90 VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS 



to i 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 hsemolytic 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 haemolytic substance (0.32 gm. out of 

 0.45 gm. of lecithin) . Its hsemolytic activity was 0.00002 gm. per 1 c.c. 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 0.00002 gm. = 

 complete 0.0000 1 gm. = partial haemolysis. After a long standing 0.00004 

 gm. = total and 0.00002 gm. partial haemolysis against 1 c.c. 5 per cent ox 

 corpuscles. 



Heating of 1 per cent solution for 3 hours at ioo° C. did not cause any 

 appreciable loss of activity, while 0.1 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, 1 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 debris. 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 2 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. 



