The, dried venom is very similar to that of the cobra, the proteid. (From the Physiological Laboratory, University College, London.

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1 ON THE NATURE AND ACTION OF THE VENOM OF POISONOUS SNAKES. II. A NOTE UPON THE VENOM OF THE INDIAN VIPER (DABOIA RuSSELLII). BY R. NORRIS WOLFENDEN, B.A. M.D. Cantab. (From the Physiological Laboratory, University College, London.) I HAVE by the courtesy of the Surgeon-General of India received a small portion of Daboia venom. The Daboia Russellii or Russell's viper is the most typical of the Indian viperine snakes, and it is of great interest to compare the constitution of this venom with that of the colubrine snakes, or cobras. The, dried venom is very similar to that of the cobra, the proteid scales, if anything, being a little yellower in colour, and finer and more glistening than the dried cobra venom. It is, like cobra venom, soluble in water, the solution not being quite clear, (containing epithelial scales), and frothing on shaking like an albuminous fluid. It is also, like cobra venom, acid in solution. On boiling the solution, there is first an opalescence followed by coagulation. When the coagulum is filtered off, a solution is left which still contains some proteid. There is in Daboia venom therefore some proteid other than globulin or serum albumin. I will refer to this again. Passing a stream of CO' through Daboia venom well diluted, produces a fine precipitation in the fluid of globulin, preceded by opalescence. Upon dialysing the venom, there is at the end of three or four days a whitish yellow deposit of globulin on the dialyser. Neutral salts, MgSO4, NaCl, and ammonic sulphate, produce precipitates in Daboia venom. These precipitates are, like those obtained from cobra venom, very fine anid difficult to remove

2 358 R. NORRIS WVOLFENArDEN. I have obtained clear evidence of three proteids in Daboia venom, which I will proceed to discuss. 1. Globulin. Saturating the Daboia solution with MgSO4, produces at once a precipitate. The fluid was shaken for three hours and allowed to stand till the precipitate had fallen in a compact cloud. It was then filtered off, through a filter well wetted with concentrated MgSO4, and the filtrate was quite clear. The precipitate was washed with concentrated MgSO4, and finally dissolved up in water. It yielded a proteid solution, coag,ulating at 750 C. (b) A second method I have used, was to dialyse the solution for a few days and collect the small amount of precipitate from the dialyser and centrifugalise it. This is not so good a method as the saturation method. (c) I also precipitated all the proteids by ammonium sulphate added to saturation with subsequent shaking, dissolved up the precipitate in water, and submitted it to dialysis. After two or three days dialysis there was a greater amount of globulin on the dialyser than after the previous method. (d) By passing a stream of CO' through the solution diluted times with water, I obtained a small amount of globulin. There is nothing in this globulin to distinguish it from other bodies of the same name. In the three last mentioned methods the globulin was collected by centrifugalising and throwing on to a filter. Temperature of coagulation of the globulin. This is the same as that of other globulins, viz. 750 C. EXPERIMENT I. 10 c.c. of the solution of globulin prepared by saturation with MgSO4 were taken, acidified with 3-4 drops of 20/0 acetic acid, and submitted to coagulation'. Opalescence 680 C. Coagulation EXPERIMENT II. 10 c.c. of an untreated solution of Daboia venom were acidified with 3 drops of 2 0/0 acetic acid. Opalescence 680 C. Coagulation 750 C. EXPERIMENT III. 10 c.c. of the same solution were acidified with 5 drops of 2 0/n acetic acid, making a very acid solution. I used Professor Schafer's apparatus for this purpose.

3 VENOM OF DABOIA Opalescence 650C. Coagulation 730C. Excess of acidity always lowers the coagulation points of proteid solutions. At the risk of repetition I may state the reasons for inevitably concluding the presence of globulin in Daboia venom. 1. Its precipitation by MgSO4, NaCl, and (NH4)2, SO4. 2. Its precipitation by CO'. 3. Its coagulation at 750 C. 4. Its beino thrown out of solution by dialysing. 2. Serum albumin. After removing the MgSO4 precipitate and resaturating and shakina with MgSO4 in order to remove the last trace of globulin, the addition of Na'SO4 in bulk to saturation and subsequent shaking brings down a small quantity of another proteid. The amount of it is very small, just as in cobra venom, and owing to this and the difficulty of collecting small quantities of proteids, I was unable to determine the coagulation point accurately. After taking three samples of this proteid dissolved up in water (the sodic precipitate thrown on to a filter, washed with sodic sulphate and dissolved in water), I could not determine a coagulation more definitely than that there is distinct opalescence between 750o800 C. in specimens made acid with a few drops of 2o/o acetic acid. That this body is serum albumin I conclude from 1. The precipitation of the magnesic filtrate by Na2SO4. 2. The occurrence in the solutions of the soda precipitate of an opalescence on boiling, within the range of coagulation temperature of serum albumin ( C.). 3. Albumose or Syntonin. After boiling solutions of Daboia venom to 950C. and removal of the coagulated proteid there is still some proteid left in the filtrate which is not globulin or serum albumin, since I boiled the solutions three times and filtered twice or three times through double folds of filter paper. As this proteid gives a precipitate witli acetic acid and potash ferrocyanide it cannot be peptone. It will be convenient here to refer to the results of dialysis of the venom. Dialysing, a watery solution of Daboia venom, I examined the dialysate at intervals for trace of proteid. At the end of 41 hours, the outside fluid was faintly acid, but yielded no evidence of proteid, even after concentration (xanthoproteic, biuret, acetic acid and ferrocyanide, picric acid, and tannic acid tests). After 10 hours dialysis, examined again after concentration, there wvas still no evidence of proteid.

