236 



THE VENOM OF HELODEKMA. 



ful biuret reaction. After neutralizing with sodium carbonate 0.75 c.c. was 

 injected into a mouse of 30 gm. at 4'' 10" p. m. The effect was noticeable 

 almost at once. Besides the usual symptoms there seemed to be a good deal of 

 pain and irritation at the site of injection. At 5^ W^ p. m. the hind legs were 

 completely paralyzed. At S*" 40"" p. m. respiration was feeble, though the ani- 

 mal still struggled occasionally. At 6^ 30™ p. m. the respiration was still more 

 feeble. The animal was not observed during the night, but was found dead the 

 following morning in the position in which it had last been seen the evening 

 before. 



The alcoholic filtrate was treated with an equal volume of 95 per cent alco- 

 hol. A second precipitate formed, apparently, about half as voluminous as the 

 first. This was separated from the alcohol and dissolved in 1 c.c. of normal 

 saline solution. It gave a rather weak biuret reaction. After neutralization 

 with sodium carbonate 0.5 c.c. was injected into a mouse of 21 gm. Symptoms 

 set in almost at once and ran a rapid course, the mouse dying within an hour. 

 Paralysis set in so soon there were almost no convulsive symptoms. 



The alcoholic filtrate was then treated with an equal volume of ether. A 

 slight precipitate formed, which was allowed to settle overnight. It was then 

 removed by filtration and dissolved on the filter in 10 c.c. of normal saline solu- 

 tion. While the previous fractions obtained by precipitation with alcohol did 

 not dissolve readily in water, this fraction was very soluble, yielding a perfectly 

 clear, colorless, and faintly acid solution. This was concentrated in vacuo for 

 14 hours to a volume of 1.5 c.c; of this, 0.25 c.c. was injected into a mouse of 

 23 gm. The mouse was affected almost at once; there was no struggle, but a 

 rapid progressive paralysis, the animal dying in 85 minutes. This experiment 

 was repeated upon a second mouse with quite similar results. The remainder 

 of the solution was examined with great care for biuret-giving substances, but 

 the reaction was quite negative. 



The alcohol-ether filtrate was concentrated by an air-current until all the 

 ether had been removed. Water was added to the alcoholic solution remain- 

 ing. An appreciable amount of precipitate was formed, soluble in ether and 

 capable of being extracted from its suspension in weak alcohol by ether. It 

 was removed in this fashion: the ether evaporated, and the residue suspended 

 in normal saline solution. When injected into a mouse it produced no untoward 

 effects. There seems to be present in the venom an appreciable amount of 

 fatty, or at least of ether-soluble, material. Whenever a solution of the venom 

 was precipitated with alcohol an appreciable amount of material remained 

 dissolved. 



The entire experiment with glacial acetic acid was twice repeated with 

 somewhat larger quantities of venom (0.5 gm.). In each instance the results 

 were essentially the same. After all the material that could be precipitated 

 from the glacial acetic-acid solution with alcohol had been removed a further 

 slight precipitate could be obtained by the addition of ether. This precipitate 

 was always very small in amount — not more than a few milligrams. In one 

 of these experiments it did not give the biuret reaction; in the other it did. 



