CHAPTER V.

_THE CHEMICAL STUDY OF SNAKE-VENOMS._

In the condition in which they are received on issuing from the glands, venoms always present the appearance of a thick saliva, of an oily consistency and more or less tinged with yellow, according to the species of snake by which the poison has been produced. They are entirely soluble in water, the addition of which renders them opalescent. Tested with litmus they exhibit a slightly acid reaction; this acidity, which is due to the presence of a very small quantity of an indeterminate volatile acid, disappears on desiccation, so that solutions of dried venom are neutral. The taste of venoms is very bitter. Their density, which is slightly greater than that of water, varies from 1030 to 1050.

Venoms are composed of a mixture, in variable proportions, of proteid substances, mucus and epithelial _débris_, fatty matters and salts (chlorides and phosphates of lime, ammonia and magnesia), with from 65 to 80 per cent. of water.

The elementary analysis of Cobra-venom made by H. Armstrong[7] gave the following results:--

Carbon 43·04 per cent. Hydrogen 7·00 “ Nitrogen 12·45 “ Sulphur 2·50 “ Residue Small quantities.

Not much is to be learnt from these figures; it would be of far greater importance to know the exact constitution of the proteid substances to which venom owes its physiological properties. Unfortunately, our knowledge of the chemistry of the albuminoid matters is still too imperfect for it to be possible for us to determine their nature.

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As early as 1843 it was pointed out by Lucien Bonaparte that in the venom of _Vipera berus_ the most important principle is a proteid substance to which he gave the name of _viperin_ or _echidnin_, and which he compared to the digestive ferments. Later on Weir Mitchell and Reichert, and subsequently Norris Wolfenden, Pedlar, Wall, Kanthack, C. J. Martin, and MacGarvie Smith, showed that venoms, like diastases, exhibit a great complexity in composition; that all their characteristic toxic constituents are precipitable by absolute alcohol, and that the precipitate, when redissolved in water, recovers the properties possessed by the venom before precipitation.

According to Armand Gautier,[8] venoms contain alkaloids. The latter may be obtained, in very small amounts, however, by finely pulverizing dried venom with carbonate of soda, and systematically exhausting the mixture with alcoholic ether at a temperature of 50° C. These alkaloids have yielded crystallized chloraurates and chloroplatinates, and slightly deliquescent crystallized chlorhydrates. The latter produce Prussian blue when treated with very dilute ferric salts, and mixed with a little red prussiate. They therefore represent reductive bodies analogous to ptomaines.

Norris Wolfenden did not succeed in extracting these alkaloids from Cobra-venom, whence they had nevertheless been isolated by Armand Gautier. Wolcott Gibbs, and afterwards Weir Mitchell and Reichert, likewise failed to find them in _Crotalus_-venom. The toxicity of these bases is, moreover, but very slight, for the totality of the alkaloids extracted by A. Gautier from 0·3 gramme of Cobra-venom did not kill a small bird.

It is therefore to the _toxalbumins_ that the toxic properties of venoms are essentially due.

All venoms are not equally affected by heat. The venoms of COLUBRIDÆ (_Naja_, _Bungarus_, _Hoplocephalus_, _Pseudechis_) and those of the HYDROPHIIDÆ are entirely uninjured by temperatures approaching 100° C., and even boiling for a short time. When the boiling is prolonged, or when venoms are heated beyond 100° C., their toxic power at first diminishes, and then disappears altogether. At 120° C. it is always destroyed.

The venoms of VIPERIDÆ (_Lachesis_, _Crotalus_, _Vipera_) are much less resistant. By heating to the coagulating point of albumin, _i.e._, to about 70° C., their toxic properties become attenuated, and they are entirely suppressed between 80° and 85° C. _Lachesis_-venoms are the most sensitive; their toxicity is lost if they be heated beyond 65° C.

On separating the coagulable albumins of the venoms of COLUBRIDÆ, by heating to 72° C., followed by filtration, we obtain a perfectly limpid liquid, which is no longer injured by boiling, and in which the toxic substance remains wholly in solution. The albuminous precipitate, when separately collected and washed, is no longer toxic. The clear liquid, after being filtered, is again precipitated by absolute alcohol, and the precipitate, redissolved in an equal quantity of water, is just as toxic as the original filtered liquid.

The venoms of VIPERIDÆ, when coagulated, by heating them to a temperature of 72° C., and filtered, are almost always inert. The albuminous coagula, if washed, redissolved in water, and injected into the most sensitive animals, produce no harmful effect whatever.