4 360 R. NORRIS WOLFENDEN. Examined at the end of 24 hours the same result was obtained. After three days dialysis there was a slight trace of proteid in the dialysate, which was also faintly acid. At the end of ten days dialysis, there was very evident proteid in the dialysate, which after concentration gave not only the xanthoproteic, but the acetic acid and ferrocyanide test and could not therefore have been peptone. The dialysate was distinctly acid. There is no peptone in Daboia venom for the following reasons: 1. Saturation of the fluid by MgSO4 and subsequently by Na2SO' removed all trace of proteid. Double saturation by these salts will not renmove peptone from solution. 2. Boiling a solution of Daboia venom after Hofmeister's method with ferric oxide, removed all trace of proteid from the solution. 3. The proteid which dialyses after prolonged dialysis is not peptone, since it is precipitated by acetic acid and ferrocyanide of potash. With regard to the proteid that is left in solution on boiling Daboia venom and removing the coagulum, it is possible for it to be either acid albumin or albumose. Unfortunately I have not yet had sufficient Daboia venom to make this clear, but I am of opinion that some portion of it at any rate is albumose. I find that the solution is i. Precipitated by acetic acid' and this precipitate is not completely soluble on adding excess. ii The addition of HNO3 produces a slight precipitate which is dissolved on warming and comes down again on cooling. iii. It is precipitated on adding NaCl to saturation thoug,h slightly. iv. It is precipitated by acetic acid and potassic ferrocyanide. These characters make it bear a strong resemblance to the albumose group. I have in a previous communication2 referred to the probability of the bodies called by Weir Mitchell and Reichert peptones, being albumoses. These bodies were found in the venoms of certain American snakes, notably Crotalus and Toxicophis (mocassin) venom3. The physiological effects of the poison. Having had only a very small quantity of this venom to work with I am not able to say very much at present on this head. I have how- 1 The precipitate produced in boiled or unboiled and filtered specimens by strong acetic acid and which is Mucin must not be mistaken for this. 2 See preceding paper. 3 Med. News, April 28, 1883.

5 VENOM OF DABOIA. 3a1 ever made some experiments with the globulin venom, which I will refer to. The ordinary symptoms of poisoning by Daboia venom are not constant and generally fall under one or other of the following descriptions. 1. Death from primary convulsions. 2. Death from convulsions and paralysis. 3. Death from blood poisoning. The local effects of Daboia poison are usually very great. In fact viper poisoning usually produces greater local injury than colubrine poisoning and severe local effects are often the result of poisoning by such a comparatively weak viper as our common English Pelias berus. Injections of a syringeful (hypodermic) of the solution of Daboia venom I used for the foregoing experiments failed to produce the customary and typical convulsions which usually follow Daboia poisoning, even in birds (which are said to be unusually susceptible to this convulsive power in the poison). Injections of the globulin were followed by severe local symptoms and chronic poisoning, followed by death and with some implication of the respiratory system. ExPERIMENT. The magnesium sulphate precipitate was dialysed for several days, the opalescent solution from the dialyser centrifugalised. The white precipitate collected and dissolved up in water. This formed a solution strongly proteid. M xv of this were injected under the skin of the back of a healthy rat at 3.10 on Nov. 10th Quiet and seems sleepy Trying to lick wound Keeps leg drawn up Quiet and drowsy Licking the wound vigorously-seems rather weak on its legs, probably because one leg kept drawn up to the body Has had one or two spasmodic jerks of the whole body, but runs about at intervals Breathing natural. Apparently well, except for the wound hurting. The animal was left and not seen until next day. Nov The animal is quiet, coat staring, breathing rapid and shallow, the leg and foot on the injected side black and quite gangrenous. It died during the afternoon. P.M. There was gangrene for considerable distance round the site of puncture of the needle, with bloody extravasations into the connective tissue of the back and the other signs were the same as detailed in the next case.

