1918] 
BONNS—ETHERIZATION AND ENZYME ACTIVITY 269 
volumes of the enzyme dispersion previously autoclaved at a 
pressure of 15 pounds. 
The sucrose substrate was acidified with 0.1 се. of deci- 
normal hydrochlorie acid, since invertase is known to act best 
in acid medium. The solutions of albumin, casein, and pep- 
tone were made neutral, and for the alkaline series of these 
substrates 2 cc. decinormal sodium hydrate added to each 
flask, with an equal volume of water to the neutral ones. The 
digestion tubes were made by coagulation in hot water of a 
10 per eent aqueous solution of albumin in glass tubing of 
2 mm. diameter. These tubes were placed in 50 сс. of dis- 
tilled water plus 0.5 ec. toluol, and 2 сс. decinormal hydro- 
chlorie acid or sodium hydrate added to the acid and alkaline 
series respectively. All flasks were placed in the incubator 
and maintained at 40-419 C. throughout the several periods 
of ineubation. At the close of such periods they were placed 
in the autoclave, subjected to 15 pounds pressure for a few 
minutes, and upon cooling were used in the quantitative de- 
terminations hereinafter discussed. 
The period of incubation varied with the substrate studied. 
The time for checking enzyme action was approximately de- 
termined by the use of trial flasks of substrate with enzyme 
extraet from the etherized series. Such flasks were used only 
for the carbohydrates. In the ease of starch, drops from the 
trial flask were tested on a spot-plate with iodine solution at 
intervals after the beginning of incubation until the end point 
appeared to be approaching; at this point the flasks were 
autoclaved. For dextrin, sucrose, maltose, and inulin, 10-сс. 
samples were taken from similar test flasks at one-half or one 
hour intervals, and the relative amounts of cuprous oxide 
precipitate obtained with Fehling’s solution were noted. On 
this basis the carbohydrates were incubated for the following 
periods: starch, 45 minutes; dextrin and maltose, 5 hours; 
sucrose and inulin, 9 hours. The time of incubation for all 
the other flasks in the experiment,—31 days,—was purely 
arbitrary, and was based on the extremely slow action of 
enzymes other than carbohydrases, as observed in previous 
work in this laboratory. 
