WHAT ARE ENZYMES? 81 



protein is left behind by the use of this 50 per cent, 

 alcohol.] Allow this preparation to stand 5-10 minutes, 

 then filter, keeping the temperature below 20 C. (This 

 takes from I to 2 hours.) Pour the filtrate into 7 times 

 its volume of a mixture of i part of alcohol to 4 parts 

 ether (more protein and other impurities are here sep- 

 arated). Within 1015 minutes the enzyme (including 

 certain impurities) separates as an oily solution. Decant 

 the supernatant liquid. Dissolve the precipitate in the 

 smallest amount of pure water at a temperature of 10-15 

 degrees Centigrade and reprecipitate at once by pouring 

 into 5 volumes of absolute alcohol. Allow it to settle, 

 keeping temperature low ; filter, dissolve in 200-250 cubic 

 centimeters of 50 per cent, alcohol containing 5 grammes 

 of maltose. Pour the solution into a collodion sack of 500 

 cubic centimeters capacity, and dialyze against 2,000 cubic 

 centimeters of 50 per cent, alcohol at not above 20 C, 

 and preferably not below 15 C. Replace dialyzate twice: 

 after 15 hours and a second period of 8-9 hours with 

 fresh 50 per cent, alcohol. Continue dialysis 40-42 hours. 

 Filter. Pour clear filtrate into an equal volume of a 

 mixture of alcohol and ether (equal parts). Filter in the 

 cold, and place the precipitate in a vacuum desiccator. 4 

 The powder obtained is so active that it can digest 20,000 

 times its own weight of starch. And still we are not at 

 all certain that this is an enzyme uncontaminated with 

 foreign bodies ! 



Of the three or four representative workers in attempts 

 to isolate a pure enzyme, the substances obtained by Pro- 

 fessor Sherman and Dr. Osborne (of the Connecticut ex- 

 periment station) showed decided protein characteristics ; 

 whereas the two German investigators, Lentner and 

 Fraenkel, both agree in proclaiming their products as 

 carbohydrate in nature. How near or how far from the 

 truth is either group? To begin with, no proof that any 



4 A vessel (containing a hygroscopic substance to take up 

 moisture) from which the, air has been exhausted. 



