PHYSICAL AND CHEMICAL PROPERTIES OF SNAKE VENOM 91 



METHOD A: The precipitate was then washed into a flask of suitable size and 

 was shaken thoroughly to obtain a minute suspension. Afterwards a strong cur- 

 rent of H 2 S gas was introduced which split the copper compound of the venom 

 and took away the dark-bluish color of the precipitate. The excess of H 2 S was 

 then driven off the fluid by a strong air-current. The copper sulphite was next 

 filtered and separated from the clear, biuret-reaction-free filtrate. This last filtrate 

 was able to produce a similar paralytic effect in the frog and respiratory cessation 

 in the rabbit. The solution was weaker than the native venom solution and sug- 

 gests the possible alteration of the active substance while being treated by alkalies. 

 Copper sulphite did not retain in it any amount of the active principle, because 

 extraction of it by means of 96 per cent alcohol, acetic ether, chloroform, acetone, 

 petroleum ether, or methyl alcohol did not succeed either in a cold or in a hot state. 



The filtrate has shown a great tendency to lose its activity, especially when dried. 

 In many instances the dried materials were completely inactive. After drying it 

 becomes less soluble in water, and not at all soluble in the usual organic solvents. 

 In every case it contained no nitrogen. In solution it produced a foam. 



METHOD B: This is accomplished by taking up the copper precipitate with 

 alcohol and preserving the same under 96 per cent alcohol for some time in order 

 to get rid of water from the precipitate. Then add alcoholic hydrochloric acid 

 to the supernatant alcohol and shake thoroughly. The copper is quickly com- 

 bined with chlorine and goes into solution in alcohol, while the ophiotoxin remains 

 in a pure state undissolved in the same. Then decant the copper solution and re- 

 peatedly wash the precipitated fine flocculence in alcohol, until the washing fluid 

 shows no chlorine in it. Then the precipitate is dissolved in water and tested for 

 its strength. It shows that ophiotoxin is always weaker than the native venom 

 solution. Also that ophiotoxin, which is not found to be protein-molecules, as in 

 the native venom, is easily rendered inactive by various temperatures and alkalies. 



As these methods did not give satisfactory results Faust devised the fol- 

 lowing method based on observations of earlier investigators. In this he 

 utilized the non-coagulable nature of the active principles of cobra venom by 

 higher temperature. 



Ten grams of dried cobra venom were dissolved in 100 c.c. of water and very 

 weakly acidified with acetic acid, then heated 15 minutes at 90 to 95 C. in a water- 

 bath, sodium chloride being added to saturation in the meantime. The greater 

 part of the proteins contained in the native venom is then thrown down as coarse 

 clumps and the fluid is easily filtered. The coagula on the filter was found to be 

 wholly inactive. 



In this process magnesium sulphate and ammonium sulphate can not be 

 used, as both of these salts precipitate the main quantity of ophiotoxin along 

 with the proteins. The light-yellowish nitrate is found to be just as active 

 as the original cobra-venom solution. It contains also ophiotoxin and some 

 other substances which give biuret reaction. The color produced by adding 

 cupric sulphate and sodium hydrate, especially on warming, was deep violet, 

 characteristic of albumoses and peptones. The ophiotoxin does not diffuse 

 or dialyze. 



Faust dialyzed the above filtrate through membrane until chlorine reaction 

 was no more obtained. If a more prolonged dialysis is made the biuret 

 reacting substance may also disappear, but this is always associated with 



