1921] 
KARRER—H-ION CONCENTRATION AND AMYLASE ACTION 75 
A 5 per cent soluble starch solution, which was to be added 
to the above as a substrate, was prepared by mixing the requisite 
amount of Lintner’s soluble starch (Merck) and water and 
refluxing for about 3 hours. One per cent of toluene was added 
to the stock solution as a preservative. 
Extracellular amylase.—1t was found in preliminary experi- 
ments that the total volume of the nutrient solution remaining 
in the various flasks after the growth of the fungus averaged 
about 40 cc. in all the series, so it was unnecessary to bring the 
total volume of the solution to a definite volume. 
In studying the extracellular amylase, 39 cc. of each of the 
above-described solutions were placed in 100-cc. Erlenmeyer 
flasks. To each of these, 1 cc. of the starch paste and 10 cc. 
of culture solution were added, thus making the total volume 
50 ec. One per cent toluene was used as a preservative. The 
solutions were then incubated at 28° C. for 24 hours. A smaller 
quantity than 10 cc. of culture solution containing the excreted 
enzyme was insuffieient to produce marked activity in some 
cases, and a greater amount often caused too much variation in 
the final H-ion concentration of the solution. Further, an 
incubation period of 6-12 hours was found to give such low 
diastatic values that a longer period of incubation was employed. 
A control series was set up by adding 10 cc. of the culture solu- 
tion, which had been inactivated by heating in a boiling water- 
bath for 15 minutes and then made up to the original volume, 
to solutions of the above H-ion concentrations. In this manner 
the effect of the H-ion eoncentration upon the reagents, and 
also the reducing power of the enzyme solution was determined. 
A determination was made of the amount of reduction occurring 
in the enzyme solution alone. 'This was found not to vary 
during the course of the experiment. 
Method of determining the enzymic action.—At the end of 24 
hours, the saccharifying power of the enzyme was tested accord- 
ing to the following method. Ten cc. of each of the buffered 
solutions and distilled water were accurately pipetted into each 
of two 50-cc. centrifuge tubes and made neutral to phenol- 
phthalein by adding either NaOH or H;PO,. The tubes were then 
plunged into boiling water for 10 minutes in order to inactivate 
the enzyme. The same procedure was followed for the inacti- 
