BIOCHEMICAL STUDIES. 235 



removed by nitration. The filtrate was practically devoid of toxicity. The 

 kaolin on the filter paper was then extracted on the filter with a little very weak 

 acetic acid. The extract gave a distinct biuret reaction and was quite toxic, 

 though not as powerful as the original solution. 



Since the only precipitate which did not carry down the toxic principle was 

 the copper precipitate; and since this was formed in alkaline solution, it seemed 

 possible that a method by which protein is removed from an alkaline solution 

 might not carry down the toxic principle. Such a method is the uranyl acetate 

 method used by Kowalewsky,* Jacobyf, Glaessner,^ Abel and Ford, and 

 others. The procedure is to render the solution faintly alkaline with sodium 

 carbonate and then to precipitate the protein with a saturated solution of 

 uranyl acetate. A solution of venom, prepared as in the first experiment, was 

 treated in this way. It was rendered weakly alkaline with sodium carbonate, 

 and uranyl acetate was added till all the biuret-giving material had been pre- 

 cipitated. The precipitate was then removed by nitration. The filtrate was 

 dialyzed till free from uranium and then concentrated in vacuo to the original 

 volume; 1 c.c. injected into a mouse did not produce any characteristic symp- 

 toms. The precipitate produced by the uranyl acetate was then dissolved with 

 the aid of acetic acid and dialyzed till free from uranium. It was then concen- 

 trated to a volume of 15 c.c. 0.25 c.c. was injected into a mouse of 18 gm. 

 The symptoms set in very rapidly and death ensued within an hour. It is there- 

 fore evident that uranyl acetate is not a suitable reagent for purifying the 

 venom. 



Thereupon 0.2 gm. of venom finely powdered was treated for a few hours 

 with 10 c.c. glacial acetic acid. Only a portion was dissolved. The residue 

 was removed by filtration. It was gelatinous and semi-translucent. Most of 

 it dissolved readily in water. The solution was filtered, the filtrate being quite 

 opalescent. The filtrate was precipitated with twice its volume of alcohol. 

 A relatively scanty precipitate formed. This was separated by filtration and 

 thoroughly washed with alcohol. It was then dissolved in 1.5 c.c. of normal 

 saline solution, neutralized with sodium carbonate and 0.5 c.c. injected into a 

 mouse of 26 gm. beneath the skin of the abdomen. The injection was made at 

 4 p. m. The animal showed no special symptoms that afternoon, but was very 

 quiet; the next morning it was found dead, lying flat on its abdomen, much in 

 the position in which it had been last observed on the preceding afternoon. 

 Evidently while the glacial acetic acid extracted most of the toxic principle, 

 some of it remained undissolved. 



The glacial acetic-acid solution of the venom was treated with twice its 

 volume of 95 per cent alcohol. The flocculent precipitate formed was allowed 

 to settle over night and then removed by filtration. After washing thoroughly 

 with weak alcohol (alcohol 95 per cent 2 parts, water 1 part) it was dissolved 

 in 1.75 c.c. of normal saline solution. It was slightly acid and gave a power- 



Zeitschr. f. anal. Chem., Bd. 24. S. 551 (1895). 

 fZeitschr. f. physiol. Chem., Bd. 30, S. 135 (1900). 

 tBeitrage (Hofmeister) z. Chem. Physiol, u. Path., Bd. I., S. 1. 

 Journ. Biological Chemistry, vol. vn, p. 273 (1907). 



