野口英世著

Journal of Experimental Medicine

所収論文

こ の PDF は Journal of Experimental Medicine に 掲 載 さ れ た 論 文 を Rockefeller
University Press の許可（2020 年 3 月 18 日付）を得てアップロードしています。

A STUDY OF T H E PROTECTIVE ACTION OF SNAKEVENOM UPON BLOOD CORPUSCLES.
BY HIDEYO NOGUCHI, M.D.,
ASSISTANTOF THE

ROCKEFELLER

INSTITUTE.

(From the Rockefeller Institute for iVIedical Research, New York.)

The present article deals with certain new facts concerning
the action of venom upon blood. For the purpose of defining
the scope of this study, it will be advisable to review briefly themore important contributions to our knowledge of the action ofsnake venom upon the blood.
The hitherto well-established effects of venom upon the blood are
the hmmolytic and ha~magglutinative action upon the corpuscles,
and the inhibitory and accelerating action upon the coagulation of
the plasma. The reducing action upon oxyh~emoglobin has also
been recognized. It was Mitchell and Reichert,l probably,who first
definitely described the phenomena which are now known as hmmolysis and agglutination, and these same investigators emphaticallycalled attention to the loss of coagulability of the venomized
blood. Martin and Smithfl Phisalix, 3 Kanthack, 4 Mitchell 'and
Stewart, 5 and Stephens and Myers 6 pursued the subject further
and, while confirming the studies of Mitchell and Reichert, succeeded, at the same time, in extending our knowledge of venom
activity. Martinfl indeed, found that large doses of the venomof a certain Australian snake produce extensive intravascularclotting, a fact which Lamb s also made out for the venom ofan Indian viper, Daboia russelli; and Flexner and Noguchi 9
pointed out that the blood clots which are found in the rightside of the heart and the pulmonary artery after the intravenous
infusion of daboia venom contained agglutinated red blood corpuscles. It was Lamb 1° who first demonstrated the power ofvenom to cause coagulation of citrated blood in vitro. Cunningham 11 observed the discolorizing effect of venom upon blood
191

192

Protective Action of Snake Venom

corpuscles, from which he erroneously concluded that venom
exerts its chief toxic action upon blood, and the various phenomena commonly observed in poisoned animals are merely theconsequences of the loss of the respiratory function by the altered
red corpuscles. Plexner and Noguchi 12 found the mechanism ofvenom hmmolysis to be analogous to serum hmmolysis, and demonstrated the occurrence in venom of haemolytic amboceptors
in Ehrlich's sense. Calmette 13 discovered the important fact, destined to have very far-reaching consequences, t h a t one of thevenom activating principles is thermostable, the other being of thenature of the usual serum complements. This observation of Calmette led immediately to the brilliant discovery of lecithin-venom
activation by Kyes, 14 who also succeeded in preparing a crystalline compound of venom hmmolysin and lecithin which possesses
all the native haemolytie strength of the venom. The independence of the haematoxic and other toxic principles, the n u m b e rof which is surprisingly large, has been shown by the later studies
of Flexner and Noguchi. is
The properties of venom already enumerated depend on principles which, with the possible exception of the haemolysin in thelecithin compound, have not been separated in a state approaching purity. Mitchell and Stewart 16 described another propertyof crotalus venom which had apparently escaped the attentionof previous investigators. They observed t h a t in a mixture ofblood and fresh venom, in equal parts, the corpuscles, instead ofundergoing haemolysis, were actually preserved from disintegration for a period considerably greater than in the control specimens to which no venom had been added. On the other hand,
if the a m o u n t of venom employed was less than i o per cent of
the mixture, hmmolysis occurred in the usual way. Mitchell and
Stewart were led by their experiments to regard the once dried
venom as being a less effective preservative t h a n the fresh secretion, and they noted that, of the corpuscles tested, those of therattlesnake were most perfectly protected by erotalus venom.
Stephens and Myers 17 described a similar phenomenon in the caseof cobra venom and h u m a n blood, although they do not state
how long the corpuscles can be preserved in this way. PlexnerHideyo Noguchi

193

and Noguchi 18 in their first studies noted a protection of rabbit
corpuscles by means of rattlesnake venom, and, somewhat later,
Kyes 19 and Kyes and Sachs 2o encountered the same phenomenon
in the course of their studies with cobra venom, and Lamb 29
with daboia venom.
While the earlier investigators merely recorded their results,
Kyes and Sachs 21 endeavored to explain the phenomenon. They
accounted for it by supposing a complement (lecithin) deviationor "side-tracking" through the action of the large excess ofvenom h~emolytic amboceptors which were present in the mLxture of blood and venom. Their crucial experiments were conducted as follows: Blood corpuscles were treated with a large
excess of cobra venom and thoroughly washed to remove theexcess of venom. Since, as Flexner and Noguchi 22 had shown,the venom amboceptors became attached to the corpuscles, thelatter could now be ha~molyzed by contact with suitable com~plements (serum, lecithin). In the control experiments the excess of venom was not removed by washing, and in them thetubes containing the larger amounts of venom either failed
entirely to show hmmolysis after addition of complement, or theamount of hmmolysis was greatly diminished.
In the course of a recent study of the influence of venom inpromoting the h~emolysis caused by physical and chemical agents,
this protective action of strong solutions of venom was given
special attention, and the present communication deals with theresults of this investigation. It may now be regarded as wellestablished that when the optimum of the h~emolytic action ofvenom is exceeded, the degree of hmmolysis which it is capableof producing diminishes progressively as the dosage of the venom
increases. Among the natural biological h~emolysins, venom alone
is known to possess this property, but, in the course of a study ofbacteriolysis by certain immune sera of high potency, Neisser andWechsberg 2a observed an inhibition of bactericidal effects whenan excess of amboceptors, relative to the complement content, wasbrought into the bacterial suspension. A similar although probably a distinct phenomenon has been described by Detre andSellei 24 in their studies of h~emolysis caused b y bichloride of194

