almonds well in a mortar and transfer to a flask. Add about 

 200-300 c.c. of ether and allow the mixture to stand for 2-12 hours. Filter off the 

 ether and extract again with fresh ether. The greater part of the fat will be removed 

 in this way. Then dry the residue from ether and, as rapidly as possible, extract 

 twice or three times with boiling 90-98 % alcohol which removes the amygdalin. 

 The residue, after ether extraction, contains both amygdalin and emulsin, and, if 

 allowed to stand, the emulsin will hydrolyze the amygdaliu : hence the necessity for 

 rapid extraction with alcohol. Evaporate the filtered alcoholic extract on a water- 

 bath or, better, distil in vacua to a small bulk. Then add an equal volume of ether 

 and allow the mixture to stand for a time. The amygdalin separates out on standing. 

 Filter off the precipitate, dissolve in a little hot water and allow to crystallize in a 

 desiccator. 



Expt. 142. Preparation of emulsin (Bourquelot, 10). Weigh out 25 gms. of 

 sweet almonds. (Bitter almonds can also be used. The sweet variety is preferable ; 

 since from them the emulsin can be more readily prepared free from amygdalin.) 

 Plunge them for a moment into boiling water and remove the testas. Pound 

 thoroughly in a mortar, and extract the bulk of the oil with ether as in the last 

 experiment. Then grind up the residue with 50 c.c. of a mixture of equal parts of 

 distilled water and water saturated with chloroform and allow the whole to stand 

 for 24 hours. Filter by means of a filter-pump, and to the filtrate add glacial 

 acetic acid (1 drop to 15 esc. of the filtrate) whereby the protein is precipitated. 

 Again filter, and to the filtrate add 3-4 times its volume of 96-98 % alcohol. The 

 emulsin is deposited as a white precipitate. Filter off the precipitate and dissolve it 

 in about 100 c.c. of cold distilled water. 



Expt. 143. (a) To demonstrate the hydrolysis of amygdalin by emulsin. Into 

 each of two flasks put 50 c.c. of a 1-3 % solution of amygdalin. To one flask add 

 25 c.c. of the emulsin solution prepared in the last experiment. To the other flask 

 add 25 c.c. of enzyme solution after it has been well boiled, and again boil the 

 mixture after adding the enzyme. Fit each flask with a cork and sodium picrate 

 paper. The paper in the flask containing the unboiled enzyme will rapidly turn red, 

 the control remaining yellow. Unless both the enzyme and the amygdalin solution 

 are well boiled in the case of the control, the paper may show reddening in time on 

 account of traces of prussic acid present in both solutions. 



(b) Simplified method for extraction of amygdalin and emulsin, and demonstra- 

 tion of hydrolysis of amygdalin by emulsin. Take 1 2 bitter almonds. Remove the 

 testas by immersing them for a short time in boiling water. Then pound up the 

 almonds well in a mortar and transfer to a flask. Add about 50 c.c. of alcohol and 

 heat to boiling on a water-bath. Filter off the extract, and evaporate it to dryness 

 on a water-bath. The residue will contain amygdalin. 



102 