6 362 B. NORRIS WOLFENDEA. EXPERIMENT. Globulin prepared by precipitation with (NH4)'SO4 and subsequient dialysis, was dissolved up in water. A syringeful (M xx) was injected under the skin of the back of a healthy rat at 2.45 on Nov Respiration hurried-pain at the spot The animal develops a number of laryngeal spasms, and rapid breathing, which is obviously einbarrassed. This lasted until 4 p.m. when it was left. Death occurred during the night. P.M. Great pulmonary congestion. Heart contracted. Blood dark colouired and fluiid. Liver congested. Trachea containing blood-stained fluid. Round the spot of injection most remarkable baemorrhages for a great distance, of dark venous blood, and a little frothy exudation (not so nmuieh of this as in Cobra globulin). Small intestine very much stained with bile. No htemorrhage in the intestine. The globulin in these two cases did not produce the convulsions typical of Daboia poisoning, but that might have been because it was not a sufficiently strong solution. There is evidence of embarrassment of the respiratory mechanism and strong evidence to shew that it produces not only great local effects, even gangrene, but a congested condition of internal organs, as well as most rernarkable local haemorrhages. I cite these two experiments, not because I as yet claim any particular action for the globulin, but because I think they are of interest. Daboia venom has not before, I believe, been chemically examined. It appears to be difficult to get, as I have only succeeded in obtaining, one small specimen of it from India during the year. Comparison of Deboia and Cobra Venom. In my previous paper I have shewn that Cobra venom coutains three proteids, 1. Globulin, 2. Acid Albumin, 3. Serum Albumin, the latter only in small quantities however. I have mentioned also the occurrence, which was exceptional, of an albumose. It is interesting to observe that in Daboia poison we have three proteids also. In both a globulin predominates. Both contain a small quantity of serum albumin, the one, Cobra venom, contains besides an acid albumin, and the other, Daboia, a nearly allied proteid, which I am inclined to think is an albumose. The results of Weir Mitchell

7 VENOM OF DABOIA. and Reichaart in examining Crotalus, Copperhead, and Mocassin poison, point to the presence of globulins and albumoses in these venoms. True peptones do not occur in these venoms (Cobra and Daboia), and the bodies described by the American observers in their venoms, under the name of " peptone " are, as I have said before, in all probability, albumoses. The power of the venom in all these snakes appears to reside in the proteids. Why should proteids in these cases be so strongly poisonous? It would not be desirable to discuss this question now, but I may suggest a few reflections. The proteid molecule is still little understood and it is not unreasonable to suppose that in some cases its arrangement may produce a poisonous body. In peptone itself we have an example of a toxic proteid, since injections of this body into the circulation reduce the blood pressure rapidly and remarkably'. At least some viper poisons appear to do the same. Albertoni' experimenting with viper poison from Vipera Redi, concluded in the following manner: 1. Blood pressure is lowered by viper poison; and this may be both quick, and great in amount. 2. Between these blood pressure changes and quickness of death there is a certain relation. 3. If the blood pressure sinks to about 50 mm. death follows in a few minutes. Viper poison has some considerable effect upon the blood itself, and many competent observers (Sir Joseph Fayrer and others) have noted the fluid condition of the blood after death by Daboia poisoning. It is of interest to call to mind the effect of ordinary peptone when injected into an animal. The blood loses its coagulability for some time and blood pressure is notably and rapidly lowered. If one albumin can be poisonous, there is no reason why others should not be. It is not to be contended that all albumins, or all globulins, or all albumoses should be exactly alike, and there is no antecedent improbability in the supposition that a rearrangement of the albumin molecule, without altering the ultimate composition of the body, and without depriving it of any of the characters making it typical of the class to which it belongs, should make it a poison. Until we know better the meaning of the terms 'proteid,' 'poison,' we can not deny the possibility of proteids being, under certain conditions, poisonous, or harmful to the economy the I Schmidt Mulheim. Archivf. dnat. u. Physiol. 1880, Albertoni. Molesch-Untersuch. Bd. xii

8 364 B. NORRIS WOLFENDEN. balance of which hangs on such delicate and little understood factors. If we once get rid of the idea that every 'poison' must be an 'alkaloid,' we may the more easily comprehend how proteid secretions like snake venoms may become physiological poisons to the system. There is no doubt that certain albumoses are more or less poisonous. Politzer's experiments with the albumoses separated after Kiihne's methods are instructive.