BIOCHEMICAL STUDIES. 237 



The quantities which had to be used for this reaction were unfortunately of 

 necessity so small that this does not finally prove the toxic principle to be non- 

 protein, though it renders it exceedingly probable. The material was exceed- 

 ingly toxic, though the quantity available was insufficient for quantitative deter- 

 minations of toxicity. The impression obtained, however, was that it was very 

 much more toxic than any of the other preparations. 



The efforts to purify the venom by this method were not pursued further, 

 because of the insufficient yields. Since the toxic principle withstood the 

 action of glacial acetic acid it was thought that it might perhaps be purified by 

 means of peptic digestion. Therefore 0.1 gram was dissolved in 10 c.c. of 0.15 

 per cent hydrochloric acid. It was filtered and 5 mg. of pepsin added. It was 

 placed in the thermostat at 38° C. for 19 hours. During this time a few fine 

 floccules had separated (nuclein?) . These were removed by filtration; 0.3 c.c. 

 of filtrate was rendered neutral and injected into a mouse of 18 gm. without 

 producing much effect in 2 hours; but on the following morning the animal was 

 dead. The material was replaced in the thermostat for 5 days. Then 0.3 c.c. 

 was neutralized and injected into a mouse of 20 gm.; it did not become very 

 sick and recovered. The experiment was repeated with the same result. 



The experiment was repeated with tryptic digestion. After 18 hours of 

 digestion the injection of 0.25 gm. into a mouse of 28 gm. produced a severe 

 intoxication with paralysis, but ultimate recovery. 



It is therefore evident that while the venom withstands peptic and tryptic 

 digestion for a considerable time, the resistance is not great enough to warrant 

 the use of digestion in the process of purification. 



The best results, after many trials, were finally obtained by a modification 

 of one of the methods used by Faust in his recent paper on the venom of the 

 rattlesnake.* The filtered venom solution was treated with acetic acid as 

 long as a precipitate formed. This precipitate was removed by centrifugation 

 and repeatedly washed. There is at this stage a considerable loss of activity, 

 for, in spite of thorough washing, this precipitate, which is either mucine or 

 nuclein, retains a high degree of activity. This phenomenon was also observed 

 by Santesson and led him to conclude that the active agent was in part nuclein. 

 The filtrate, which was active, was treated very carefully with weak sodium 

 carbonate till only very M^eakly acid. It was then rapidly heated to boiling 

 over a free flame to coagulate the small amount of coagulable protein. It is 

 very important that the acidity be right. If it is not, clear filtration is exceed- 

 ingly difficult and tedious. As soon as the solution had come to a boil it was 

 rapidly chilled with ice-water and the very sUght coagulum removed by filtra- 

 tion. The clear filtrate was then dialyzed till free from chlorine. Dialysis 

 should not be continued beyond this point, for there is a distinct loss of activity 

 in the process. The dialyzed solution was then concentrated over sulphuric 

 acid in vacuo, saturated with ether, and transferred to a centrifuge tube. It 

 was then put in a centrifuge placed in the cold-storage room at a temperature of 

 — 18° F. The centrifuge was set in motion. From time to time it was stopped 



*0p. cit. 



