16 
(7) On standing in contact with glycerin, a portion of the glycerin 
became dark coffee-brown in color. The glycerin extract gave splendid 
reactions with guaiacum, both alone and on mixing with water. The 
fungus itself became brownish black in color, and on maceration with 
water it gave a reddish-brown extract, which was found to give a 
very decided reaction with guaiacum. 
(8) On standing in contact with equal amounts of water and toluene 
until August 22. 1904. the haver of toluene became clear lemon yellow, 
whereas the aqueous layer became clear reddish brown. Some of the 
toluene was carefully drawn off by means of a pipette and shaken up 
with a small amount of water in a test tube. The water at once became 
reddish brown in color, and on adding guaiacum it developed a deep 
blue coloration at once. Some of the toluene was then added to an 
equal amount of water in a test tube and the mixture shaken. On 
standing* for a short time the mixed liquid separated into two layers, a 
reddish-brown aqueous layer and a clear lemon-yellow layer of toluene. 
Some of the lower layer was withdrawn by means of a pipette and 
tested with guaiacum. It also gave a deep indigo-blue coloration 
immediately. On the other hand, when a large amount of water was 
employed to extract the oxidase from its solution in toluene, the aque- 
ous solution became pink in color, but failed, for some reason unknown 
to me at present, to give the guaiacum reaction. The aqueous portion 
of the original solution was found to be exceedingly active — one drop 
of its solution giving a good guaiacum reaction when diluted to 3 cubic 
centimeters with water. 
Several conclusions may be drawn from these results: 
(1) That the oxidase of Lepiota americana is much more readily 
soluble in water than in any other solvent. 
(2) That even in solution the oxidase is not destroyed by 40 per cent 
formic aldehyde. 
(3) That while it does not dissolve in ethyl, amyl, or allyl alcohol, 
it is not destroyed by contact with these substances. 
(4) That it is soluble in glycerin and apparently, to some extent at 
least, in toluene. Whether the perfectly dry oxidase is soluble in 
toluene remains to be proved. It may be, of course, that it is the 
water which is dissolved in the toluene which really takes the oxidase 
into solution. This point requires further investigation. It is certain, 
however, that when water and toluene are both present a part of the 
oxidase passes into the toluene layer. 
(5) That certain substances, such as chloral hydrate, destroy the 
oxidase completely. 
REAGENTS FOR THE OXIDASES. 
Several vears ago O. M. Shedd and mvself a called attention to the 
i c5 
fact that phenolphthalin could be employed advantageously as a reagent 
«Am. Ckem. Jour., XXVI, 1901, 526-539. 
