238 THE VENOM OF HELODERMA. 



and the tube examined. Gradually three layers were formed an upper layer 

 of fine ice-crystals ; a middle layer rather cloudy or even milky from the sepa- 

 ration of material due to the chilling; and a sediment consisting of fine precipi- 

 tate formed by the chilling. This process was allowed to go on until the layer 

 of ice-crystals had filled about half the tube. Then the tube was removed, a 

 rift made with a file at the lower limit of the ice layer, and the tube held over a 

 dish and cracked with a hot wire at the level of the file-scratch. The part of 

 the tube containing the ice was thus removed. As much of the liquid above 

 the sediment as possible was removed and the sediment rinsed into a fresh cen- 

 trifuge tube with as little water as possible. It was warmed till the solution 

 became clear and the freezing and centrifuging repeated. After the fourth 

 freezing a solution was obtained which was exceedingly active, yet gave not the 

 slightest trace of biuret reaction. The amount of material in it was very 

 slight, for when working with small quantities of material, as was unfortunately 

 necessary, the losses are relatively considerable, due to the imperfect separa- 

 tion of the active principle by freezing. By using larger quantities these losses 

 would undoubtedly be lessened. The precipitate obtained in the first freezing 

 was never free from biuret-giving material. Faust reports that in his experi- 

 ments this was sometimes the case. The solutions which he subjected to cold 

 seem to have contained less protein than the heloderma solution similarly 

 treated, because colloidal iron removes protein better than coagulation. The 

 heloderma solutions still contained much biuret-giving material and a part of 

 this is precipitated on freezing. On the second freezing, the dilution of the 

 biuret material being greatly reduced, less or none is frozen out. Finally, the 

 yields seem to be less with this method for heloderma venom than for rattle- 

 snake venom. A good deal of the active principle is adsorbed in the acetic-acid 

 precipitate; not all is precipitated in the chilling process; the clear liquid was 

 always quite toxic. The solution finally obtained contained very little mate- 

 rial in solution. The doses injected could not have contained more than a 

 small fraction of a milligram of the toxic principle. The animals were affected 

 almost immediately. There was usually a short convulsion followed by rapid 

 progressive paralysis and death in from 14 to 60 minutes. 



Though many of the experiments performed in the effort to isolate the 

 active principle yielded negative results so far as the purification of the toxine 

 is concerned, it was still thought advisable to describe the typical experiments 

 in some detail, because they furnish information concerning the behavior of the 

 venom to various reagents. While this investigation, unfortunately, has not 

 accomplished all that was expected, it is believed that it has made some con- 

 tributions to the chemistry of heloderma venom. Many of the observations of 

 Santesson have been confirmed. It has been shown that the toxic principle is 

 very readily adsorbed by all kinds of precipitates.* The observation of San- 

 tesson that the venom contains a good deal of a protein precipitable by acetic 

 acid has been confirmed. This precipitate, which Santesson classed with the 

 nucleins, but which may be a mucin, is toxic. Santesson believed this "nuclein" 



*Cf. the chapter on adsorption for a more detailed account. 