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The results of dialysis likewise differ when we experiment with the venoms of COLUBRIDÆ and VIPERIDÆ. The former pass slowly through vegetable membranes, and with greater difficulty through animal parchment. The latter do not dialyse.

Filtration through porcelain (Chamberland candle F) does not sensibly modify the toxicity of the venoms of COLUBRIDÆ; on the contrary, it diminishes that of the venom of VIPERIDÆ by nearly one-half.

By using a special filter at a pressure of 50 atmospheres, C. J. Martin has succeeded in separating from the venom of an Australian _Pseudechis_ two substances: a non-diffusible _albuminoid_, coagulable at 82° C., and a diffusible, non-coagulable _albumose_. The former produces hæmorrhages; the second attacks the nerve-cell of the respiratory centres.

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All venoms exhibit most of the chemical reactions characteristic of the proteids:--

_Millon’s reaction._

_Xantho-proteic reaction_ (heating with nitric acid and subsequent addition of ammonia = orange coloration).

_Biuret reaction_ (caustic potash and traces of sulphate of copper).

_Precipitation by picric acid_, disappearing on being heated, reappearing when cooled.

_Precipitation by_ saturation with _chloride of sodium_.

_Precipitation by_ saturation with _sulphate of magnesium_.

_Precipitation by_ saturation with _ammonium sulphate_.

_Precipitation by a 5 per cent. solution of sulphate of copper._

_Precipitation by alcohol._

According to C. J. Martin and MacGarvie Smith, the albumoses of the venoms of COLUBRIDÆ are _hetero-albumoses_, _proto-albumoses_, and perhaps _deutero-albumoses_ in small quantities. They can be separated in the following manner:--

The solution of venom is heated to 90° C., and filtered in order to separate the albumins coagulable by heat. The filtrate, saturated with sulphate of magnesium, is shaken for twelve hours. By this means there is obtained a flocculent precipitate, which is placed upon a filter and washed with a saturated solution of sulphate of magnesium. The filtrate is dialysed for twenty-four hours in a stream of distilled water, and then concentrated, likewise by dialysis, in absolute alcohol. Thus we obtain a few cubic centimetres of liquid, which contains a small quantity of _proteids_ in solution. These _proteids_ can be nothing but a mixture of _proto-_ and _deutero_-albumoses with peptones. That there is actually no trace of the latter can easily be ascertained.

Neumeister[9] has shown that it is impossible to precipitate all the _proto-albumoses_ of a solution by saturation with neutral salts, and, since the filtrate becomes slightly turbid when a few drops of a 5 per cent. solution of sulphate of copper are added to it, we must conclude that it contains a small proportion of these _proto-albumoses_.

The deposit retained upon the filter after washing with sulphate of magnesium is redissolved in distilled water, and dialysed for three days. An abundant precipitate then becomes collected in the dialyser. This is centrifuged. The clear liquid is decanted with a pipette, then concentrated by dialysis in absolute alcohol, and finally evaporated at 40° C. until completely desiccated. The solid residue is washed and centrifuged several times in distilled water, after which it is dried on chloride of sodium.

This method enables us to separate two albumoses, both precipitable by saturation with sulphate of magnesium, and belonging to the class of _primary albumoses_: one of these, _proto-albumose_, is soluble in distilled water, the other, _hetero-albumose_, is insoluble; but the latter can be dissolved in dilute solutions of neutral salts. These bodies are respectively identical with those obtained by the pepsic digestion of proteids.[10]

In order to study separately the local and general effects of these different albumoses, C. J. Martin and MacGarvie Smith performed the following experiment:--

They introduced beneath the skin of the belly of a guinea-pig, previously shaved and rendered aseptic, two small pieces of sterilized sponge, about 2 c.mm., one of which was impregnated with the solution of proteid, while the other served as control. The two small incisions, one on either side of the median line, were then sutured and covered with collodion. In this way the maximum of local effect and the minimum of general effects was obtained. The solutions of albumoses introduced by this method into the organism produced an enormous œdema, which, in from six to eight hours, extended along the whole side of the abdomen containing the sponge charged with poison.

To test the general toxic effects, the solutions were injected into a vein or into the peritoneal cavity. It was thus found that the _proto-_ and _hetero-albumoses_ killed the animals in a few hours.

It must therefore be concluded from these facts that the active principles of venom are _proto-_ and _hetero-albumoses_, the albumins that it contains being devoid of all toxic power.

