86 VENOMOUS SNAKES AND THE PHENOMENA OF THEIR VENOMS 
Starting from the biological observations made by Flexner and Noguchi, 
Kyes, under the direction of Ehrlich and partly in association with Sachs, 
finally made a very important discovery on the hemolytic constituent of snake 
venom. Flexner and Noguchi found that venom becomes hemolytic when 
there is at the same time the serum of a susceptible species, and thought it 
to be regular complements of blood serum which activate venom. Calmette 
discovered that the heated serum contains more activating principles, and all 
serum becomes, no matter whether originally venom-activating or not, com- 
plementary for venomon boiling. From these observations Kyes was led to 
discover the nature of the venom-activating principles of heated serum. It 
was found to be lecithin. Later Kyes obtained a definite compound of 
venom and lecithin, which is hemolytic by itself. This is called lecithid 
of venom. His original method of preparing venom-lecithid is given below: 
4o c.c. of at per cent solution of cobra venom in 0.85 per cent NaCl solution are 
mixed with 20 c.c. of a 20 per cent chloroform solution of lecithin in a flask of about 
100 ¢.c. capacity and then shaken in an apparatus for this purpose for about 2 hours. 
Then the whole mixture is centrifugalized for 45 minutes — 3,600 revolutions per 
minute. When the process is successful the watery portion of venom separates 
sharply from the clear chloroform portion below with a compact, whitish, narrow 
layer between the two portions. The clear chloroform portion has about 19 c.c. 
as a rule, and can be easily separated by fine pipette. Then mixing it with 5 
volumes of ether, there appears a precipitate which is the lecithid. The ee 
lecithin remains in solution in ether. 
As a rule Kyes washed the precipitate with original volume of ether 
ro to 20 times repeatedly, in order to remove all trace of lecithin from the 
lecithid. The lecithid can be preserved a long time under ether, without 
change, or it can be dried carefully, but in the latter case its solubility is 
observed to undergo change, although its hemolytic activity remains intact. 
From 1 gram of dried cobra venom about 5 grams of dried lecithid has been 
prepared. 
A very important finding is that after the preparation of lecithid, the watery 
portion lost its hemolytic property almost completely, while the neurotoxic 
principle still remained undiminished in the solution. The cobra lecithid is 
found to be hemolytic, but not fatal to animals. When injected into a 
mouse in a quantity which could dissolve 200 c.c. of the blood in vitro 
it produced only an infiltration of the site of injection. In a rabbit ro c.c. 
of a 1 per cent solution of lecithid caused an extensive infiltration when 
injected subcutaneously. 
The properties of cobra lecithid: The primary product, which is obtained 
by repeated washing in ether and compressed between filter papers, is insolu- 
ble in acetone and ether, but soluble in chloroform, cold alcohol, and warm 
toluol. From solution in chloroform and alcohol it is precipitated by ether 
and acetone. Lecithid which still contains some ether, or lecithid rapidly 
freed from ether by air-current, dissolves in water without cloudiness, and 
presents a clear, slightly yellowish solution. Its solubility in various lipoid 