Protective Action of Snalce Venom

mercury. But excepting the few experiments made by Kyes
and Sachs, upon which they base their notion of complement
deviation, no really extensive study of this property of venom
has been made.
The explanation offered by Kyes and Sachs involves the belieft h a t the principle, which is responsible for the protection, is
identical with that which causes, under favorable conditions,dissolution of the corpuscles. If it can, therefore, be shown that
it is not the h~emolysin which is protective, but some other principle, the notion that lecithin deviation explains the appearances
can not be maintained. It is possible by means of suitably
arranged experiments to test fully the assumption of a protective action exerted by venom and due to h~emolysin, and, further,to study other features of this action with a view of acquiring amore complete understanding of it.
The experiments to be related were devised to determine
whether the protection is effective only against complements,
or whether it operates as well against other hmmolytic agencies
from which the biological factors are entirely eliminated. These
several aspects of the subject will be dealt with in the following
pages.
Preliminary Remarks upon the Action o] Venom upon Blood
Corpusdes.--In the experiments to be given, the blood of thehorse was chiefly used, and it is meant unless another kind ofblood is specified. Unwashed horse corpuscles are fairly susceptible to venom h~emolysis, while the corpuscles after washing(three washings in fifteen or twenty times their volume of salt
solution at each operation) are insusceptible to venom. The
horse has the advantage of yielding small or moderate quantitiesof blood at not too frequent intervals without causing perceptiblechanges in the resistance of the corpuscles. By using a single
horse and establishing the resistance of its red corpuscles a standard can be obtained that, within small limits, is constant. In
carrying out the experiments on resistance this condition wasessential.** T h e o b a l d S m i t h (The .Journal oi Medical Research, i9o4, xii, 385) h a s described a r e d u c t i o n in r e s i s t a n c e to salt s o l u t i o n s of corpuscles of horses s u b -Hideyo Noguehi

195

The blood corpuscles of the horse present another advantage
in their greater resistance to the mechanical injury involved indefibrination and washing.
The defibrinated blood in 0.9 per cent salt solution is susceptible to cobra, crotalus, and moccasin venoms, the varieties whichwere employed. A mixture of blood suspension and cobra venom,
in which the venom is contained in greater quantity than 5 percent of the dry poison, will remain undissolved when kept forseveral hours at 2o° C. ; but at the end of twelve hours complete
hmmolysis will have taken place.
In general it may be stated that rapidity and completeness ofha~molysis depend upon the proportional amounts of venom andblood in the mixtures: the greater the quantity of venom andthe smaller the amount of blood, the slower and less perfect thehmmolysis. There is, however, one factor which controls to alarge extent the rapidity and degree of h~emolysis, namely, thepresence of serum constituents in the mixture. So long as a
trace of serum remains, complete dissolution can not be prevented by any practicable concentration of venom. An equal
mixture of defibrinated blood and 25 per cent cobra venom*
solution will undergo hmmolysis in one or two hours.
It has already been stated that the thoroughly washed horse
corpuscles are not hmmolyzed by venom no matter what thedegree of concentration. If cobra venom be used in strengths
above 4 per cent with a corpuscular suspension of 5 per cent, nochange m a y take place in the mixture in m a n y weeks; whilewith a quantity of venom as small as o. i per cent the corpuscles
will disintegrate rather more quickly than in the control tubes
which are not entirely sterile.
Corpuscles which had been brought into contact with thestronger solutions of venom were tested for resistance to salt
solutions of varying tonicity.
These tests disclosed theunexpected fact that corpuscles thus venomized, and in thejeered to r e p e a t e d bleedings in t h e p r o d u c t i o n of d i p h t h e r i a a n t i t o x i n . Our
practice was to t a k e relatively small q u a n t i t i e s of blood a t i n t e r v a l s of a n u m b e rof days from a s t r o n g horse t h a t h a d n o t previously been used for a n y experiment.
* The p e r c e n t a g e of dried v e n o m is always m e a n t .

196Protective Action of Snake Venom

presence of an excess of venom, are not hmmolyzable even b ywater.
At the same time their susceptibility to heat is changed. I t
has been found t h a t control tubes of blood corpuscles alone arehmmolyzed completely in from one hundred and seventy-five to
one hundred and eighty minutes when kept at the constant temperature of 53 ° C. In the presence of venom of a concentration
not exceeding i per cent, complete hmmolysis will take place atthis temperature in from five to fifteen minutes; with a concentration of venom as low as o.oi per cent thirty minutes will be
required; with a concentration of i o per cent there is no perceptible change in the corpuscles for the first twenty minutes,
after which laking commences. This last laking is not, however,typical. A bright hmmoglobin color does not appear in the fluid,
but the cells undergo disintegration, the color of the mixture
becomes coffee-like, and in about one hour a turbid precipitateof cell particles and venom granules can be made out under the
microscope.
Strong solutions of venom are capable of protecting the corpuscles from destruction by water, but venom solutions below
2 per cent in strength render the corpuscles more sensitive tosalt solutions, as measured by the tonicity of the fluid for corpuscles; and as the strength of the venom falls from thislimit the susceptibility, as measured by the degree of tonicity,diminishes.
Is the Protective Action Due to Venom H~emolysin f - - T h e firstquestion to be considered is whether the protective action ofstrong solutions of venom is in any way dependent on the hmmolytic amboceptors. Since this protection is exhibited against
water and heat, it is safe to assume that it bears no necessary
relation to complement deviation. However, even these reactions do not prove that the hmmolysin is unconcerned in theprocess. Some other explanation, one, namely, in which analteration in solubility of the protoplasmic constituents of thecells is concerned, has more likelihood of fitting the facts observed, but nevertheless it is necessary to exclude by suitablemeans any possible participation of the hmmolysin.

Hideyo Noguchi

197

There is a marked difference between the effects produced b ycomplements present, in an available form, before the venom
has acted on the corpuscles and those produced by complements
added after the treatment with venom. Should even a trifling
quantity of serum be left with the corpuscles, it is not practicableto obtain the protective effect of venom, while if the effect isonce produced and the excess of venom allowed to remain, verylarge amounts of serum or of lecithin fail to bring about
hmmolysis.
The first set of experiments relates to the difference in effectproduced by two concentrations of venom. In each experiment
the washed corpuscles were in 5 per cent suspension, and wereallowed to remain in contact with the venom for two hours before
any complement was added. The mixtures were all made in
0. 9 per cent solution of salt. The two strengths of venom were
io and 2 per cent, Table I expresses briefly the result.
TABLE I

5 per cent horse corpuscles.

