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 haemolytic constituent of snake 

 venom. Flexner and Noguchi found that venom becomes haemolytic 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 venom on 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 haemolytic by itself. This is called lecithid 

 of venom. His original method of preparing venom-lecithid is given below: 



40 c.c. of a I 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.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 unaltered 

 lecithin remains in solution in ether. 



As a rule Kyes washed the precipitate with original volume of ether 

 10 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 solubiHty is 

 observed to undergo change, although its haemolytic activity remains intact. 

 From I 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 haemolytic property almost completely, while the neurotoxic 

 principle still remained undiminished in the solution. The cobra lecithid is 

 found to be haemolytic, 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 10 c.c. 

 of a I 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 



