240 THE VENOM OF HELODEEMA. 



Trtptic Treatment. 

 To test the venom solution for tryptic enzyme it is only necessary to give 

 it the requisite alkalinity and then let it digest itself, since the venom itself con- 

 tains a mucine-like substance which is precipitable on acidifying with acetic 

 acid. The following experiments were performed: 



No. 1. 0.5 c.c. venom suspension, 0..5 o.c. 0.4 per cent sodium carbonate solution, 0.1 



c.c. toluol. 

 No. 2. The same. 

 No. 3. The same. 



All the tubes were stoppered and set in the thermostat. After 4 hours 

 they were filtered and 4 per cent acetic acid carefully added. A good precipi- 

 tate formed. A second set of similar tubes was prepared and digested 12 hours. 

 On addition of acetic acid to these a precipitate also formed. 



The original 1 per cent venom solution was then treated with just enough 

 weak acetic acid to precipitate the mucine-like substance. This was filtered 

 off. The filtrate was carefully neutralized and tested for tryptic enzyme bj' 

 the casein method of Gross, Fuld, and Michaelis.* To this end a solution of 

 0.1 gm. casein in 200 c.c. water containing 10 drops of 10 per cent sodium car- 

 bonate was prepared. The following tubes were then prepared: 



No. 1. 0.5 c.c. venom suspension, 0.5 c.c. casein solution, 0.1 c.c. toluol. 

 No. 2. The same. 

 No. 3. The same. 



These were incubated in the thermostat for 12 hours. They were then 

 carefully acidified. Casein was precipitated in two of the tubes. 



Neither of the two methods may be very reliable. The method bj- which 

 the acetic-acid precipitable protein of the venom is used as indicator may have 

 been negative because there is too much of the protein present. If only a small 

 amount of it had been digested this would have escaped detection. The casein 

 method is exceedingly unsatisfactory because of the difficulty of avoiding an 

 excess of acidity. Therefore further experiments were tried. Gelatin plates 

 were poured in Petri dishes with 2 per cent gelatin. Small areas of the gelatin 

 surface were covered with the venom suspension rendered alkaline with sodium 

 carbonate. After considerable lengths of time no digestion of these areas 

 could be detected; they were not depressed below the rest of the surface. Ex- 

 actly similar experiments were tried, using blood-serum coagulated at 56° C. in 

 place of the gelatin. In these dishes there seemed to be a slight solution of the 

 serum surface over the areas covered with venom suspension; the effect was 

 very slight. It is, therefore, taking all these experiments into consideration, 

 exceedingly doubtful whether any tryptic enzyme at all occurs in the venom. 



Invertase. 



This experiment was performed by Dr. C. S. Hudson. He was unable 

 with the polariscope to detect any effect on the rotation of cane-sugar solution, 

 the medium being 2 per cent acid with acetic acid. 



*Cr. Abderhaldon, E. Op. cit., S. 19. 