Io % Cobra venom

A m o u n t ofcorpusLnl-

lar suspension.

Aanount of lecithin andfresh serum.

C.C.

C,C.

0.:

-q- 0. 9 % s a l t sol. 0. 7
T r ~ N. l e c i t h i n o. 7
T ~ N.
"
0.7-[- h o r s e s e r u m

2 % Cobra venom

O.:
i&
•4

venom

O.

= No hsemolysis
No hmmolysis
Doubtful result, owing
to the formationofg r a n u l e s , b u t n o h~emolysis after 6 hours.
= No hmmolysis; strong
discoloration after somehours.

+ 0 . 9 % s a l t sol. 0. 7-t-~
N. l e c i t h i n 0. 7
-I- h o r s e s e r u m+ horse serum

Contro!
0.9 % S a l t sol. ; n co. 7Result, at zo° C.

= No hmmolysis.
Complete hmmolysis
m 5 minutes.
o. 7 = C o m p l e t e h ~ e m o l y s i sin 2 h o u r s .
0.0 7 = C o m p l e t e h ~ e m o l y s i sin 4 h o u r s .

+ 0. 9 % s a l t sol. 0. 7
-t- T ~ N. l e c i t h i n o. 7
+-r~

N.

"

0. 7

+ horse serumo.7= No hmmolysis.
= Complete hmmolysis
in 8 h o u r s .
Complete h~emolysis
in x h o u r .
= No hmmolysis.

198

Protective Action of Snake Venom

E x a m i n a t i o n of the table shows t h a t the corpuscles which area c t e d on b y xo per cent v e n o m are not hmmolyzable b y quantities of fresh horse serum and lecithin, which far surpass thea m o u n t s theoretically required to bring a b o u t solution; whilewith the v e n o m in 2 per cent solution the corpuscles were perfectly dissolved b y lecithin and serum. The q u a n t i t y of complements used in the experiments given in the table was in great
excess, and the excess in the tests with io per cent v e n o m wast e n times greater t h a n in the tests with 2 per cent venom, andone hundred times greater than actually required under optimal
conditions.
Before discussing the participation of the venom-hmmolysin
in the protective action, it will be necessary to describe theinfluence of heat upon the reaction. As a preliminary step, theinfluence of high temperatures u p o n the hmmolytic action of
cobra v e n o m was made. It was found t h a t the filtrate from thecoagulum of v e n o m heated for five minutes at ioo ° C. was stillh~emolytic, and, indeed, quantitatively in the same degree as theu n h e a t e d venom.
Tubes containing a 25 per cent solution of cobra v e n o m in o.9
per cent salt solution were heated to 95 °, ~oo °, and ~35 ° C., respectively. The temperatures of the first two tubes were maintained for five minutes, while the last t u b e was kept at the hight e m p e r a t u r e in the autoclave for one hour. W h e n v e n o m insolution is heated to 95 ° C. it becomes milky in appearance,
while at ~oo ° C. coarse particles of coagulated m a t t e r separate.
At the t e m p e r a t u r e of ~35 ° C. the coagulum shrinks to a solid,
spongy mass, leaving the fluid portion clear. The heated v e n o m
was now tested and it was found t h a t the soecimen heated to
95 ° C. was practically unaltered in its ha~molytic and protective
action; while at ~oo ° C. it had lost a portion of its effect only,
and at ~35 ° C. it had lost entirely b o t h of these properties.
The next series of tests was made with the separated portions,coagulum and filtrate. For this purpose the specimen heated to
95 ° C. was employed, and to obtain a perfectly clear fluid, filtration through a Berkefeld bougie was resorted to. The resultsof this test were conclusive ; the filtrate had no protective action,

Hideyo bloguchi

199

while the coagulum when suspended again was protective. The
filtrate from the specimen heated to too ° C. was also non-protective. The filtrates from both of these fractions contain thehmmolytic principle in undiminished amounts.
These experiments prove definitely that the protective principle is distinct from the hmmolytic amboceptors, and it is interesting to note t h a t the coagulum from the specimen heatedto 95 ° C. is still capable of exerting protective influence uponblood corpuscles. It seems safe to state t h a t the coagulated
proteid is not the protecting body; and hence it is a fair assumption t h a t in process of coagulation the protective principle ismechanically carried down under conditions of union which still
permit its entering into combination with blood cells.*
Although the filtrate from a solution of venom heated to 95 °or ioo ° C. has no protective action, such a filtrate exerts an injurious action on the corpuscles, whereby they are rendered moreeasily hmmolyzed by salt solution. The washed corpuscles of thehorse are laked by 0.44 per cent salt solution, b u t after treatm e n t with the filtrates mentioned they are instantly laked bysolutions of o. 5 per cent. The temperature of r35° C. destroys
not only the protective b u t also the injurious body; and at
t h a t temperature the filtrate is without h~emolytic action evenin the presence of suitable complements.
Our experiments lead us, therefore, to regard corpuscular protection by strong solutions of venom as a peculiar effect which
is opposed to h~emolytic action, and one which has thus far received neither adequate attention nor probable explanation.
Moreover, it would seem that the h~emolyzing principle in venom
is directly injurious to corpuscles even in cases in which suitablecomplements are absent. Until a special injurious agent which
predisposes corpuscles to laking by physical agents is discoveredin venom, we are inclined to believe that the injurious action of
the filtrates is due to venom hmmolysin.
* In view of the fact that in his experiments which led to the view of complement deviation by v e n o m amboceptors Kyes employed rabbit corpuscles, I
h a v e m a d e a series of tests with these cells. My results show t h a t rabbit
corpuscles are somewhat less perfectly protected t h a n some other cells, butProjective Action of ~S'nake Venom

200

A passing consideration should be given to venom agglutinins,since it is at least conceivable that the agglutinated masses ofcorpuscles arising from venom treatment are less readily affectedby laking agents than the separated corpuscles. But since thethey exhibit unmistakable evidences of protection to heat, saponin, and water.
Moreover, t h e y also show the paradoxical reaction to salt solution, since t h esuper-venomized corpuscles show at one point a greatly lessened resistance tosolutions of sodium chloride. A table is appended in which these results m a y
be observed.
I C. C. OF 5 ~O S U S P E N S I O N OP R A B B I T BLOOD (washed)

Amountof 5 %
cobra
v e n o m solution c. c.

