504 
BULLETIN OF THE BUREAU OF FISHERIES 
Dialysis was then used as a method of preparing the green substance and concen- 
trating it with the hope of obtaining some crystals when sufficiently concentrated. 
From three to four hundred green oysters were ground very fine through a meat 
grinder and extracted with water by pressing through several layers of cheesecloth. 
The extract was first dialyzed in collodion sacs impermeable to congo red, then con- 
centrated in vacuo. A thick sirup was finally obtained, but no crystals were formed 
because of the large amount of impurities. 
Some of the properties of this concentrated protein-free extract are: Heating to 
boiling destroys the color. Addition of alkali gives the blue-purple color of the biuret 
test. Heating of the alkaline solution causes a precipitate of cuprous oxide, indicat- 
ing the presence of sugar or other reducing substance. H 2 S decomposes the pigment 
readily with the formation of CuS. A steel spatula immersed in the green liquid soon 
becomes copperplated. This indicates that the compound exists in a highly dis- 
sociated state. One drop of HC1 to about 5 cubic centimeters of extract causes a 
discharge of the green color. It apparently is not a simple copper salt as CuCl 2 , Cu, 
Br, or Cu(C 2 H 3 0 2 )2, since addition of ammonia does not greatly deepen the color, as 
is the case with such copper salts. Boiling the extract discharges the color, thus 
necessitating vacuum distillation when concentrating. 
An attempt was made to determine whether the compound existed as a copper 
ammonium complex. Aeration to remove ammonia before and after decomposition 
with H 2 S failed to reveal evidence of a copper ammonium complex. 
While the evidence cited indicated that the compound was not identical with or 
even remotely related to hemocyanin or other copper protein complex, it was never- 
theless thought possible to exist in the oyster as a copper protein complex and possibly 
suffer decomposition on treatment with water used in the extraction. To settle this 
point, the following experiment was carried out. Normal white oysters were ground 
up and the oyster fluids obtained by pressing through a cloth. Green oyster fluids 
were similarly obtained and the one dialyzed against the other. Some of the green 
pigment diffused through the collodion sac tested previously for tightness with congo 
red, leaving no doubt that the green pigment exists in the oyster as a simple, readily 
diffusible compound. 
The green pigment is quite readily soluble in methyl alcohol, less so in ethyl alcohol, 
and quite insoluble in butyl or amyl alcohol. It is insoluble in such fat solvents as 
chloroform, ether, acetone, or benzene. It is soluble in pyridine. 
On standing about three or four months, the green color disappears and the extract 
turns to a reddish chocolate color. It was thought that it might represent a reduced 
form of the green pigment. Bubbling air or oxygen through the extract does not 
bring back the green color. Upon shaking with methyl alcohol, ethyl alcohol, or 
pyridine, the color returns in these solvents. 
OXYGEN CONSUMPTION OF GREEN OYSTERS 
The deposition of large quantities of copper in the oyster was regarded by Boyce 
and Herdman (1897) as a degenerative reaction which may be due “to a disturbed 
metabolism, whereby the normal copper of the hemocyanin, which is probably pass- 
ing through the body in minute amounts, ceases to be removed and so becomes stored 
up in certain cells.” It was thought that the determination of the metabolic rate of 
green and normal oysters would supply evidence in favor of or against this suggestion. 
Green oysters differing widely in depth of pigmentation were obtained from various 
sections of Long Island Sound. Inasmuch as previous work of Herdman and Boyce 
