The Journal of Physiology - 1892 - Kanthack - The Nature of Cobra Poison

THE NATURE OF COBRA POISON. By A. A.
KANTHACK,
John Lucas Walker Student in Pathology, Cambridge University.
PART I.
IT has been known for some time past that the active principle of cobra
poison is an albumose. But, so far as I am aware, no systematic inves-
tigation of this albumose has ever been made. A good opportunity
having offered itself, I began the following experiments, applying the
methods which were so successful in the hands of Dr Martin and
Mr Hankin, to separate the poisonous substance of the snake-poison.
The latter was obtained directly from the snake by applying
pressure to the head and thus squeezing the poison out of the glands.
By alternately pressing first one side and then the other, both glands
may be emptied separately. The first drop was always rejected, so as
to avoid the poison which had stagnated in the ducts. The snakes were
fresh and active and brought up to the laboratory immediately they
were caught; for it is believed that captivity materially lessens the
secretion and toxicity of the poison. The black variety of Naja Tripu-
dians was used, and other varieties have not been employed. I need
not remark, that in these matters I was assisted by a native snake
charmer whom I took into my service.
The poison is a "clear, transparent fluid varying in colour from
a yellow or straw tint to complete colourlessnessl." According to Wall
it has an acid reaction. I found, however, that if the first flow be
carefully avoided and rejected, the reaction is neutral or faintly
alkaline. It is very viscid and soon dries, " leaving a yellow substance,
easily pulverizable, resembling gum arabic or dried egg albumen,
behind2." It gives a beautiful biuret reaction, and with nitric acid a
1 A. J. Wall. Indian Snake Poison.. Chap. v.
2 A. J. Wall, loc. cit.
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NATURE OF COBRA POISON. 273
precipitate, almost wholly soluble on heating and reprecipitated on
cooling, showing the presence of other proteids besides albumoses. On
dialysis a portion of the toxic albumoses passes through the membrane,
but very slowly. Prolonged boiling destroys the activity of the poison,
but this will be more fully discussed at another place.
Preparation of the Albumose.

The first step was to prepare the albumose in a pure state and to
enquire whether one or more albumoses were present. For this
purpose four methods were used.
(1) Martin's method'. The poison was slightly diluted with steri-
lised water and then thrown into a large excess of alcohol, and allowed
to stand for a week. The alcohol was separated off and the white
precipitate washed with absolute alcohol, dissolved in sterilised dis-
tilled water and once more precipitated by alcohol. The resulting pre-
cipitate was allowed to stand under alcohol for a week, then washed
with alcohol and dissolved in water, to which a little thymol had been
added to prevent putrefaction. The solution was found to contain no
other proteids besides albumose.
(2) Hankin's method2. The solution of cobra poison was saturated
with neutral ammonium sulphate and the mixture allowed to stand for
several days. The white precipitate was dissolved in water and
dialysed, until all trace of the salt had disappeared. The solution was
then concentrated by dialysis against absolute alcohol and subsequently
poured into a large excess of alcohol and treated as under (1).
(3) As Wall3 has shown, that cobra poison is destroyed by
prolonged boiling only, the albumin was almost entirely got rid of by
momentary boiling and filtering. The filtrate was precipitated by
ammonium sulphate and treated according to Hankin's method.
(4) The solution of cobra poison was placed into a dialyser, thymol
having been added to the water to counteract decomposition, and after
ten days most of the albumose had passed through. The solution was
concentrated by dialysing against absolute alcohol and then a large
excess of alcohol added. The precipitate which resulted was treated as
under (1). Of these four methods Martin's is the safest and quickest,
and also gives the greatest quantity of albumose.
1 XIX. Annual Report of Local Government Board. 1889-90, p. 238.
2 Brit. Med. Journ. July 12, 1890. 3 Op. cit.
18-2
274 A. A. KANTHAACK.
Nature of the Albu.rnose.

(a) The fresh proteid precipitate, whether obtained by the addition
of absolute alcohol or ammonium sulphate, is amorphous and white, but
when dried slowly, it forms a translucent colourless mass.
(b) Its solution is colourless or slightly opalescent, and neutral or
alkaline to litmus-paper.
(c) It is very soluble in water, and quite insoluble in alcohol.
(d) It gives a brilliant biuret reaction with caustic potash and a
trace of cupric sulphate.
(e) Nitric acid gives a precipitate, soluble on heating, and coming
down on cooling, if the solution is of sufficient concentration. With
dilute solutions a precipitate is not obtained until a trace of sodium
chloride has been added.
(.f) Picric acid gives a precipitate, dissolved on heating, but
coming down again on cooling.
(g) Boiling produces no physical changes.
(h) Saturation with ammonium sulphate gives a precipitate.
Letting the fluid stand for forty-eight hours and filtering off the pre-
cipitate, the clear filtrate gives no biuret reaction, while a solution of
the white precipitate reacts beautifully. On adding acetic acid to
the filtrate no precipitate is obtained after the further addition of
ammonium sulphate.
(i) Saturation with sodium chloride causes a precipitate. The
filtrate gives no biuret reaction, nor a precipitate on the addition of
acetic or nitric acid.
(le) The solution gives the ordinary proteid reactions, the colour
with Millon's reagent being less bright, and more of a pinkish yellowish
hue.
From these reactions there can be no doubt, that the substance in
question is an albumose, and that only one, viz. a primary albumose,
is present.
Does Cobra poison contain an alkaloid?
The next point to settle was whether besides the proto-albumose
the poison contains an alkaloid. The best procedure is to dry the fresh
poison at 400, powder it carefully and extract it with alcohol (95°0I).
The powdered dried poison was put into a test-tube and by means of a
mechanical contrivance shaken, more or less uninterruptedly, for forty-
NA TURE OF COBRA POISON. 275
eight hours. Then the mixture was filtered, and the filtrate, which gave
no biuret reaction, evaporated to dryness at 400 C., and the slight
residue redissolved in alcohol (95 o/0). This was repeated two or three
times, so as to remove as much of the proteid as possible. The final
solution in alcohol gave none of the albumose reactions and with
platinic chloride a turbidity and slight precipitate, which was very
light and on filtering completely absorbed by the filter-paper, so that no
further examination could be made. Other tests for alkaloids were not
performed, partly because it was impossible in Simla to procure the
necessary reagents, partly because the animal experiments with this
substance proved to be negative, as will be shown below. Altogether
I am inclined to deny with Norris Wolfenden' the presence of an
alkaloid.
Physiological Action of the Albumose.
The effect of cobra poison is so well known, th.'(need not dwell
on it here. Experimenting with the albumose it was found that
(1) A solution of the sarne acted in exactly the same manner as
the natural poison;
Amount injected. Site of injection. Result.
(1) Min. iii. of a solution peritoneal cavity died in

of albumose of rabbit 15 min.
(2) ditto ditto died in
(3) ditto ditto 30 min.
~~subcutanieous
ditto tissue died
died in
in
(4) ditto
(3)(4) ~~~~~ofa rabbit 60 mmn.