Blood No. I.
T e m p e r a t u r e of 53 ° C.

i

0.5
o.25
O.I
0.075
0.05
0.025

Blood No. I I .
i % saponin sol.

N o h~emolysis, precipitate formed, Slight h~emolysis
cells disintegrated in two hours.
No heemolysis, precipitate formed,
cells disintegrated in t w o hours.
"
Complete h~emolysis in 9 m i n u t e s Partial
Complete "

o.ei

o.OO5

"

42
I30

35

0.43

In repeating
Kyes'sexperiments
coincided with the points of so-called~ T e n o m SO~

0,025
o.oI

0-005

I founddeviation

%

"

that
the points of protectionof complement
as is shown intable.

A m o u n t of
5 % cobra
lution c . c .

Complete hmm,o,lysis in o.5% salt sol.
0.53 % . . . .
. . . .
0.68 %. . . . .
0.566 %"
o.s5 %"
o.535 % " "0.52 v~ ,, 0.48 % " "0.458 % " "oControl(no v e n o m )the next

Slight h~emolysis in w a t e r .
P a r t i a l hmmolysis in w a t e r .

~ 7½

o.ool
0.0005

i
o.5
o. 25
o. I
0.075
0.05

Reaction to w a t e r a n d NaC1 solution.

i c. c. oF 5 % RABmT BLOOD (defibrinated)
Blood No. I.

Blood No. I I .

No hmmolysis
No hmm2lysls
Trace "
Marked ',',
Trace
Moderate hmmolysis
A l m o s t complete hmmolysis ! Comp,lete h~em21ysis
Complete h~em,o,lysisI
A l m o s t complete h~emo,lysiso.ooi
A l m o s t complete hmmolysis ~Much hsemolysis
No0.0005
Trace hmmolysis
Control(no v e n o m ) No"

Blood No. I I I .
No hmm,o,lysis
Slight "Moderate h~emolysis
eomp, lete
A l m o s t complete haem,olysls
M a r k e d h~emplysis
No

"

,,

Hideyo Noguehi

201

venom agglutinins are destroyed at relatively low temperatures
their participations can be quickly eliminated. I have also
tested abrin and rlcin, which are strongly hmmagglutinative,
without finding a corresponding protection. Indeed, corpuscles
treated with abrin are slightly reduced in resistance, while corpuscles treated with ricin show about the normal reaction to salt
solution.
The serum of the rattlesnake is highly agglutinative and h~emolytic for corpuscles of the horse, and yet it does not protect t h e min any degree. The corpuscles, after a contact of twelve hours
with the inactivated rattlesnake serum in excess, were h~molyzed
by salt solution of 0.45 per cent strength.
That other coagulated proteids and mucin do not contain any
such protecting principle is shown by the negative results obtained with coagulated serum of the horse and of the rattlesnake,and with salivary mucin.

The Effect o~ Washing the Corpuscles a~ter Venomization.--Attention has been drawn to the fact that the property of venom
which is being described is operative only in the presence of anexcess of venom. Corpuscles once protected can be diluted withwater indefinitely without any laking taking place, so long as theexcess of venom is not removed by washing, and other evidencesof protection, such as increased resistance of the corpuscles toheat and to various chemical laking agents, can be demonstrated.
If, however, the venomized corpuscles are subjected to repeated
and thorough washing with 0. 9 per cent salt solution, it is foundt h a t the protection disappears and, moreover, the corpuscles are
now more easily laked t h a n normal blood cells.
Four mixtures of washed corpuscles and venom were prepared.
The corpuscles were used in 3 per cent suspension in salt solution, and the venom strengths were xo, i, o.i, and o.oi per cent.
The period of contact was six hours. A portion of each mixture
was removed and washed in o. 9 per cent saline four times, t w e n t ytimes the volume being used at each washing. Two sets of tubes,
after dilution four times with saline solution, were exposed to thetemperature of 53 ° C. and the results noted. They are reproduced in Table II.

Protective Action of Snalce Venom

202

TABLE I t
S t r e n g t h of venom
solution.

Washed venomized corpuscles.

Unwashed venomized corpuscles.

~o %Complete h0emolysis in ue No typical laking, but general
minutes
disintegration of ceils in 60
minutes ; the fluid presented
a light brownish hue.

1%Complete h~emolysis in 25 Complete h~emolysis in 6 minminutes
utes.

O,I ~0Complete hmmolysis in 25 Complete h~emolysis
minutesminutes.

in

12

O.OI

Complete hmmolysis in 3° Complete hsemolysis
minutesminutes.

in3°

Control (no venom) Complete hmmolysis in i7o Complete hmmolysis inminutesminutes.

~75

Table II shows that horse corpuscles suspended in the venom
solutions of strengths above o.~ per cent absorb about the same
quantity of venom irrespective of the actual concentration of thesolutions. The quantity of venom absorbed sufficed to bringabout laking in twenty-five minutes, the mixtures having been
kept during this period at 53 ° C. ; and this a m o u n t of venom isapparently the m a x i m u m quantity which the corpuscles are able
to retain after repeated washings with the saline solution.
There is no m e t h o d of ascertaining with absolute accuracy
whether this quantity was originally absorbed, or whether it is
the part of the original quantity fixed to cells and retained withgreat avidity. An approximate estimation could, of course, bemade of the a m o u n t of venom remaining in the fluid part of the
mixture after simple contact of the blood-cells and venom; b u t
since, in carrying out this estimation, no account can be taken of
the venom which is loosely and mechanically held by the corpuscles, figures approaching accuracy are impossible.
T h a t venom directly injures blood corpuscles has been mentioned, b u t Table II is valuable in showing that this injury canbe expressed in quite definite figures. I know of no series ofexperiments in which laking by means of heat has been used toHideyo Noguehi

