The anticoagulant substance in the venoms of Colubridæ and Ancistrodon is precipitable by alcohol, like the coagulant substance in the venoms of Viperidæ and like the neurotoxins, from which it is difficult to separate them. The separation can nevertheless be effected by the aid of heat, if we make use of certain venoms that are particularly resistant to high temperatures, such as those of the Cobra or the Krait. These latter venoms, when heated for one hour at 70° C., cease to be anticoagulant, and preserve their toxicity unimpaired. It is, however, impossible to suppress the toxicity without at the same time destroying the anticoagulant substance.
Antivenomous serum completely protects citrate- or chloridate-plasmas against the anticoagulant action of venoms. It is sufficient to mix ½ c.c. of 4 per cent. saline antivenomous serum with 1 c.c. of 4 per cent. saline plasma to ensure that the subsequent addition of 1 milligramme of Cobra-venom to this mixture remains without effect upon the coagulability of the latter. If, after a contact of two hours or more, 2 c.c. of distilled water be added, coagulation is produced just as in saline plasma without venom.
B.—Effects of Venom upon the Red Corpuscles.
(1) Hæmolysis.—The hæmolytic properties of venoms, that is to say, their faculty of dissolving the red corpuscles, have been the subject of very important researches on the part of a number of investigators during the last few years (W. Stephens,[45] Flexner and Noguchi,[46] Calmette,[47] Phisalix,[48] Preston Kyes and Hans Sachs,[49] Noc[50]).
The different venoms are all hæmolytic, but in very variable doses. It is possible to make a very precise comparative study of them from this special point of view by taking as a base for each venom, as was done by Noc, the unital dose of 1 milligramme (or one-tenth of a cubic centimetre of a 1 per cent. solution freshly prepared and not filtered, the filtration through porcelain retaining an appreciable part of the active substance), and noting the time strictly necessary for this dose of 1 milligramme to dissolve completely, in vitro, 1 c.c. of a 5 per cent. dilution of red corpuscles of the horse in physiological saline solution.
It is very important, before allowing the venom to act on the red corpuscles, to first wash the latter by means of several successive centrifugings with 8 per 1,000 physiological saline solution.
It is also better to choose the corpuscles of the horse in preference to those of other species of animals, since they exhibit a nearly constant mean sensitivity. The corpuscles of the ox, goat, sheep, and rabbit are less sensitive. Those of man, the guinea-pig, and the rat, on the contrary, are more so.
On experimenting with washed corpuscles, it is found that venom alone is incapable of dissolving them. In order that dissolution may take place, we are obliged to add to the mixture either a small quantity of normal horse-serum, preferably heated, and, consequently, deprived of alexin (Calmette), or ½ c.c. of a 1 in 10,000 solution of lecithin in physiological saline water (P. Kyes).
Venom, therefore, is capable of hæmolysing red corpuscles only when it is quickened, either by heated normal serum, or by lecithin. The solution of lecithin employed for this purpose should be prepared by dissolving 1 gramme of lecithin in 100 grammes of pure methylic alcohol. Taking 1 c.c. of this dilution we add it to 9 c.c. of 8 in 1,000 saline solution, and make a second dilution of 1 c.c. of the foregoing mixture in 9 c.c. of saline water. This latter dilution of 1 in 10,000 is utilised as the reagent.
Let us now see how the serum or lecithin acts. It has been shown by P. Kyes that with either of these substances the mechanism of the hæmolytic action is the same, for the serum quickens the venom only through the agency of the free lecithin it contains. The lecithin takes part in the reaction by combining with the venom to form a hæmolysing lecithide more resistant to heat than its two components, for it may be heated for several hours at a temperature of 100° C., without the loss of any of its properties.