(5)dditto
itto(5) ditto ~~~~~~died
~~~~~70 in,
min.
(6) Min. iii. of natural peritoneal cavity died in
poison (diluted) of rabbit 30 min.
(7) (7 ditto subcutaneous tissue died in

ditt. Wolfndn Thisof a rabbit mViII
90
1R. N. W oIf e n d en. This Journal, Vol. vii.
276 A. A. KANTHA CK.
(2) injected into the peritoneal cavity of rabbits it acted much
more rapidly than when injected subcutaneously.
Thus three minims of a solution of the albumose injected into the
peritoneal cavity of three rabits, killed them respectively in 15, 20 and
30 minutes, while injected subcutaneously into two rabbits, it did not
kill them in less than from 60 to 70 minutes. The same result was
obtained with the natural poison. A dilute solution of poison was made
and three minims injected subcutaneously into a white rabbit which
died in 90 minutes, while three minims injected into the peritoneal
cavity of a rabbit killed it in 30 minutes.
The most noticeable fact was, that on injecting the poison or
albumose into the peritoneal cavity paralysis of the extremities,
especially the hind legs, set in with great rapidity, while on injecting
the poison or albumose subcutaneously, these symptoms seldom
appeared, the animal simply dying of asphyxia.
(3) The accumulative action of the poison or albumose is well
marked. A weak solution of cobra poison was made by adding a
solution of thymol ( ) to it. Eight minims had no effect on a
rabbit, when injected subcutaneously at one sitting. A rabbit was
taken and on the first day two minims were injected, on the second day
four minims, on the third day six minims and on the fourth day eight
minims, when the animal died after eight hours. To another rabbit
eight minims were given and after twenty-four hours again eight
minims, and it died in five hours. Lastly two and a-half minims were
injected into the subcutaneous tissue of a fowl, and after half an hour
this was repeated. The fowl died 30 minutes after the second dose, or
one hour after the first injection, while on giving five minims at one
injection death did not occur under two hours.
Making a solution of the albumose, it was found that four minims
killed a fowl in 84 minutes, while if given in separate doses of two
minims with an interval of 10 minutes, the animal died within 35
minutes after the first injection.
These experiments establish, that a more toxic action is produced
by injecting at intervals smaller doses. This has also been established
by Sewall' with regard to rattle-snake venom. A similar fact was
demonstrated by Martin' with regard to the anthrax albumose. The
difference between his albumose and the cobra albumose is, that the
latter is extremely active, very mjinute doses sufficing to kill an animal
without fail.
1 This Journal, Vol. viii. *2Loc. cit
Animal. Operation. Result. Control experiment.
lst day Min. ii. of dilute cobra poison well To control animal gave
(1) Rabbit 2nd day Min. iv.
3rd day Min. vi. ,,
,, ,,
,, ,,
,, well
well Min. viii. ofone
solutionat same
thesitting:
4th day Min. viii. ,, ,, ,, died in 8 hours alive and well
To control animal gave

lst day Min. viii.
(2) Rabbit 2nd ,,,, ,,,, ,,,, well Min. viii. of the same
day Min. viii. died in 5 hours solution atonesitting:
alive and well
To control animal gave

Min. ii. s. at 11.30 a.m. , , died at Min. v. of the same
Min. ii. ss. at 12 ,, , , 12.30 p.m. solution at one sitting:
died in 2 hours
(4) Fowl Min. ii. ss. at 11.35 a.m. , l died at cf. above
Min. ii. ss. at 12.5 p.m. ,, ,, 12.25 p.m.
Effects of Physical Influences on Cobra Poison.

(1) Effects of heat. It was shown by Martin that anthrax albu-
mose boiled for twenty minutes loses its toxic properties, and Dr
W. Hunter has demonstrated, that the action of the tuberculin
albumose is lessened, if exposed to a temperature above 70° C. in a dry
condition. Wall has shown "that a temperature of 100i C. has a dis-
tinct effect in lessening the strength of cobra poison, but that a very
great heat continued for a very long time is required to completely
destroy the properties of a concentrated solution. Dilute solutions,
however, are destroyed with much greater ease." It was found, that
concentrated solutions withstood boiling in a water-bath for one or two
hours, before they lost their poisonous action entirely, though 40-60
minutes sufficed to lessen it considerably. A weak solution, however,
could be destroyed in half an hour or twenty minutes. The same
results are obtained with a solution of the pure albumose. If the latter
be free from any other proteid, no precipitate falls on even prolonged
boiling, though the solution in some instances becomes slightly more
opalescent. When, however, a solution of cobra poison is heated, it
first becomes cloudy and then a white precipitate falls. This, of course, is
due to the albumin contained in the poison, as it issues from the glands.
Heat then lessens the action of the albumose, but it requires
prolonged boiling to destroy the pure and concentrated albumose. The
278 A. A. KANTHA UK.
remarkable point is, that though the poison loses its toxic properties
after boiling for two or four hours, its solution still gives the biuret
reaction at the end of this time. Boiling ordinary cobra poison, diluted
with water, momentarily, and filtering, the clear filtrate is as toxic as
before, showing that the albumin is only an impurity. Again after
heating a solution of natural poison and filtering, acetic acid gives no
precipitate, whether caustic potash has been previously added or not.
Animal. Experiment. Result. Control.
Min. v. of unboiled so-

(1) Ordinary Min. v. of concentrated
solution of albumose
alive
and lution killed a rabbit
rat boiled two hours well in 50 mins.
Min. v. of concentrated
(2) ditto solution of albumose do. cf. (1)
boiled one hour
Min. v. of concentrated died in

(3) ditto solution of albumose 65 min. cf. (1)
boiled up at once
These three examples may suffice.

(2) Diffused light has no influence on a solution of the albumose or
the natural poison, as they may be kept for 8 weeks before a light
window, without losing any of their deadly power. They thus differ
materially from tetanin'.
(3) The effect of bright sunlight could not be tried on account of
the season of the year, and this must be reserved for a future time.
Effect of Chemical Reagents on Cobra Poison and its Albumose.
Most of these experiments were performed with the natural snake-
poison. A standard solution was made by diluting with thymol the
fresh poison squeezed out of the glands.
In this manner the result of various reagents can be better gauged,
as the solution may be made of a certain strength, so that a certain
number of minims kill an animal in a certain time. The solution was
made of such strength that one minim killed a rabbit in four hours.
To effect this, about 50 to 100 volumes of thymol must be added to one
1 B. Kitasato. Zeitschr. f. Hyg. x. No. 2.
volume of poison, demonstrating well the extreme toxic nature of the
albumose.
(1) Chlorine water (freshly prepared): Cobra poison was diluted
with chlorine water so as to contain the same percentage of poison as
the thymol solution. Letting the mixture stand for 24 hours and
injecting 1 minim into a rat, the animal died after 24 hours, while the
control rat died after an hour. The remainder was allowed to stand for
further four days, and after this interval had lost its toxic properties, as
two and four minims injected into two rabbits respectively had no
effect on them, and a rat remained perfectly well after so large a dose
as four minims.
