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On the Nature and Action of the Venom of Poisonous Snakes: I. The

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On the Nature and Action of the Venom of Poisonous Snakes: I. The ON THE NATURE AND ACTION OF THE VENOM OF POISONOUS SNAKES. I. THE VENOM OF THE INDIAN COBRA (NAJA TRIPUDIANS). BY R. NORRIS WOLFENDEN, B.A. M.D. Cantab., F.C.S. (From the Physiological Laboratory, University College, London1.) Introductory. MANY of the hitherto known facts concerning cobra venom suggest, at least, the possibility of its toxicity being partially or wholly due to one or more proteid principles. There is, however, the possibility of its dependence upon some alkaloidal body or bodies, a view which has been taken by some experimenters. In the Report of the last Snake Commission2-an exceedingly valuable and instructive report-Mr Pedler sums up the results of his chemical examination into the nature of the poison thus: "It is quite impossible to draw any deductions as to the nature of the poison. It is more than possible that the poison is a mixture of albuminous principles with some specific poison." Sir Joseph Fayrer and Dr Lauder Brunton compared the action of cobra poison to the alkaloid conia3. It is not surprising that the albuminous con- stituents of cobra venom should have escaped examination for so long, and that attention should have been more directed to other possibly poisonous principles. Blyth, in a paper to which I shall refer further on, distinctly claims to have separated from cobra poison an extremely toxic body, which is not an albumin. Bearing on the question of the nature of this poison, I may quote the following from the Indian Snake Report: "Coagulation of the venom by alcohol does not destroy its activity, as we have shewn in our former communication. The coagulum thrown down by the alcohol is innocuous, or nearly so; but the poisonous principle remains in solution, and the alcoholic extract 1 An abstract of this paper was read before the Royal Society, Dec. 17, 1885. 2 Report of Commission on Indian and Australian Snake Poisoning. Calcutta, 1874. 3 Same report, p. 71, in course of a very valuable paper by these gentlemen in the report. 328 R. NORRIS WOLFENDEN. possesses similar properties to the poison itself. A specimen of poison was received from India in a coagulated state; but we are uncertain whether this occurred spontaneously or was produced by the action of reagents" (it was mixed with alcohol before being sent)--" it was active"'. The toxic effect of this solution was however due to the incomplete coagulation of the albumins by the alcohol; a fact which Wall's experiments prove2. "If to a solution of cobra poison absolute alcohol be added, a white precipitate is thrown down. After the precipitate has been thoroughly washed with alcohol it can be redissolved in water, and the solution produces all the effects of cobra poison." If dried cobra poison in a state of fine powder be added to absolute alcohol, and the mixture be frequently agitated, the alcohol will derive no poisonous property from the cobra poison. If, however, instead of absolute alcohol, rectified spirit be emnployed, the water in the rectified spirit is capable of taking up a certain amount of the poison. So if absolute alcohol be added to liquid cobra poison as it comes from the snake a precipitate will fall which is poisonous; but the supernatant fluid is also poisonous from some of the active agent being held in solution by the natural fluid of the venom. That this is so, can be proved by evaporating this clear supernatant fluid to a small bulk, when a further addition of absolute alcohol will produce a further precipitate of the poisonous agent. In other words, the active agent of cobra poison is precipitated by, and is totally insoluble in, absolute alcohol; but mixtures of alcohol and water are capable of dissolving a certain amount of the poison in proportion to the quantity of water present. This is precisely what we should expect of an albuminous fluid, and is of itself a strong argument in favour of the proteid nature of cobra venom. Of the earlier literature of snake poisons I do not propose to speak. Anyone interested in this will find an exhaustive and very interesting paper embodied in the report, and written by my friend Dr Vincent Richards, a member of the last Snake Commission. The extremely valuable labours of Sir Joseph Fayrer and Dr Lauder Brunton have scarcely dealt with the chemico-physiological side of the question. Drs Weir Mitchell and Reichert in America, working chiefly with the venoms of American snakes, Crotalus adamanteus, Toxicophis 1 Indian Snake Commission Report, Calcutta, 1874, p. 67. 2 Indian Snake Poisons, p. 131. COBRA VENOM. 329 piscidorus (the Mocassin), and Agkistrodon contortrix (the Copper-head), have made a preliminary report of a very interesting nature. They find that Crotalus venom is destroyed at 800C.