232 THE VENOM OF HELODERMA. 



The part of the venom insoluble in water could not be freed completely 

 from toxic material without very long-continued washing, though it is likely 

 that the toxic material is merely adsorbed or otherwise mechanically held in 

 the insoluble portion. The aqueous solution, which was slightly yellow and 

 faintly opalescent, was treated with a little chemically pure neutral copper ace- 

 tate and then with weak potassium hydrate, drop by drop, till the reaction be- 

 came distinctly alkaline and no further precipitate formed. The liquid and 

 the precipitate were then separated by centrifugation. The precipitate was 

 dissolved in water acidulated with acetic acid and then carefully made alkaline 

 again. The precipitate was again separated by centrifugation and washed 

 repeatedly. It was finally freed from copper by first washing with 95 per cent 

 alcohol; then with alcohol containing a little hydrochloric acid, till all the cop- 

 per was removed. There remained only an insignificant precipitate, which 

 was washed free from chlorine with alcohol. 



Only a few milligrams of material remained. The adherent alcohol was 

 allowed to evaporate spontaneously and the residue was dissolved in 0.75 c.c. of 

 normal saline solution. Of this 0.5 c.c. were injected into a mouse of 22 gm. 

 weight beneath the skin of the abdomen. The mouse was uncomfortable for 

 several hours, but within 24 hours was apparently quite normal again. Hence 

 either the treatment destroyed the toxic substance or else the copper precipi- 

 tate did not carry it down. The venom of Heloderma must therefore be differ- 

 ent from that of the cobra and the rattlesnake. According to Faust's observa- 

 tions, the toxic principles of the latter are precipitated with copper hydrate. 



Further observations showed that in all probability no appreciable quan- 

 tities of the venom were destroyed by the copper treatment. The clear solu- 

 tion decanted from the precipitate of copper hydrate showed a strong typical 

 biuret reaction. It was carefully acidified with acetic acid. An abundant 

 white precipitate formed, which was separated by centrifugation from the 

 liquid. To remove any traces of copper it may have contained it was carefully 

 washed with a little water containing a trace of acetic acid; then dissolved with 

 the aid of sodium carbonate and again precipitated with acetic acid and washed. 

 It was finally washed with an abundance of weak alcohol. The precipitate, 

 when finally quite free from copper, was dissolved in 10 c.c. of normal saline 

 solution with the help of very little very weak sodium carbonate. 0.25 c.c. was 

 injected into a series of white mice of 20 to 25 gm. weight; all died with charac- 

 teristic symptoms. The protocol of one experiment may serve as an example ; 



White mouse weighing 20 gm. received 0.25 c.c. beneath the skin of the 

 abdomen at 4 h 23 m p. m.; became uncomfortable and sick within a few min- 

 utes; at 4 h 41 m p. m. it fell over on one side for a moment and then had convul- 

 sions, then recovered somewhat. This was followed by dyspnea and paralysis, 

 the mouse lying on one side. Finally it was quite paralyzed, dying at 5 h 15 m 

 p. m. 



It is therefore evident that the copper method of Faust is not applicable to 

 heloderma venom, since the copper precipitate carries down little, if any, of the 

 toxic principle. 