(1) Ordinary Mi. i. of chlorine mix- died after died after an

rat ture which
24 hours had stood 23 hours hour
Min. of chlorine
ture iv.which mix-
had stood . and
(2)ra alve died n
rat 6 ds well 30 mdy.
Min. ii. of chlorine mix- alive and died in
(3) Rabbit ture which had stood well 1 hour
4 days
Min. iv. of chlorine mix- alive and died in
(4) Rabbit ture which had stood well 50 min.
5 days
Chlorine water, therefore, if allowed to act sufficiently

long on cobra poison, destroys its poisonous action com-
pletely. The unfiltered chlorine mixture always gave a brilliant
biuret reaction.
(2) Trichloride of Iodine (10 per cent.). To five minims of the
standard solution 2 c.c. of trichloride of iodine were added and the
mixture allowed to stand for 24 hours and injected into a fowl. The
latter remained perfectly well, while a control hen died quickly. On
account of the yellow colour due to the trichloride, the mixture was not
tested for albumose by means of the biuret reaction. While, however,
chlorine water causes a well-marked turbidity, the solution remains
perfectly clear on adding the trichloride.
(3) Carbolic Acid (10 per cent.). On adding carbolic acid to a
solution of the natural poison, a distinct cloudiness was observed. The
resultant liquid was filtered, after being allowed to stand at least 24
hours. The filtrate was harmless. The addition of carbolic acid to the
pure albumose solution as a rule caused no turbidity or precipitate, and
delayed the toxic power.
Min. iii. of a strong pure died in 2 hours;

albumose solution mixed (also symptoms died in
(1) Fowl with carbolic acid (10 per of carbolic acid 35 un.
cent.) and allowed to stand poisoning)
48 hours
(2) Fowl Min. iii. of the same carbolic died in died in

acid mixture free from acid 21 hours 30 min.
Min. v. of natural poisonI

diluted with 10 per cent. alive and died in
(3) Rabbit solution of carbolic acid and well 55 mm.
allowed to stand for 48
hours (filtered)
Carbolic acid thus delays the action of the poison con-

siderably, and if the latter be not too concentrated destroys
it altogether. It does not, however, interfere with the biuret
reaction.
(4) Permanganate of Potassium' added to the standard solution of
cobra poison destroys its toxic property after 24 to 48 hours. A brown
precipitate appears, and the filtrate, which is almost colourless-unless
an excess of permanganate has been added-does not give a biuret
reaction.
(5) Solution of Nitrate of Silver causes a precipitate, when added
to a solution of pure albumose, and the filtrate obtained, after letting
the mixture stand for 24 hours, is harmless.
(6) The same result is obtained on adding a solution of corrosive
sublimate to cobra poison and allowing the mixture to stand for
24 to 48 hours. The filtrate injected into an animal is not toxic.
(7) Tannic Acid was added to a solution of the pure albumose and
the resultant mixture kept for 48 hours and then filtered. The filtrate
Cf. A. J. Wall, loc. cit.
was quite harmless. If, however, the solution is not filtered the lethal
property is not destroyed, though it may be delayed.
(8) Caustic Potash. The effect of caustic potash has been care-
fully and accurately described by Wall. His experiments have been
repeated, the natural poison as well as the albumose being used.
Five minims of the standard solution were mixed with eight minims
of pure caustic potash and injected at once into a fowl: however with no
effect. On adding four minims of acetic acid to this alkaline mixture
the toxic properties are re-established, though there is some delay of
action. Caustic soda may be employed instead of potash. " Thus
cobra-poison mixed with a solution of potash can be injected
into the system without producing any symptoms, but when acetic
acid is added to this solution all the powers of the poison are
restored." "This change is not due to the fact that the solution is
rendered alkaline, for it is not necessary to completely neutralize
the alkaline mixture by acetic acid." Wall remarks, that by adding
ammonia to snake-poison no alteration in the symptoms is produced.
To test this, five minims of a solution of the pure albumose were taken
and ammonia added, and after 48 hours injected into a fowl without
any bad effect. Taking a strong solution of natural cobra poison (three
minims) and adding three minims of the pharmacopeial liquor
ammonia, fortior and injecting the mixture at once into a fowl, death
did not occur before 4 hours and 45 minutes, while without the
ammonia a fowl died in less than an hour. My results therefore differ
from those of Wall. Again mixing a concentrated solution of the pure
albumose with ammonia and allowing it to stand for 48 hours and
injecting it into a fowl, the latter died after three hours, while the
control hen succumbed in 35 minutes. Ammonia therefore has a
decidedly retarding influence over the active principle of cobra-poison.
On the other hand, as Wall rightly pointed out, if the caustic alkali
is allowed to act for a long time, from 24-48 hours, the poisonous
power is permanently lost and not restored by acetic acid. He says:
"the loss of power is found to be accompanied by a physical change;
for when the solution is no longer poisonous, acetic acid fails to throw
down any longer a white precipitate." He argued, that the poisonous
agent of cobra-venom is an albumin, and that the venom remains
active, as long as it is possible to recognize the albumin by chemical
tests. This explanation, however, cannot be correct, as there is no
doubt whatever that the active principle is an albumose and as such
not thrown down by acetic acid. He worked with natural poison only,
282 A. A. KANTHACK.
and as this also contains ordinary albumin, he was led to a wrong
conclusion. Experiments were made to prove the error of his observa-
tions. 0
A solution of natural cobra-poison was heated, until no further

precipitate appeared, and the fluid was filtered. Acetic acid caused no
precipitate or turbidity in the clear filtrate. The filtrate was highly
poisonous, but caustic potash counteracted the toxic effect completely.
Adding caustic potash to the fresh filtrate and then also acetic acid, no
precipitate appeared and yet the fluid was poisonous after the addition
of the acid. The appearance of a precipitate with acetic acid cannot
therefore be considered a criterion of the persistence of the activity of
the poison.
Likewise on adding acetic acid to a solution of the pure albumose,
before or after the caustic potash has been mixed with it, a precipitate
is not obtained and yet the effects are the same, that is, the alkali
immediately destroys the toxic power, but the latter may be restored
by acetic acid, if added before 12-24 hours have elapsed.
A few animal experiments will now be given in illustration.
Ainimal. Experiment. Result. Control.
Min. v. of solutioni of cobra-

(1) Fowl poison + Min. viii. of KOH alive and well
injected immediately
(2) Fowl Ditto, but also + Min. iv. of died in 6 hrs. and died in 1 hour
acetic acid 20 min. and 15 min.
Min. x. of same solution of
(3) Rabbit poison + 2 c.c. of KOH al- alive and well
lowed to stand 24 hrs.; then
added 1 c.c. of acetic acid
Solution of albumose (Min.
(4) Fowl v.) + KOH injected imme- alive and well
diately
(5) Fowl Ditto, but also + acetic acid died in 4 hours died i
Solution of albumose (Min.

