BIOCHEMICAL STUDIES. 233 



Thereupon the metaphosphoric acid method was tried. The aqueous 

 venom solution prepared as in the previous experiment was saturated with 

 sodium chloride,weakly acidulated with acetic acid, and the vessel containing the 

 solution immersed for 1 5 minutes in a water-bath heated to 90 to 95 C . It was 

 then quickly chilled by immersion in ice-water and filtered. The precipitate 

 was washed on the filter with distilled water till the greater part of the chlorine 

 was removed. It was then dissolved in 10 c.c. distilled water with the help of 

 a little sodium carbonate and the alkalinity partially neutralized with hydro- 

 chloric acid. Complete neutralization was not feasible because of the forma- 

 tion of a precipitate. 0.5 c.c. of the faintly alkaline solution, which contained 

 some sodium chloride, was injected into a mouse of 20 gm. beneath the skin of 

 the abdomen at 3 p. m. The mouse was uncomfortable and restless at first, 

 at 3 h 45 m it became quiet and plainly quite sick; at 3 h 55 m it had a convulsion, 

 and thereafter it became evident that there was the beginning of paralysis with 

 dyspnea, paralysis being most pronounced in the hind leg; at 6 p. m. the para- 

 lysis was far advanced, though the mouse still reacted to pinching; it died 

 between 6 and 8 p. m. 



The filtrate obtained from the above precipitate was dialyzed till all the 

 sodium chloride was removed. Through the addition of wash-water and 

 through the dialysis the original volume was much increased. It was therefore 

 concentrated at a pressure of 2 mm. of mercury, barium oxide being used as 

 drying agent. Within 12 hours it was thus reduced to about the original vol- 

 ume of 15 c.c. In 1 c.c. of this solution, which gave a strong biuret reaction, 

 8 mg. of sodium chloride were dissolved to make it approximately isotonic with 

 a normal saline solution, and 0.5 c.c. injected into a mouse of 18 gm. The injec- 

 tion was made at 10 h 30 m a. m. ; at 10 h 50 m the mouse was somewhat affected 

 but still restless; at ll h 30 m a. m, paralytic effects began to be noted; at 12 noon 

 the effects were marked; at 12 h 15 m the mouse was lying on its side; at 12 h 45 m 

 it was dead. Evidently heating and dialysis had not destroyed the activity, 

 though it would appear that the solution had lost somewhat in strength. This 

 is undoubtedly due, in part, to the occlusion in the precipitate obtained with 

 acetic acid, sodium chloride, and heat of some of the toxic principle. It is 

 probably also due in some measure to the concentration of the solution and to 

 the dialysis. Other experiments have shown that concentrating solutions of 

 the venom, freed from the greater part of their protein, causes them to lose tox- 

 icity; and that dialysis also causes some loss of activity. As the investigations 

 of Cooke and Loeb have shown, the toxic principle is able to dialyze slowly 

 through membranes.* 



The remaining dialyzed solution was then treated with small quantities of 

 freshly prepared 10 per cent metaphosphoric acid solution, avoiding an excess; 

 a slight, very fine, flocculent precipitate formed, which could be removed com- 

 pletely only with the greatest difficulty. The ordinary, fine, ashless filter paper 

 used in quantitative analysis was quite ineffective. A clear, non-opalescent 

 solution was finally obtained by filtering many times through the same small, 



*Cf. the paper by Elizabeth Cooke and Leo Loeb. 



