90 VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS 
to 1 per cent venom solution in saline and then separated with ether and 
chloroform. <A previous addition of alkali was necessary for precipitation of 
lecithid from chloroform by ether. Acetone also did not precipitate without 
alkalization. Yet haemolytic substance was already formed, and evapora- 
tion of the fluid in vacuum, washing with acetone and ether (of the residue), 
gave a large proportion of the pure hemolytic substance (0.32 gm. out of 
0.45 gm. of lecithin). Its hemolytic activity was 0.co002 gm. per I C.Cc. 5 per 
cent suspension of ox corpuscles. 
Preservation of these lecithids for a longer period produced reduction in 
their activity. Immediately after precipitation with ether o.co002 gm. = 
complete o.cocor gm. = partial hemolysis. After a long standing 0.00004 
gm.=total and 0.00002 gm. partial hemolysis against 1 c.c. 5 per cent ox 
corpuscles. 
Heating of 1 per cent solution for 3 hours at roo° C. did not cause any 
appreciable loss of activity, while o.1 per cent solution became weakened 
after 2 hours’ boiling. 
The lecithid preparations were not poisonous, except in causing swelling 
of the site of injection. 
That the active principle of cobra venom can be prepared in a form free 
from any proteid substance has been shown by Faust,’ who prepared a neuro- 
toxic substance free of nitrogen from Naja tripudians and named it ophiotoxin. 
The methods of preparation were as follows: 
Ten grams of dried cobra venom were mixed with 500 c.c. of water and allowed 
to stand over night. The next morning the fluid was filtered and separated from 
the undissolved portions, which are mainly the epithelia and cellular débris. Com- 
bustion of the same leaves a little ash. If the insoluble portion of cobra venom 
is mixed with hydrochloric acid, a gas evolves which blackens the lead acetate paper 
and smells of H,S. 
The clear, light-yellowish filtrate was mixed with a neutral solution of cupric 
acetate or with a chemically pure (namely, free of iron) solution of copper chloride, 
and was, after some time, diluted with KOH or NaOH solution by dropping, until 
the fluid showed a weakly or distinctly alkaline reaction. Simultaneously the fluid 
showed an intense biuret color and threw out a precipitate consisting chiefly of 
copper oxyhydrate. 
In the alkaline reaction, if no further precipitate comes out upon addition of 
NaOH, the precipitate is separated from the solution by filtration. The filtrate, 
which is of a deep violet color, throws out another lot of precipitate of the nature 
of protein or protein-like substances upon addition of a dilute acetic acid. But 
this fraction was found to be entirely inactive. The filtrate thus obtained was also 
entirely inactive when tested for its action after removal of the copper salt by 
dialysis. 
The first precipitate was then redissolved in a weak acetic-acid solution, then 
precipitated again by carefully adding drops of KOH or NaOH solution. The 
precipitate was again obtained, while the fluid once more showed biuret reaction. 
The precipitate was rapidly filtered after settling. Usually, but not in all cases, the 
filtrate has still some weak biuret color. If any biuret reaction still occurs, the 
redissolution in acetic acid solution and reprecipitation with alkalies will have to be 
repeated. 
1 Faust. Ueber das Ophiotoxin aus dem Gifte der ostindischen Brillenschlange. Leipzig, 1907. 