(6) Fowl v.)+KOH,allowedtostand alive and well
45 hrs., then + acetic acid
NATURE OF COBRA POISON. 2W83
It is very difficult to explain this action of caustic potash or soda on
the cobra-poison, for even after having allowed the albumose to be
acted upon by the alkali for 24 hours, the biuret reaction is still
obtainable, and it is also present after 48 hours, though fainter. Again
adding caustic potash to a solution of pure albumose and then also
nitric acid, the typical reaction for proto-albumose is well marked,
whether acetic acid has been previously added or not.
(9) Ammonia. The effect of ammonia has already been mentioned
above, and a few words will suffice.
Min. v. of solution of pure

(1) Fowl albumose+ammonia and al- alive and well died in 3 hrs.
lowed to stand 48 hours
Min. iii. of strong solution of died in 10 min.
poison + Min.subcu- in
(2) Fowl natural
of ammonia injected xxx. undertosymptoms
due the am- diedmin.
35
taneously at once monia
Min. iii. of same solution as died in
(3) Fowl (2) + Min. iii. of ammonia died in 5 hrs. 35 mm.
injected at once.
(4) Fowl Min. xxx. of ammonia with- died in 10 mm.

out poison
Min. iii. of same solution as died in
(5) Fowl (2) + Min. iii. of ammonia, died in 3 hrs. 30 mi.
allowed to stand 48 hours
Min. iii. of same solution as
(6) Fowl (2) + Min. xxx. of water al- died in 50 min.
lowed to stand 48 hours
Ammonia therefore materially lessens the toxic power of cobra-

poison or its albumose, and if the solution be weak enough, it destroys
it altogether.
(10) Acetic Acid when added to a solution of pure albumose, as
was mentioned above, causes no precipitate, and so far as I could find
out, also did not affect the toxicity of it. For even after allowing the
mixture to stand, it killed a fowl in two hours; and the control animal
also died in two hours.
284 A. A. KANTHAACK.
(11) Alcohol precipitates the albumose, and it is found, that the
filtrate is quite harmless. But if injected unfiltered, the mixture is as
toxic as a pure solution of albumose.
(12 and 13) Citric ancl Lactic Acids caused no change in the
physical and toxic properties of the poison or its albumose, though the
former seemed to delay the effect somewhat, if it be permitted to judge
from a single experiment. For without citric acid a fowl died in 40
minutes, while after the addition of a crystal of acid to the solution of
the poison and allowing the solution to stand 24 hours, a hen succumbed
after an hour and 45 minutes.
(14) Pancreatin. Twenty minims of a solution of poison, of such
strength that two minims would kill a rabbit in 2-4 hours, were diluted
with 8 c.c. of sterilised distilled water and a little pancreatin (Burroughs
and Wellcome) added to it, and the mixture kept at 400C. for 24
hours; 4c.c. of the filtrate injected into a rabbit were quite harmless.
The remainder of the filtrate gave a beautiful biuret reaction and a
precipitate with nitric acid, dissolved on heating and reprecipitated on
cooling. To check this result three minims of a strong solution of
poison were treated with pancreatin for 24 hours and injected subcu-
taneously into a hen, which died after 4 hours and 30 minutes, while
the control animal succumbed in 45 minutes. Pancreatic digestion,
therefore, has a decided effect on the poison.
(15) Pepsin (pharmacopeial), on the other hand, delays the effect
of the albumose only to a slight extent.
These chemical bodies were tried partly empirically, partly with the
idea of deciding whether this destroying action, if such exist, was due
to a specific effect on the albumose. The mineral acids were not tried
as in concentrated solutions they cause a rapid decomposition and
severe local changes. The bodies which destroy the toxicity of cobra-
poison evidently act in two ways: (a) by precipitating the albumose, so
that the filtrate is free from the toxic agent, (b) by causing molecular
changes in the albumose, which, as is well known, is a highly sensitive
body.
A list will now be given of the bodies tried in the above experi-
ments, arranging them according to their destructive influence over the
poison.
Reagent8 which destroy or lessen the toxic power of cobra-poi8onl.
Reagents. Effect. Remarks.
(1) Caustic potash destroys the toxic power } on addition of

(2) Caustic soda rapidly acetic acid the
toxic power is
restored
(3) Chlorine water destroys the toxic power,
if allowed to act long
enough
(4) Trichloride of iodine destroys the toxic power,
if used strong enough
(5) Permanganate of destroys the toxic power,
potash if allowed to act 24 hours
(6) Carbolic acid destroys the toxic power
of weak solutions
(7) Pancreatin destroys the toxic power
or delays the toxic effect
considerably
(8) Nitrate of silver destroys the toxic power
by precipitating the al-
bumose
(9) Corrosive sublimate ditto
(10) Tannic acid ditto
(11) Alcohol ditto
(12) Ammonia lessens the toxic power,
if allowed to act long
enough
(13) Citric acid lessens the toxic power
somewhat
(14) Pepsin lessens the toxic power
slightly
EXPERIMENTS TO ESTABLISH A PROTECTION OR CURE AGAINST

COBRA-POISON.
A. Protection and Immunity:
(1) By means of Tolerance. Sewall has shown, "that it is possible
by injection of a few minute doses to give pigeons such tolerance of
rattle-snake venom, that three months after the treatment, they were
This table should be compared with a similar one given by B. Ki4asato in his
" Experimentelle Untersuchungen uiber das Tetanusgift," loc. cit.
able to stand what would otherwise be seven times the lethal dose."
It is also known, that animals may be protected against tetanus by
means of establishing a tolerance against tetanin, which probably
is also an albumosel.
Animals may in like manner be accustomed to withstand large
doses of cobra-poison. But in such experiments it is necessary to allow
a sufficiently long interval to elapse between successive doses, as the
accumulative effect of the cobra-albumose is well marked. This is
clearly shown by the following experiments:
EXPERIMENT I. Eight minims of a solution of poison were not sufficient
to kill a rabbit, when injected subcutaneously. But on giving it another
eight minims after an interval of 24 hours, it died within 5 hours.
EXPERIMENT II. Eight minims of the same solution were injected
subcutaneously and again after a week. The rabbit remained well. This is,
of course, not an instance of tolerance, as the dose given was, under ordinary
circumstances, not sufficient to kill the animal.
Again it is necessary to commence with small doses and increase
them gradually.
EXPERIMENT III. Of the above solution, at intervals of 24 hours, the
following doses were injected: two minims, four minims, six minims, eight
minims. After the last dose the rabbit died within 8 hours.
With the experience gained by these preliminary experiments,
three animals were prepared and accustomed to tolerate large doses.
The whole treatment occupied ten weeks. Two rabbits and a hen were
employed. It will suffice to give details of one case only to demon-
strate the method employed.
EXPERIMENTS IV.-VI. (Two rabbits and a hen.)
Days.
1-4. A daily dose of two minims of same poison as was used in the
above three experiments for four days.
5 & 6. Two days' rest.
7-9. A daily dose of three minims of same poison for three days.
10-13. Four days' rest.
14-16. A daily dose of four minims of same poison for three days.
21 & 22. A daily dose of five minims of same poison for two days.
