Enzyms of Apples 107 



PREPARATION OF MATERIAL FOR EXAMINATION 



The first problem was naturally that of securing an extract of the cell 

 contents of the apple pulp which would contain the enzyms in active 

 form. Since it was not known whether any or all of these enzyms 

 would be diflfusible through the cell walls (extracellular), a preliminary 

 mechanical rupturing of the cells or rendering of them permeable by 

 drying, according to well-known methods of technique in enzym study, 

 was necessary. Several methods were tried, as follows: 



(i) Whole apples were run through a horse-radish grater and the 

 resulting pulp pressed in an ordinary laboratory hand press. The 

 resulting juice was thick, with small particles of pulp, and attempts 

 were made to clarify it by filtration. These were unsuccessful because 

 of the clogging of the filter by the pectin bodies of the juice. 



(2) Apples were rasped and pressed as before and the juice allowed 

 to stand for some time, during which the suspended solids settled fairly 

 well, and the supernatant clear juice was decanted. Precautions against 

 enzymic activity during the settling were taken by keeping the settling 

 jars in an ice box. 



(3) An attempt was made to secure a dry powder of the apple pulp 

 by drying thin slices in a vacuum desiccator over sulphuric acid; but 

 the large proportion of sugars and pectin bodies in the tissue made 

 this impossible, the slices being gummy and impossible to grind into a 

 powder even after six weeks' exposure in the desiccator. 



(4) Thin slices of apple pulp were treated by the acetone-ether method 

 first used by Buchner, Albert, and Rapp (i) in the preparation of 

 Dauerhefe, or active dry yeast powder. This process was very satis- 

 factory, the apple slices, after the treatment and exposure to the air 

 overnight, becoming so dryand brittle that they could easily be powdered 

 between the fingers and very easily reduced to a fine powder in a mortar. 

 Several investigators have reported that the enzymic activity of the 

 dry powder so prepared is not less than that of the original tissue, and 

 the writer's observations confirm this. This appears to afford an 

 excellent means of preparation of sugary or gummy materials of this 

 kind for enzym extractions. 



(5) Apples were peeled and cored, and the flesh cut into small blocks. 

 These were then mixed with an equal weight of sharp quartz sand and 

 the mixture rubbed gently in a mortar until uniformly disintegrated. 

 The mixture was then transferred to a fine silk cloth and pressed gently. 

 By this means a limpid juice could be obtained which was nearly free 

 from pectin materials, although slightly cloudy with suspended particles 

 of pulp. Experience has shown that harsh grinding and severe pressure 

 result in diminished activity of the juice, particularly in its oxidase 

 activity, but with gentle manipulation, as above, very active juice can 

 be obtained. 