203

determine the degree of decreased resistance of blood cells. Them e t h o d seems to promise a fair degree of accuracy, and it also
would seem to promise the discovery of substances of primary
injurious nature, which until now have been assumed to be inactive except in the presence of suitable complements.
In order t h a t this primary injurious action m a y be expressed,
the concentration of the venom solution must be kept between
o. i and i per cent. Higher concentrations either begin to beprotective or do not increase the injury, because they are not soentirely absorbed. In any case an excess of venom would appear
not to be present in the unheated mixtures, for hastening of heath~emolysis is not observed in the venomized corpuscles which havepreviously been washed. Hence the question arises whether at
the high temperature more venom is not absorbed t h a n at 2o ° C. ;from this increased fixation and injury perhaps the greater susceptibility to laking proceeds. No answer to this question isa t t e m p t e d at present; b u t I shall now proceed to the next seriesof experiments which are concerned with the effect of repeatedwashings in saline solution upon the tonicity of the venomized
corpuscles, as measured by their resistance to salt solutions ofdifferent strengths.
TABLE IIIS t r e n g t h of venom solution.

Venom removed.

Venom not removed.

io%~%0"5I ~00.49 %
0.48 %
0.465 a~o0.44 ~oNot h~emolyzable in water.
0.48 ~ooo.465
o.44 o'~o.44 ~Y~O.I ~0O.OI ~0Control (no venom)

The striking difference in the m a n n e r of reaction to hypotonic
solutions between the corpuscles acted upon by strong vertom
solutions and by solutions of weaker concentration is well illustrated in this table. After the action of io per cent venom, thecorpuscles can be diluted indefinitely with water without showing any tendency to dissolve, while the same corpuscles, after204

Protective Action of Snake Venom

repeated washing with 0. 9 per cent salt solution, show a higher
hypotonic index than normal--o.51 per cent as compared with
0.44 per cent. The significance of this behavior is quite plain.
Strong venom solutions react in some manner with the corpuscles
so that they are rendered insoluble in water; the insoluble compound is, however, capable of being broken up by certain chemic a l s - i n this case sodium chloride. But whether it is the sodium
chloride as such, or the free ions in the mixture, the experimentdoes not show. It may be remarked here that the protective
property is not quickly removed by the saline solutions, but several washings are required to remove it. It would seem, therefore, that an excess of sodium chloride alone does not suffice,but some other factor is concerned.
Since it has now been shown that the entire protective effectof the venom, as regards water hmmolysis, can be removed bywashing the corpuscles with the saline solution, the question
arises whether this process restores the susceptibility of the corpuscles to lecithin and serum haemolysis. It has already beenstated that the presence of serum in the mixture will preventthe strongest solutions of venom from causing protection; andnow it may be said that the protection once removed by salt
solution the corpuscles become susceptible to serum and lecithin
haemolysis.
The next table gives the results obtained with lecithin andhorse serum. The conditions of the experiment were similar to
those of the preceding experiments. The period of contact ofvenom and washed corpuscles was two hours; and the washing
was always done with 0.9 per cent salt solution. The volume ofsuspended corpuscles was 3 cubic centimeters, to which o. 5 cubic
centimeter of T ~ N. lecithin or ~ cubic centimeter of horse
serum was added. The observations in the case of the horse
serum were made at the end of four hours.
It appears that washing with salt solution exposes the protected corpuscles to laking with serum and lecithin. This susceptibility to laking is due to the fixation of venom ha~molysinby the corpuscles. When the activity of very strong solutions,a f t e r removal of all unattached venom, is compared with theHideyo Noguchi

205

activity of weak solutions, in which subsequent washing isomitted, a m a r k e d difference in the rapidity with which hmmolyr
sis occurs is apparent. The difference m a y depend, possibly
on one of two causes, first, t h a t the corpuscles in the absence ofcomplement actually fix an insufficient q u a n t i t y of h~emolysin
to produce rapid laking; or, next, the washing removes a partof the hmmolytic amboceptors already attached.

The Action of Certain Substances upon the Venornized Blood
Corpusdes.--The effects of water, salt solution of different concentrations, and heat upon the venomized corpuscles have been
TABLE I V

I

Strengtho~ v e n o m I

solution.

zb %LECITHIN.
~~[o RS E SERUM.

• l

Venom r e m o v e L I -

Venom

not

removed.

Venom removed.

V e n o m notremoved.

Complete h~emolysi~ No h,qemolysis
Complete h~emolysis No hsemolysis.
in 28minutes /Complete h~emolysis.
Complete hmmolys[~ Complete hmmolysis[. . . .
C in 28 minutes.
[ in 7 minutes{o.x %Complete hsemolysi~Complete h~molysisjPartlal h~emolysls
J in 29 minutes.
| in 9 minutes,
Iii,to.ox %] Complete hmmolysi~ Complete h~molysis]. . . .
]
in 30 minutes. ) in 28 minutes.
Control (n~ Complete h~emolysi~/ No hzemolysis
venom) 1 in 8 hours.
|
t
•

%

/

recorded. The next series of experiments deals with the actionof still other definite agents, such as acids, alkalies, etc., upon theprotected cells. The m e t h o d of procedure was as follows: Fourtubes, containing 4 cubic centimeters each of 5 per cent cobra
venom in o. 9 per cent salt solution and 6 per cent of washedhorse corpuscles, were allowed to stand for two hours at 2o ° C.
Tube No. i received 4 cubic centimeters of water, and tube No. 2
received 20 cubic centimeters of water; the corpuscles were perm i t t e d to subside when the clear fluid was decanted and thevolume of the residue brought up to 8 cubic centimeters. Tube
No. 3 received an additional 4 cubic centimeters of salt solution,
while the residue in tube No. 4 was washed once in 2o cubic
centimeters of salt solution a n d suspended in 8 cubic centimetersof saline solution. The actual tests were made by measuring

206

Protective Action of Snake Venom

cubic centimeters of each mixture into a test-tube, to which thesolutions of chemicals were added gradually until an amount
was introduced which produced instantaneous hmmolysis. The
chemicals were dissolved either in water or in saline solution,according as they were to be added to blood cells diluted in one
or the other of these fluids. The results are given in the nexttable.
TABL~ V
Venomized corpuscles.