23. One day's rest.
24. Six minims of same poison.
25. One day's rest.
Tizzoni und Cattani, Centralbl, d. Bakteriologie und Parasitenkunde, 1891. 6.
26. Six minims of same poison.
27& 28. Two days' rest.
29. Twelve minims of same poison.
34. Two minims of poison ii. (i.e. sufficient to kill a rabbit in 1-4 hrs.).
39. Three minims of poison ii.
44. Three minims of poison ii.
49. Four minims of poison ii.
54. Five mimims of poison ii.
59. Seven minims of poison ii.
64. Eight minims of poison II.
69. Eight minims of poison ii.
74. Twelve minims of poison ii.
79. Twenty minims of poison ii.
84. Twenty-five minims of poison ii.
85-98. Two weeks' rest.
99. Five minims of strong cobra poison solution-less than a snake
would inject with a bite. The animal died after a few hours.
It is therefore impossible to establish an immunity against the bite
of a cobra in this manner, as all the three animals, treated by the same
method, succumbed when tested with a strong solution of poison.
They can however be accustomed to resist large doses, for all the
control animals succumbed to poison Ii.
(2) By means of Cobra-Blood or Serum. Waddell, has shown
that a cobra is unaffected by its own poison. Judging from the
successes of Behring and Kitasato, Hankin and others, it was
thought just possible that in the previous treatment with cobra-
serum a protection against the venom might be found. The results
of all experiments, however, were negative, whether the serum
was given simultaneously with, or some time before, the poison.
L. A. Waddell, Scientific Memoirs by Med. Officer of the Army of India, 1890. Iv.
pHI. XIII. 19
The following experiments will elucidate this.
EXPERIMENT VII. A small quantity of a solution of poison was mixed
with 10c.c. of fresh cobra-blood and injected into a white rat: it died in
37 minutes: the control animal died in 55 minutes.
EXPERIMENT VIII. 10 c.c. of cobra-serum were injected into a rabbit
and 24 hours later ten minims of the same poison, as used in the previous
experiment, injected subcutaneouisly; it died in 45 minutes: the control
animal died in 45 minutes.
EXPERIMENT IX. To another rabbit 5 c.c. of serum were given in daily
injections for three days, and on the fourth day twelve minims of poison I.;
it died in 4 hours 30 minutes: the control animal died in 6 hours.
EXPERIMENT X. Another rabbit was prepared in the same manner as in
the previous experiment, and after 48 hours two minims of poison ii. injected
subcutaneously; it died in one hour: the control animals died in 2-4 hours.
EXPERIMENT XI. A rabbit was prepared in the same manner and after
24 hours one minim of poison II. injected; it died in 41 hours: the control
animal also died in 41 hours.
EXPERIMENT XII. is a repetition of the last one, with the difference that
a rest of 48 hours was given. The result was the same.
This treatment, therefore, holds out no hope for success, as it does
not even prolong life.
Kitasato has shown that the toxic powers of tetanin may be
modified and destroyed by mixing it with the serum of an inmmunized
animal. Similar experiments were made with cobra poison and serum,
but the result muist be regarded as negative. Four experiments were
Experiment. Proportions of serum and poison. Result. Control.
XIII. c.c. of aserum + Min. xv. of

5 poison died in died in
12 hours 6 hours
XIV. 5 c.c. of serum + Min. iv. of died in died in

poison b 21 hours 50 min.
XV. 5 c.c. of serum + Min. i. of alive and died in

poison II well 5-34 hours
XVI. 9 c.c. of
poison II
serum + Min. ii. of died in died in
1-2k hours
41 hours
made, and in each the mixture allowed to stand 24 hours before being
injected into a rabbit.
At first sight it appears that the serum actually has an appreciable
influence over the toxic power of cobra poison. It was, therefore,
necessary to test, whether or no this effect is due to so great a dilution,
as the addition of 5-9 c.c. of serum to a few minims implies. There can
be no doubt, from the following experiments, that the above results are
not due to any inherent power of the serum; for with plain water the
same delay in the action of the poison may be obtained.
Experiment. Proportions of.water and poison. Result. Control.
XVII. 10 c.c. of water + Min. ii. of survived died in

poison ii 1-21 hours
xviii. 5 c.c. of water + Min. iii. ss died in died in

of poison ii 3 hours 1 hour
xIx. 5 c.c. of water +

strong poison Min. iii. of died in
1 hour 30died in
minutes
(3) Effect of Blood of an Iguana (Varanus Bengalen.sis or

V. salvator) on Cobra Poison.
The native snake-charmer having asserted that some species of
Varanus, (vernacular Goh), are not affected by cobra poison, three such
animals were procured. As their cuticle is impenetrable to the fangs
of a snake, I regarded the statement with scepticism. The " Gohs"
were in a starved condition, not having had any food for twelve days
when given to me, and up to the hour of death they refused any food.
Ten minims of undiluted poison, freshly extracted from a cobra, were
injected into the peritoneal cavity of a large " Goh." After two hours
it was sleepy and lazy, but did not die until the fourth day, having
starved all the time. To another "Goh," a young and small animal,
half dead from huniger, twelve minims of a slightly diluted solution
of the same poison were given, and it did not die before 20 hours had
elapsed. It never presented any signs of asphyxia or dyspncea and
apparently died from hunger and cold. Three minims of the same
diluted solution killed a fowl in thirty minutes. Without claiming
19-2
290 A. A. KA NTIIA aK.
that the "Goh" is resistent against cobra poison, it may be asserted
in all fairness, that it is considerably less susceptible to it than the
warm-blooded animals experimented on, and it therefore seemed worth
while to investigate the influence of this animal's blood on the poison.
Animal. Proportion of poison Method employed. Result. Control.

and Goh's blood.
xx. Min. iii. of poison injected immediately, died died in

Fowl +Goh's of fresh
2 c.c.blood without allowing
mixture to stand the after
28 hours 35 min
Min. iii. of poison

XXI. + 2 c.c. of glycerin .. . died in died in
Fowl extract of Goh's mijected immedately 35 min. 35 min.
blood
... ..
xx.Mm.5 ix. ofof poison
XXII.
Fowl
cx.
Goh's blood
f iis
n mected
j after allowing
it to stand 24 hours
died in
1 hour
died
quickly
It seems from these experiments, that the blood of the " Goh" has
some positive influence, especially if it be remembered that three
minims of the poison used, diluted with 5 c.c. of water, killed a fowl in
50 minutes. The number of experiments, however, is hardly sufficient
to allow us to speak with certainty, and an opportunity did not offer
itself of repeating the experiments. The following experiments do not
throw much light on this question:
5 c.c. of Goh's blood into died after 2 (a) died after an

XXIII. peritoneal cavity, and 28 hours and hour and 10 min.
Rabbit hours later Min. iii. of the 15 min (b) died after 30
same poison min.
5 c.c. of Gooh's blood into

XXIV. peritoneal cavity and 48 died after an died in 15 to
Rabbit hours later Min. v. of the hour 20 min.
same poison
These experiments are nevertheless suggestive enough to demand
repetition.
