1905. | Studies on Enzyme Action. 597 
its behaviour with phenylhydrazine, this contained a biose, together with 
much glucose. The manner in which isomaltose and maltose were detected 
will be apparent from the following descriptions :— 
Proof of the Presence of Isomaltose—To remove the unchanged glucose, 
about 20 c.c. of boiled yeast-water were added to the liquid, which was then 
sterilised and inoculated with a quick-acting pure yeast, S. intermedians, 
Hansen. After fermentation had gone on during 10 days at 25°, the solution 
was filtered, mixed with a little calcium carbonate to neutralise any acid 
which had been formed during the fermentation, boiled to expel alcohol and 
then reinoculated with yeast under sterile conditions. These operations were 
usually repeated a second time, experience having shown such repetition 
to be the only way in which the last trace of fermentable sugar can be 
removed. 
The solution finally obtained, when clarified with charcoal, was almost 
colourless ; the total volume was 150 c.c.; its rotatory power in a 1-decimetre 
tube was ap = 4°20. 
When a mixture of about 15—20 e.c. of the solution with 2—3 ce. of 
almost colourless phenylhydrazine, dissolved in 3 cc. of 50 per cent. acetic 
acid, was heated in a flask in a boiling water bath during 1—14 hours, no 
separation of osazone took place from the hot liquid, which was an indication 
that no glucosazone was formed. On cooling the liquid, a light yellow 
flocculent osazone separated out slowly; this was filtered off, well washed 
with cold water and redissolved in boiling water, in which it was entirely 
and easily soluble. The osazone only crystallised out when the solution was 
nearly cold, differing in this respect from maltosazone, which erystallises out 
while the solution is still hot. After a second crystallisation from water, the 
osazone was crystallised from dilute alcohol; it then formed a light brownish 
yellow crystalline powder, which decomposed when heated at about 120°. To 
complete the purification, it was next crystallised from wet ethylic acetate. 
The slightly yellow micro-crystalline powder thus obtained was very soluble in 
boiling water; on heating, it melted at about 156°, forming a brown liquid, 
which decomposed at 198°, behaving in every way exactly in the manner 
described by E. Fischer. 
It was to be supposed that if it were a derivative of Alneods) it would be 
hydrolysed by emulsin: 30 c.c. of the solution were therefore mixed with a 
small quantity of active emulsin and a few drops of toluene; the liquid was 
then set aside in a closed flask during several days at 38°, along with a similar 
quantity of the solution to which no enzyme had been added. To test 
whether glucose had been formed, the filtered solutions were heated side by 
side with phenylhydrazine. In the one case, separation of an insoluble 
28 2 