I. Dilution with watero. i 2 5

0.75

1.75

2. S e d i m e n t e d

a n d res u s p e n d e d in w a t e r

o.~2

0.7

I.

with

saline

o,33

x.5

2.

4. W a s h e d w i t h a n d res u s p e n d e d in saline
solution

0.25

0.8

0. 3

5. Control, n o v e n o m

0.5

1.75

0.4

3. D i l u t i o no. x % saponin.

~ N. HC1 ~a N.NaOH ~ N. NH~
e.G.
o.O,
¢.c.

solutionc.o.

No hmmolysis.

iC o m p l e t e h m m o l y s i s , o. 3

"

"

0,45

An analysis of this table shows an essential agreement between
the manner of action of hydrochloric acid and sodium hydroxide.
These substances cause hmmolysis in smaller quantities when
added to the watery suspensions than with suspensions of corpuscles in saline solution, the difference being approximately asi : 2 or i : 2.5. On the other hand, after removal of the excess ofvenom by washing in the saline solution, the corpuscles show agreatly reduced resistance, since they are about twice as readily
laked by these chemicals as the control without venom. On
the other hand, in the watery medium the super-venomized
corpuscles are about four times as susceptible to hydrochloric
acid and twice as susceptible to sodium hydrate as the control
specimens. This apparent paradox is probably explained by
the nature of the medium, for, the insoluble compound of venom
and proteid once dissolved, the previously protected corpuscles

Hideyo Noguchi

207

immediately become the victims of water-laking. The reduction of resistance exhibited by the venomized cells suspended
in salt solution is an expression of the injury inflicted by thevenom directly upon the corpuscles in the manner alreadydescribed.
On the other hand, ammonia permits unmistakable evidencesof protection to be exhibited. In the first place, in the presenceof excess of venom, ammonia proved to be relatively a weaksolvent for the protecting compound, and, in the next place, inthe case of the washed venomized cells the susceptibility of the
corpuscles to the solvent action of the ammonia is increased
beyond the control, although this difference is again less marked
than with hydrochloric acid and sodium hydrate.
Another remarkable fact is the inability of saponin to cause
laking of the protected corpuscles. According to Kobert, 2s saponin is h0emolytic probably because of its property of dissolving
lecithin, which it withdraws from the corpuscles with the effectof destroying the integrity of the cells. It would seem, therefore,that the protection of the corpuscles against saponin hmmolysis
might be associated with a change in the availability of the intracellular lecithin. It is improbable that venom extracts thelecithin, for if it did it is likely that the corpuscles would bedirectly disintegrated by the venom, but such disintegration does
not occur. It is far more probable that the venom action is tobring about some physical or chemical alteration in the cells
which prevents the usual effect of saponin upon the corpuscles.
That, indeed, the effect of venom is not permanent, as would be
the case were it to abstract the lecithin, is shown by the resultsof washing the protected corpuscles in the saline solution andsubsequent exposure to saponin. Such washed cells are hmmolyzed by saponin even more readily than the controls. Hencethe evidence is in favor of the view that venom fixation of the
corpuscles is due to some change in the character of the cellularconstituents, and that the venom acts also directly upon the
corpuscles, producing an injury even though this injury is not
at once evident. We shall return to the first aspect of this subject. The strongest solutions of venom which it is practicableProtective Action of Snake Venom

208

to use, like the weaker solutions, act injuriously upon blood corpuscles. The stronger solutions, however, exert a second action- - a kind of fixation of the cells--which obscures the injurious
action. Table VI gives the results obtained with saponin andcorpuscles in the presence of an excess of venom and after its
removal. The volume of the venomized blood suspension in each
instance was 4 cubic centimeters. Saponin was employed in o.i
per cent solution, which was added in increasing quantities until
instantaneous hmmolysis was produced.
TABLE V [Strength of venom solution.

io%i%O.I ~0O.OI

C o n t r o l (no v e n o m )Washed venomized corpuscles-t- o.1% saponin.
0. 3 c . c .
0.35
0.4
0.45 "
0.45

Unwashed venomized corpuscles-I- O,1% saponm.
No hmmolysis *0.35 c. c.
0.4
it
0.45
0.45

Strong venom solutions afford no protection against formic
acid hmmolysis, and the protection against oleic acid is not absolute, but the degree of resistance against this acid was notwholly worked out. Ether in large quantities fails to lake thevenom-protected corpuscles.
The Rapidity and the Limits of the Protective Action.--In orderto determine the rapidity of the fixation of the corpuscles b yvenom a series of tests was made. A io per cent solution of
cobra venom was prepared in 0. 9 per cent salt solution, and to
this washed horse corpuscles were added in quantity of 3 percent. The mixture was allowed to stand at 20 ° C. for the periodof the experiment, and at the intervals mentioned in the table
portions were withdrawn and tested against salt solutions of different degrees of hypotonicity. A parallel series of tests was
carried on to determine what concentration of venom in o.9 percent saline solution is necessary to protect the corpuscles against
suspension in water. In the latter series a contact of five hours* 2 % saponin in the mixture caused no hmmolysis after io days.

Hideyo Noguchi
was employed.
experiments:

The following table presents the results of these
TABLE

DETERMINATION OF RAPIDITY 0F ACTION.
Pe~od ofc o n t a c t ofvenom and

corpuscles.

Reaction to sodium chloride
solution.

5 minutes

Partial

io

Trace

sol.

209

VII

DETERMINATIONOF MINIMALLIMIT OF ACTION.

ofvenom solution,

Reaction to sodium chloride
solution.

Concentration

hmmolysis in 0.3 %~%h~emolysis in o,3 %

3%

Trace

4%

No h0emolysls in w a t e r .

s%
6%

No

20

sol.
No h~emolysis in 0.3 % sol.;3060

No h~mo!,ysis in water.

t r a c e in w a t e r .