(4) As might be anticipated, a previous injection of cobra poison
boiled, until its toxic power was destroyed, was of no avail and
conferred no protection on the animal.
(5) Trichloride of Iodine, though it destroyed the poison outside
the body, was of no use in establishing a tolerance by means of previous
injections into the peritoneal cavity. It was also tried as a cure.
EXPERIMENT XXV. Three mininis of poison ii. were injected subcuta-
neously into a fowl at 12.30 p.m., and immiediately afterwards, over the site of
injection, 2 c.c. of trichloride of iodine. At 1 and 1.45 p.m. the trichloride
injections were repeated. The hen died at 4 p.m.
As a cure, therefore, this substance is a decided failure, but it
undoubtedly delays the action of the poison, as three minims of the
samne poison killed rabbits in about an hour.
(6) It is needless to remark, that all the substances which outside
the body lessened or destroyed the activity of the albumose, were tried
both as curative and protective agents, but in no case with even the
slightest prospect of success. Wall had similar experiences. The
extreme virulence of the albumose seems to baffle, at the present time,
an attempt at a cure or protection. The poisonous substances of
anthrax, tetanus, tuberculosis and diphtheria are comparatively harmless
and slow acting when contrasted with cobra poison. What is required
for the latter, is a quickly acting mnedium. From a review of the above
experiments it would seem that the best chances lie in a treatment, by
which fixed alkalies are introduced into the blood and lymph. Intro-
duced separately from the poison, immediately before or after, potash has
unfortunately no destructive power on the poison'. By long continued
injections of sodium bicarbonate the alkalinity of the blood might be
increased and the fixed alkali kept in solution, and judging from
Wall's experiments it seems not impossible that by this means a
protection, to some extent at least, against the cobra albumose might
be established. As far as I am aware the action of caustic alkalies on
other albumoses has not been tried. But if it be shown that the
anthrax albumoses are altered in a similar manner, Fodor's experi-
ments might be explained on the hypothesis, that the fixed alkali
circulating in an appreciable quantity through the tissues destroys the
1 Cf. also A. J. Wall, loc. cit.
poisonous action of the albumoses wherever it comes into contact with
them'. This matter must be reserved for future investigation.
(7) As Strychnia has been recomtmended as a cure against snake-
bite and in fact, the Indian Government addressed on this point,
experiments were made to settle this question for cobra poison. For
these experiments the pure albumose, separated according to Martin's
method,, was employed, and they were all performed on hens. Strychnia
is neither the chemical nor physiological antidote of cobra albuinose,
as the following experiments show.
EXPERIMENT I. Three minims of the albumose solution killed a fowl in
10 minutes.
EXPERIMENT II. Three minims of the albumose solution mixed with
five minims of liquor strychniae and allowed to stand for 24 hours, killed a
fowl in 10 minutes. The symptoms of strychnia poisoning masked all others.
EXPERIMENT III. Three minims of the albumose solution mixed with
three minims of the liquor and injected immediately, killed the fowl in
15 minutes under symptoms of strychnia poisoning.
EXPERIMENT IXV. On adding only one mimim of the liquor to the dose of
poison, the fowl died in 18 minutes with marked symptoms and signs of
strychnia poisoning.
EXPERIMENT V. Of a dilute solution of albumose three minims were
given at 12.15 a.m.; at 1.7 p.m. the fowl showed the first signs of drowsiness
and two-fifths of a minim of liquor strychnise were injected hypodermically,
and again at 1.17 p.m. The typical signs of strychnia poisoning set in, and
the fowl died at 1.39 p.m.
EXPERIMENT VI. Two-fifths of a minim of liquior strychnias were
injected at 1.29 p.m. and again at 1.39: the fowl died at 2 p.m.
EXPERIMENT VII. Five minims of a rather dilute albumose solution
were injected into a fowl at 3.40 p.m., .and when the first signs of drowsiness
appeared at 5 p.m., two-fifteenths of a minim of liquor strychniae were
injected at 5 p.m. and again at 5.10 and 5.20 p.m. The fowl died under
typical strychnia convulsions at 5.35 p.m.
In whatever dose strychn-ia was given, no benefit,resulted, the animal
dying in all cases. When less than one-fifteenth of a minim of liquor
strychnise was given, even with small but lethal doses of dilute albumose
solution, the animal invariably died under the typical symptoms of cobra-bite.
When more than two-fifteenths of a minim of liquor strychniae were
injected, the signs of strychnia poisoning prevailed and only quickened
death.
I T. Lauder Brunton and T. J. Bokenham. "Experiment upon the influence, etc."
British Med. Journal. July 18,1891.
NATURE O COBRA POISON. 293
Nothing, therefore, is to be expected from this treatment, and no
false hopes should be raised or fostered as to a cure by strychnia.
Two remarks shall conclude this section. It is often stated that
animals killed by a snake-bite putrefy rapidly. This statement was
not borne out by my experience, and certainly not, when the pure
albumose was employed.
Finally, the extreme local changes caused by the injection of even
small doses of dilute poison or albumose soluitions are remarkable.
Large nodules, filled with caseous pus, appeared rapidly on the abdomen
of the rabbits experimented on, so that it was at times difficult to find
a sound place in those animals which were uised for repeated injections.
EXPERIMENTS WITH THE ALCOHOmIC EXTRACT OF COBRA POISON.

" Various statements have been made by different observers about
the action of alcohol on snake-poison. 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 employed, the water in
the 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 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."'
An alcoholic extract was made in the manner described above,
being careful that such extract is quite free from albumoses, and was
injected in a concentrated form into fowls. The extract was in each
instance prepared from twenty minims of natural and undiluted poison
1 A. J. Wall, loc. cit.
294 A. A. KANPHA CK.
after having been thoroughly dried. Three hens were used and the
whole of the extract injected.
(a) Injected on July 27th: died on August 11th.
(b) Injected on July 18th: died on July 26th.
(c) Injected on July 23rd: died on July 31st.
In all these cases there seemed to be no special cause of death.
Though all the fowls died after one or two weeks, I hardly think that
this was due to some alkaloid: in any case the action of cobra poison is
not due to this body, if it exist, but to the proto-albumose, separable by
any one of the above-mentioned methods, and all experiments with a
view to establishing a cure or protection against this virulent poison
should be directed against the albumose. Wall had not recognized
the nature of the active agent of cobra poison, but his tests with regard
to the action of absolute alcohol are conclusive and required only
another step on his part to establish the chemical nature of this
substance. That this is an albumose has been settled for some time,
and the puirport of the experiments detailed above was a systematic
examination of the cobra-albumose, according to newer methods and on
the lines adopted by such careful workers as Hankin, Martin and
Hunter. Extending Wall's remarks according to our present know-
ledge of cobra poison, it must be said, that the alcoholic or spirit extract
is poisonous only so long as it contains the albumose.
I cannot conclude without alluding to the untimely death of my
friend Surgeon-Major A. Barclay, who commenced this work conjointly
with me and helped me in the first experiments. Through his exer-
tions I was enabled to have a constant supply of snakes. My thanks are
due to Surgeon-Major Ward of the Medical College, Calcutta, for his
readiness in the preparation of some of the necessary reagents.