-except in Crotalus durissus -and that this venom attacks the heart first, and lowers blood pressure very considerably, and also causes often "mortal enfeeblement" of the respiratory centres; preventing coagulation of the blood and causing local haemorrhages. From Crotalus and the Mocassin they obtain three proteids. One is not coagulated at 1000C. and is obtained by boiling and filtration, or by dialysis. These characters indicate, according to them, that it is a peptone, "of which it presents all the reactions." They further state that three of these reactions distinguish it from all hitherto known peptones. They are 1. The precipitate obtained by very dilute acetic acid, is redissolved in excess of the acid. 2. The precipitate obtained by NaCl, redissolves on adding glacial acetic acid. 3. The precipitate formed on adding Liquor Potassa, redissolves on adding HNO3, forming a yellow liquid, decolorising on further adding acid. This substance dried (from the Mocassin) at a little below 1000C. is made completely insoluble in water. The peptone of Crotalus, obtained by dialysis and dried at 400C. redissolves entirely in distilled H20, and preserves its toxicity and reactions. It is impossible not to think that these observers have been dealing with some body or bodies of the Albumose group. No peptone can possess these characters, and it is no evidence of peptone that it will dialyse, since Albumoses possess this property. I have met with a trace of a body, which being precipitated by HNO3 in the cold, and dissolving again on heating (a process which could be repeated many times) in a solution of cobra venom, suggested an Albumose in this venom. These authors also speak of a globulin in Mocassin venom, which is dissolved by boiling instead of being coagulated, and which is soluble only in concentrated acids or alkalies. This body cannot possibly be a globulin, since it is contrary to the nature of such a body to be dissolved on heating. Albumoses, like peptone, and acid or alkali albumins, are left in solution, after boiling, and removal of the coagulable proteids, and it is probable that these observers have been dealing not with globulin or peptone at all, but with some body or bodies intermediate between true albumins and peptones. 330 R. NORRIS WOLFENDEN. The essentially interesting and important conclusion to be derived from their work is, that the venom is resident in the proteid con- stituents. The fact stated by Weir Mitchell and Reichert, that the poisons of the Rattlesnake, Copper-head and Mocassin are destroyed by Bromine, Iodine and Hydrobromic Acid (330/), is explained by the fact that albumins are completely precipitated and coagulated by these bodies', and lends one more proof to the theory that those venoms are proteid in their nature. The first chemical examination of snake venom of any particular value was made in 1843 by Prince L. Bonaparte. He investigated the virus of Pelias berus, an adder, and found that the essential poison was resident iD the coagulum obtained by alcohol. The alcoholic precipitate, freed fromn alcohol, fat and salts (by ether and acidulated water), and finally dissolved in water and dried down to a solid residue, formed the body which he named Echidnin or Viperine2. Though it has been impossible for me to get a reference to this paper except at second hand, I believe however that Prince Bonaparte considered this poisonous principle to be alkaloidal. Weir Mitchell in 1861 working with rattlesnake venom obtained an "albumi- noid body," coagulated by heat, and from the filtrate an "albuminoid body," which he named Crotalin, and which he obtained by treating the filtrate from the boiled venom, by alcohol. Dr Armstrong in 1873 made the following analysis of cobra poison3. Crude poison. Alcohol Alcohol Albumin precip. extract. for comparison. (Ralfe) Carbon 43-56 . 4576 43.04 53-5 53.5 Nitrogen 4030 ..4.3...1430 12-45 15.7 15.5 Hydrogen .6..6....O6.60 7 O 7-1 7 0 Sulphur ..... 25 ... ... 1256 Oxygen ............ ... ... ... 22-0 Phosphorus ....... ... .. ... 0 4 1 I find that chlorine gas, purified by passing through a wash bottle, or aqueous bromine, completely and rapidly precipitates all albumins from solution. Iodine does the same but less readily. Hydrobromic acid also precipitates proteids. I have employed chlorine and bromine to estimate proteids quantitatively and find it is quite as accurate as the alcohol and boiling methods. 2 Quoted from paper by Sir Joseph Fayrer in Proc. Med. Society of London, a paper of great interest (1884, pp. 156-180). In the proceedings of the same society for 1884 vol. vi. is also an interesting and important paper by G. Baddaloni on viper poison. 3 Indian Snake Commission Report, p. 42. COBRA VENOM. 331 Mr Pedlerl estimated the carbon at 52.87o/o, Hydrogen 71o/o, and Nitrogen 17 58o/%. The liquid poison contained 27.74%/o of solid matter, and the ash amounted to 6.68%/,.
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