Complete h~emolysis in 0.7 %sol,h~emolysis in o.x % sol.

''

No
No
No
~'
8go
Control (no venom) Complete hmmolysis in 0,44 %sol.

Estimation of the Protective Action o~ Moccasin and Crotalus
Venom.--The tests under this heading were made in the manner
previously given. Different strengths of the venoms were prepared in 0. 9 per cent saline, and to them washed horse corpuscles were added in 3 per cent suspension. The mixtures wereallowed to stand for six hours at 2o ° C., and were then tested
against salt solutions of varying tonicity. The removal of thevenom in some of the experiments was done by means of o. 9 percent saline.
TABLE V I I I
CROTALUS VENOM.

WATER-MOCCASIN VENOM,

C o n c e n t r a t i o n s ofvenom.

20 %
~o%
s%
i%
O.I~
0.01 ~0ControI (no venom)

Ve nO1Tl n o t
Venom removed.
removed,i Venom r e m o v e d
Tonicity of NaC1 Tonicity
of NaC1 Tonicity of NaC1
sol.
sol.
sol.

Venom n o t
removed.
Tonicity of NaCI
sol.

0.54 %
0.528 %
0.5%

o.i8 %

0.57

~0

%

02

0.2

%

o.49
0.47
0.454
0.44

0.465 %
0 - 4 5 4 °To
0 . 4 4 4 v/v

~o
%
%
%

0.25

0.3

o.48
0.47
0.47
0.465

%
%
%
~v

0.45

%

o.44

07o

0.35

%

0 . 4 6 5 6~0

0.45

%

0.445 %

The above table brings out the interesting fact that, whilemoccasin and rattlesnake venoms possess the property of210

Protective Action of Snake Venom

protecting blood corpuscles, they are in this respect much inferior
to cobra venom, With neither moccasin nor rattlesnake venom
was there produced absolute protection against water hmmolysis,
It m a y be worth while to point out in this place that in ultimateh~emolytic power with defibrinated horse and dog corpuscles
these venoms were about the equal of cobra venom, althoughcrotalus venom acts more slowly and is quantitatively a little
weaker t h a n the others,* The fact of the similarity in hmmolytic
power and dissimilarity in protective action is another proof ofthe distinction between the several principles causing the two
phenomena.
TABLE I X
P e r i o d of c o n t a c t b e f o r e h e a t i n g .

Time required for complete h~emolysis a t 53 ° C.

Immediate~o m i n u~ J t e s
3°60
2 hours6"

io minutes.
II
20

38
3°
~5

"
i,

The Influence of Small Amounts of Venom upon Heat Hemolysis.--In the course of the experiments a phenomenon cameunder m y observation which will now be described. Mixtures of
washed corpuscles and venom of low concentration were exposed,
after varying intervals of contact, to the temperature of 53 ° C.
At first the effect, as measured by the degree of heat h~emolysis,
is small, but after a time it becomes marked.
However, after astill longer interval of contact the acquired resistance undergoes
diminution. A series of tests is given in Table IX. The mixture
exposed to heat consisted of 20 per cent washed horse corpuscles* 17or complete hmmolysis under the conditions mentioned, crotalus venom
requires at 20 ° C. twenty-four hours and, in addition, occasional agitation oft h e t u b e s . If t h e c o m p a r i s o n of h ~ m o l y s i s b e m a d e a f t e r s i x h o u r s a n d a t t h e
u p p e r l i m i t of d o s a g e , t h e d i f f e r e n c e b e t w e e n c o b r a a n d m o c c a s i n v e n o m , o n t h e
o n e h a n d , a n d r a t t l e s n a k e v e n o m , o n t h e o t h e r , m a y b e a s i : 20, b u t if t h e r e a d i n g
be t a k e n a f t e r t w e n t y - f o u r h o u r s i t will b e as low as 2 : 3 or, a t m o s t , 2 : 4.

Hideyo Noguehi

211

in 3° cubic centimeters of o. 9 per cent salt solution, and o.2 percent cobra venom in x.5 cubic centimeters of the same strength
salt solution. Four cubic centimeters were removed at a time
at different intervals and tested.
We can see in these results the opposed action of the two
properties of venom when acting together. The first effect todeclare itself is apparently partial fixation of the corpuscles, butwhen the venom solution is too weak to accomplish this entirely,
the directly injurious action of the poison makes itself felt.
Hence it would seem that with such weak solutions of cobra
venom as were employed in this experiment, the injurious action
upon the corpuscles, in the absence of available complement, wasmore marked than the protective action.

The Action of Venom upon the Blood Serum, the Stroma, andthe Soluble Contents of the Blood Corpuscles.--With a view ofascertaining whether venom forms with the blood constituentsany difficultly soluble compound, it was tested separately upon
the fluid and solid portions. Horse blood being used, the serum,
the stroma, and the corpuscular contents soluble in water were
tested against various venom solutions.
The stroma was prepared by laking the twice-washed, condensed corpuscles. The final product was a gelatinous mass ofpale rose color, which could be suspended in water or in saline
solution. The contents of the corpuscles were readily obtainedby laking with water and centrifuging. They could also be
diluted to any degree with water or salt solution.
The results given in the table show clearly that venom is a
precipitant for certain of the constituents of the blood. The
chief precipitate is obtained with the water-soluble contents ofthe corpuscles, although a partial reaction is also obtained withdiluted serum. The stroma reacts in a minor degree with thevenom, but whether or not this reaction depends on the venom
agglutinin or is due to a precipitant in the real sense was notdetermined.
The precipitate formed with the contents of the corpuscles inpure water is dense, viscid, and finely divided, while in saline
solution it is coarser and less in amount. The original color ofProtective Action of Snake Venom

212

~o

~:~

~

.