SIMML, August 30th, 1891.
PART II.
DOES COBRA-POISON CONTAIN A GLOBULIN ?
In the first part of my paper on the chemistry of cobra poison it
was stated that the active principle of such poison is a proto-albumose.
No allusion was made to S. Weir Mitchell's and Reichert's or
Wolfenden's well-known researches, because at the time I was en-
gaged in a series of observations to settle the question whether cobra
poison contains a globulin or not. In my first observations I did not
ANATURE OF COBRA POISON. 295
obtain any changes on dialysis which could rouse the suspicion of the
presence of such a body, and I found it necessary to seek for an expla-
nation of the view, propounded by Weir Mitchell, that cobra poison
contains an albumose (he wrongly says peptone) and a globulin.
It is hoped that this paper will show clearly, that Weir Mitchell's
observations are by no means conclusive, and that the body which he
called a globulin, is nothing but a mixture of hetero-albumose and
dysalbumose. Whether the latter body is really present, I am not in a
position to assert, but there is some evidence in favour of its existence
under certain conditions in snake poison. In many specimens of fresh
cobra poison, the hetero-albumose is primarily not present, but ap-
parently developed under certain external and internal influiences.
The following observations disprove to my mind Weir Mitchell's
and Reichert's views as to the presence of a globulin in cobra poison.
(1) Their chief evidence of the existence of globulin in the venom
is the change occurring on dialysis. They state, that on placing a
solution of the poison into a membrane, after dialysis a white granular
precipitate appears within the skin, which is soluble in dilute saline
solutions and precipitated on saturation.
Now this does by no means always occur, in fact in my first
observations it never did. Yet it does at tires take place. Thus if
globulin be present, it is not always so. Again hetero-albumose reacts
almost in the same way, and Hunter succeeded in obtaining the
latter from tuberculin by means of dialysis. From the irregular
appearance of this change within the membranie, I was inclined to
consider it an hetero-albumose. To give an example: a little fresh
poison was diluted with a small quantity of sterilised distilled water
and dialysed in the usual manner (i.e. after the addition of a crystal of
thymol for two hours against running water and for 24 hours against
distilled water). After this period, the solution in the membrane was
milky and being kept in a well corked test-tube a white flocculent
precipitate appeared which gradually became granular. On filtering,
the fluid passed through milky and opaque. The precipitate was
almost wholly soluble in *75 per cent. NaCl solution and came down
again on saturation with NaCl as a cloudy precipitate which on
standing became granular, the solution itself remaining cloudy and
milky.
(2) In another series of observations the manner of procedure was
as follows:-The proto-albumose was separated according to a modifi-
cation of Martin's method. The cobra poison was poured into an
excess of absolute alcohol, and the mixture allowed to stand for a week.
Then it was filtered and the residue dissolved in sterilised distilled
water and again poured into absolute alcohol. This process was
repeated several times. The final white precipitate was allowed to
stand under alcohol for several weeks. It now was only partially
soluble in sterilised distilled water and had also undergone a slight
deterioration in its power'. Some of it was taken up in distilled
water and the filtrate placed in a membrane and treated as under
(1) with exactly the same result, showing that the globulin-like body
appears as dialysis goes on. Now it may in all fairness be claimed
that the repeated precipitation by absolute alcohol and the subsequent
extraction with distilled water exclude the presence of globulins. Again
this globulin-like body obtained, though easily soluble in *75 per cent.
NaCl solution, was not reprecipitated until the point of saturation with
NaCl had been reached. The solution containing this body remained
permanently cloudy and milky, whether the body was precipitated with
salt or not. On adding this solution to absolute alcohol, it became
clear, but a fine white flocculent precipitate came down. At times
while the body was in solution this milkiness was hardly appreciable
and increased as salt was added to it, finally becoming in many cases
very opaque. Heating hastened these changes and the precipitation
after saturation with NaCl. It should be mentioned, that a portion
of the white granular precipitate within the membrane was perinanently
insoluble in salt solution of any strength or weakness. All these changes
are decidedly in favour of an hetero-albumose.
(3) Somne albumose prepared according to Martin's method was
allowed to stand under alcohol for eight weeks. At the end of this
time it proved to be only partially soluble in distilled water. The
residue obtained after filtering was easily soluble in *75 per cent. of
NaCl solution, but also in any NaCl solution short of saturation, and
was not reprecipitated until the resuilting solution had been saturated
with NaCl.
(4) Cobra poison was thrown into an excess of absolute alcohol and
allowed to stand under the latter for three to four days, and was then
filtered. The filtrate was rapidly evaporated on a water-bath at 70 C.
The residue was taken up in 1 per cent. carbolic acid and was only
partly soluble. After 36 hours a flocculent granular precipitate fell
down, which was soluble in .75 per cent. NaCl solution and reprecipitated
on saturation. The supernatant fluid remained cloudy and milky. The
1 Cf. BewaIl: This Journal, Vol. ViII.
same result was obtained on using some of the albumose which was
employed for the third series of observations. But here the reactions,
if anything, were more marked.
(5) The poison extracted from two large brown cobras and diluted
with distilled water was thrown into an excess of absoluite alcohol.
Only a slight precipitate appeared, but the fluid became very opaque,
and cloudy or milky. After four days the mixture was filtered. The
alcoholic filtrate remained opaque, but after saturating with NaCl and
allowing the mixture to stand for two hours, the supernatant fluid
became clear. After filtering, the residue which contained much
undissolved salt was dissolved in as little sterilised distilled water as
was just sufficient to dissolve the salt. The resulting solution was
quite clear. The salt no doubt precipitated all the albumoses and
globulins present. As on the addition of distilled water, however, the
whole of the residue was dissolved and the concentration of the
resulting saline solution was considerably above 15 per cent., it is
hardly possible that any globulins had been precipitated. On fully
saturating the solution with NaCl it at first becanme milky and cloudy,
but soon a granular flocculent precipitate appeared, the fluid as above
remaining cloudy. As, however, this might have simply been a proto-
albumose the whole mixture of precipitate and saturated saline solution
was placed in a membrane and dialysed for three hours in running
water and for thirty hours against distilled water, until all the salt had
passed out. In the skin now there was a white granular precipitate,
soluble in dilute NaCl solution and only reprecipitated on saturation
with NaCl.
(6) Some fresh poison was dialysed and the portion which had
passed through the membrane concentrated by dialysis against alcohol.
The resulting clear solution was exposed to the sun, when it gradually
became opaque and a flocculent precipitate appeared. The latter was
insoluble in distilled water, but soluble in dilute saline solution and
reprecipitated on saturation. Here a globulin is absolutely excluded,
and the change is only explainable on the assumption that the action
of heat or light converted some of the original albumose into an
hetero-albumose. Temporary boiling had the same effect as exposure
to the action of the sun.