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Many chemical substances modify or destroy venoms, and we shall see in another chapter that several of them, by reason of their properties, may be very usefully employed for the destruction, in the actual wound resulting from a venomous bite, of the venom that has not yet been absorbed in the circulation.

Among these substances the most important are:--

A 1 per cent. solution of _permanganate of potash_ (Lacerda).

A 1 per cent. solution of _chloride of gold_ (Calmette).

_Chloride of lime_ or even _hypochloride of calcium_ (Calmette), in a solution of 1 in 12, which is augmented, at the moment of use, by 5 to 6 volumes of distilled water, so as to bring it to the standard strength of about 850 cubic centimetres of active _chlorine_ per litre of solution.

A 1 per cent. solution of _chromic acid_ (Kaufmann).

Saturated _bromized water_ (Calmette).

A 1 per cent. solution of _trichloride of iodine_ (Calmette).

All these chemical bodies also modify or destroy the diastases and the microbic toxins. The venoms, although more resistant to the influence of heat, behave, therefore, like these latter, and exhibit the closest affinity with them. Moreover, like all the normal glandular juices, they possess very manifest zymotic properties, which singularly complicate their physiological action, and upon which we shall dwell later on.

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_Electricity_, employed in the form of continuous electrolytic currents passing through a solution of venom, destroys the toxicity of the latter, because under these conditions there is always formed, at the expense of the salts accompanying the venom, a sufficient quantity of chlorinated products (hypochlorites, chlorates, &c.), and a small amount of ozone, the oxidizing action of which is extremely powerful.

With alternating currents of high frequency, Phisalix, repeating the experiments that Arsonval and Charrin had performed upon diphtheria toxin, thought that he had succeeded in attenuating venom to the point of transforming it into vaccine.[11] But it has been shown by Marmier that this attenuation was simply the result of thermic actions. When, by means of a suitable arrangement, any rise of temperature was carefully avoided, no modification of toxicity was obtained.[12]

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The influence of _light_, which has no effect upon venom preserved in a dry state, is, on the contrary, very marked upon venom in solution. Solutions of venom that are destined for physiological experiments should therefore not be employed without controls, if they be several days old. Apart from the fact that, if care be not taken to render them aseptic, they very soon become contaminated with the germs of all kinds of microbes, it is found that they gradually lose a large part of their activity, especially when they remain in contact with the air. By filtering them through a Chamberland candle and keeping them in the dark, in a refrigerator, in perfectly closed phials, they may be kept unimpaired for several months.

The addition of _glycerine_ in equal parts to a concentrated solution of venom is also an excellent means of preservation.

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Phisalix has shown that the emanations from _radium_ attenuate and then destroy the virulence of Cobra- and also of Viper-venom.

“Dry Viper-venom, dissolved in _aqua chloroformi_ in the proportion of 1 in 1,000, is put up in four tubes, three of which are irradiated, the first for six hours, the second for twenty hours, and the third for thirty-six hours. Three guinea-pigs, of equal weight, are inoculated with equal quantities of the irradiated venom; a control receives the non-irradiated venom. The latter dies in ten hours; the animal inoculated from the first tube dies in twelve hours; the one inoculated from the second tube in twenty hours, and the third proves resistant without any symptom of poisoning. A second inoculation produces a transitory lowering of the animal’s temperature by half a degree. At the end of four days it dies after inoculation with a lethal dose.”

The nature of the solvent exerts a great influence upon the action of the emanations from radium: if the same experiment be performed with venom dissolved in a 50 per cent. mixture of glycerine and water, the attenuation is merely relative after six hours.

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Auguste Lumière and Joseph Nicolas, of Lyons, conceived the idea of studying the effect upon venom of the prolonged action of the intense _cold_ produced by the evaporation of liquid air.[13] The Cobra-venom employed by these investigators was in solution at a strength of 1 in 1,000. It was submitted to the action of liquid air, partly for twenty-four hours and partly for nine days at -191° C. Its toxicity was in no way diminished.

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Lastly, I must mention the recent researches of Hideyo Noguchi,[14] with reference to the photodynamic action of _eosin_ and _erythrosin_ upon the venoms of the Cobra, _Vipera russellii_, and _Crotalus_. It was found by the scientist in question that the toxicity of these various venoms is more or less diminished in the presence of these aniline colours, when the mixtures are insolated. Cobra-venom is the most resistant, just as it is in regard to the other physical or chemical agents. That of _Crotalus_, on the contrary, is the least stable.