It seems from these observations, that the opinion of Weir Mitchell
and Reichert is not based on conclusive evidence, and that there is no
reason to assume the presence of globulin in cobra poison. In fact the
above tests show, that this body does not exist in the venom, but that
298 A. A. KANTHACZ.
under keeping, exposure to heat, sun or alcohol and even by means of
dialysis the original proto-albumose is changed in part to an hetero-
albumose. In some cases the latter body may be present in the poison,
as it issues from the glands, but this is not always so. Again, as at
times the precipitate obtained was only partially soluble in saline
solution, it seems that dysalbumose was also present. But the presence
of these two albumoses is apparently due to the manipulations to which
the poison was subjected, as dialysis by itself is capable of bringing
about the characteristic changes in cases where globulin can be
reasonably excluded. A deutero-albumose was never obtained, and it
must therefore be claimed, as was stated in the first part of this paper,
that the poisonous substance of cobra-venom is a proto-albumose. All
further trials to extract an alkaloidal body have been unsuccessful, all
the hens having survived.
An attempt was also made to explain the action of prolonged
heating on cobra-albumose. The latter was prepared according to
Martin's method and allowed to stand under absolute alcohol for six
weeks. The alcohol was then filtered off, and the albumose residue
dissolved in the least possible quantity of sterilised distilled water. The
resulting slightly mlilky solution was very virulent, ten minims killing a
rabbit in fifteen minutes. This solution was heated in a Koch's steri-
lising apparatus for five hours, and at the end of this time was as white as
milk, opaque and harmless. On filtering, this milky fluid passed almost
unchanged through the paper, a slight residue remaining behind. The
latter was almost wholly insoluble in saline solution of any strength.
The milky filtrate, on the other hand, gave a faint biuret reaction.
After boiling the above solution of albumose for twelve hours, it ceased
to give the biuret reaction.
A portion of the milky filtrate was saturated with neutral ammonium
sulphate and allowed to stand over the salt for twenty-four hours. At
the end of this time the solution was clear, and a slight white precipitate
had come down, in too small a quantity, however, for further investiga-
tion. The filtrate gave no precipitate on the further addition of
ammonium sulphate after acidulation with acetic acid.
To another portion of the milky filtrate 175 per cent. NaCl solution
was added, the solution remaining milky and opaque. On saturation
with NaCl this milkiness was increased, and a slight granular precipitate
appeared after some time, the supernatant fluid remaining opaque. It
was now placed in a sausage skin and dialysed against running water
for four hours, and after the addition of a crystal of thymol against
distilled water, which was frequently changed, for forty-eight hours.
At the end of this time a copious white granular precipitate appeared
in the membrane, but the fluid in the membrane was still cloudy and
milky, and gave no biuret reaction. On allowing a portion of the
contents within the membrane to stand in a well-corked test-tube for
twenty-four hours, a copious white granular precipitate came down and
the supernatant liquid was now clear. Another portion of the contents
within the membrane was saturated with ammonium sulphate and
allowed to stand over the salt for twenty-four hours. At the end of
this time a white granular precipitate appeared, and the supernatant
fluid, as above, was clear and colourless.
On filtering the contents of the skin, the filtrate passed clear and
had lost its milky and cloudy appearance. The white precipitate
which remained behind was almost entirely soluble in dilute saline
solution, imparting a milky appearance to the resulting solution, and
was reprecipitated on saturation with NaCl, the solution itself, at the
same time, becoming more milky and opaque. On pouring a solution
(in *75 per cent. NaCl) of the precipitate within the membrane into
absolute alcohol, a fine granular precipitate fell down on allowing the
mixture to stand for twenty-four hours, the supernatant alcoholic fluid
being clear anid colourless.
These observations confirm the opinion expressed- above, that the
experiments of Weir Mitchell and Reichert and Wolfenden cannot
be considered conclusive, and that a globulin-like body may appear in
a solution of cobra-albumose after certain manipulations. It seems that
prolonged heat decomposes the proto-albumose into hetero- and dys-
albumoses, and that these bodies are apparently harmless. Dr Hunter,
to whose paper I am indebted for many hints, remarks, that the remedial
properties of the tuberculin albumoses can be altered (or lessened) by
dialysis, and that this is due to changes occurring within the membrane.
From his and the above observations it may be suggested, that this is
due to a conversion of the proto- (or deutero-) albumose to hetero-
albumose. In opposition to Kiihne the experiments with cobra-albumose
seem to show that the proto-albumose of this venom on dialysis not
infrequently throws down a body which is indistinguishable from
hetero-albumose.
SIMLA, Sept. 23, 1891.

The Journal of Physiology - 1892 - Kanthack - The Nature of Cobra Poison

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The Journal of Physiology - 1892 - Kanthack - The Nature of Cobra Poison

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THE NATURE OF COBRA POISON. By A. A.

Preparation of the Albumose.

Nature of the Albu.rnose.

(1) Min. iii. of a solution peritoneal cavity died in

(2) ditto ditto died in

(3) ditto ditto 30 min.

(7) (7 ditto subcutaneous tissue died in

Animal. Operation. Result. Control experiment.

To control animal gave

To control animal gave

Effects of Physical Influences on Cobra Poison.

Min. v. of unboiled so-

Min. v. of concentrated died in

These three examples may suffice.

(1) Ordinary Mi. i. of chlorine mix- died after died after an

Chlorine water, therefore, if allowed to act sufficiently

Min. iii. of a strong pure died in 2 hours;

(2) Fowl Min. iii. of the same carbolic died in died in

Min. v. of natural poisonI

Carbolic acid thus delays the action of the poison con-

A solution of natural cobra-poison was heated, until no further

Ainimal. Experiment. Result. Control.

Min. v. of solutioni of cobra-

Solution of albumose (Min.

Min. v. of solution of pure

(4) Fowl Min. xxx. of ammonia with- died in 10 mm.

Ammonia therefore materially lessens the toxic power of cobra-

Reagent8 which destroy or lessen the toxic power of cobra-poi8onl.

Reagents. Effect. Remarks.

(1) Caustic potash destroys the toxic power } on addition of

EXPERIMENTS TO ESTABLISH A PROTECTION OR CURE AGAINST

XIII. c.c. of aserum + Min. xv. of

XIV. 5 c.c. of serum + Min. iv. of died in died in

XV. 5 c.c. of serum + Min. i. of alive and died in

Experiment. Proportions of.water and poison. Result. Control.

XVII. 10 c.c. of water + Min. ii. of survived died in

xviii. 5 c.c. of water + Min. iii. ss died in died in

xIx. 5 c.c. of water +

(3) Effect of Blood of an Iguana (Varanus Bengalen.sis or

Animal. Proportion of poison Method employed. Result. Control.

xx. Min. iii. of poison injected immediately, died died in

Min. iii. of poison

Animal. Experiment. Result. Control.

5 c.c. of Goh's blood into died after 2 (a) died after an

5 c.c. of Gooh's blood into

EXPERIMENTS WITH THE ALCOHOmIC EXTRACT OF COBRA POISON.

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