~d
00~
o~
~o

8

oo~

I

~o
~~ ~ ~.~~ ~oo~~ ~ ~..~'~

~o'~

~

r~ ~

.

o~-~
d

~

~o

o

'~'~

o','~ ~ o

~ ~ o ~'r~o~"-d 0)~~ ~.'~
r'~oe

e
o~

o~

.~.~,~~ ~.
o~m

~o~O

I

~.~ ~'~
d

~o
o

r~
o

~.~

~'~'~

~o

~;~

o

t~

g
0

~o

o

o

~o~

~o

o

oom

m

o. l

o

~o~

oo~

Hideyo :Noguchi

213

the solution is bright red from the presence of dissolved oxyhmmoglobin, b u t venom quickly changes it to dark brown. The pigm e n t is carried down with the precipitate in the watery mixture,b u t in the saline mixture it remains for some time in the supern a t a n t fluid. Salt, in certain concentrations would seem, therefore, to prevent, more or less, the formation of the precipitate.
Indeed, if the saline solution described is further diluted withwater a more a b u n d a n t precipitation takes place.
Since the protected corpuscles are quickly dissolved by acids
and alkalis, the question naturally arises whether the precipitateis also easily soluble in these chemicals. It was found that acids
and alkalis dissolve the precipitate in approximately the same
degree as they are capable of bringing about hmmolysis of thevenom-treated corpuscles.

The Action o] Venom upon the Separated Constituents of Serumand Corpusdes.--Venom, as has been stated, causes a dense precipitate with an aqueous extract of washed corpuscles. When,however, sodium chloride is present in the solution in a strength
equalling or exceeding 0. 9 per cent, the precipitation does nottake place, or occurs only imperfectly. Now, it is m y belief t h a tvenom owes the protective action upon corpuscles described toits ability to form a compound insoluble in water with certain
corpuscular constituents. It is, therefore, desirable to ascertain
w h e t h e r any particular constituent of the corpuscles is concernedin the formation of the insoluble substance, or whether all theproteids of the cells are fixed by the venom. Moreover, sincesalt in weak concentration is able to prevent the development of
the precipitate in the corpuscular extract, it is also possible t h a t
it prevents, in serum, a similar reaction. The next experiments
were made with serums in which the saline percentages had been
reduced by dilution with water. The results of these tests are
given in Table XI.
That simple dilution with water affects the precipitability of acompound formed by venom and serum when brought together,is shown by the results given in the table. Another way of approaching this problem is to remove the salts of the serum by
dialysis. This was done for the serum of the horse and dog, andProtective Action of Snake Venom

214

dialysis was continued until a considerable amount of globulin
had separated from the serums. It was found that only a traceof chlorides remained in the fluid. The two parts--fluid andprecipitated globulin--were tested separately. The globulin
which had separated belonged chiefly to the euglobulin fraction,
but there still remained in the fluid parts of the serums considerable globulin precipitable by one-third saturation with ammonium sulphate. During the dialysis the volume of the serums
had increased two and one-half times.
TABLE X [I~[IX T U R E a .

MIXTURE B.

MIXTURE C.

U n d i l u t e d s e r u n M i x t u r e A 0.2 c . c .
i.o c. e.
W a t e r i.o c . c .
20 % c o b r av e n o m o 2 c. c.

MIXTURE

D.

Serum diluted with 4
Serum diluted
vols. of w a t e r x.o c.c w i t h 4 vols. of o.0% s a l t solution
2o% cobra venom
0,2 C. C.

T.o C. C, 20 ~0
cobra v e n o m 0.2
c.c.

_J
Horse

serum

Rabbit

"
"
::

Rat

Guinea-pig
Dog
Pigeon"Crotalus plasma

Clear

Moderate precipitate Much
precipitate
Moderate"/ Moderate
"
Much
::
[ Much::. . . . . . /Much precipitate, re-Is e m b l i n g fibrin.
}

C!?ar

::
. . . .

R B M A R K S : Controls without v e n o m remained perfectly clear for m o r e than 24 hours, while thecontrols with v e n o m alone s h o w e d only a slight a m o u n t of precipitate in water a n d in 0.9 % salt
solution.

The dialyzed horse serum was tested as follows: First tube-i cubic centimeter of the fluid portion was mixed with o.2 cubic
centimeters of 20 per cent cobra v e n o m solution; moderate precipitation. Second tube--The same mixture plus sodium chloride to o.9 per cent strength; no precipitation. Next, theprecipitate of globulin was washed in water by centrifuging
and dissolved or suspended in salt solutions of the following concentrations :o.8, 0.6, and 0.4 per cent. In the firstthere was perfeet, in the second almost perfect, in the third imperfect solution.
To the clear filtrates the 2o per cent v e n o m solution was added,
and rapid and nearly complete precipitation of the dissolved
globulins occurred. W h e n the globulin is merely suspended, as
in w a t e r or w e a k saline, the v e n o m quickly brings down the sus-

tiideyo Noguchi

215

pended particles which, left to themselves, subside very slowly,
if at all. A comparison of the amounts of sediment obtained
from the fluid part of the dialyzed serum and the redissolved
globulin shows t h a t the globulin is more completely precipitablet h a n the other constituents.
It was now decided to fraction the serum, and for the purpose the m e t h o d of Freund and Joachim 26 was employed. The
fractions--euglobulin, pseudoglobulin, serum albumin--were
freed from a m m o n i u m sulphate by dialysis and tested with c o b r avenom. The most marked reaction was given by the first, thepseudoglobulin fraction giving a less reaction, and the serumalbumin fraction no precipitate. The results of the two seriesof tests, therefore, agree in showing the globulin to be the constituent acted upon by the venom.
The water-soluble contents of the corpuscles were also fractioned. The first fraction, yielded by one-third saturation,
was proportionately large and contained a part of the coloringm a t t e r of the corpuscles. The second fraction, obtained by onehalf saturation, was less in a m o u n t than the first, b u t contained
the greater part of the oxyhmmoglobin. The third fraction,
obtained by saturation, was small in quantity. The salts were
removed by dialysis.
The first two fractions were deep red in color. One cubic
centimeter of the solution of each was mixed with 0.2 cubic
centimeters of 2o per cent cobra venom; the result was precipitation. The second fraction gave the more a b u n d a n t precipitate,leaving the fluid clear of coloring matter. In order to obtain
similar clearing of the first fraction additional dilution with water
was required. The third fraction yielded no precipitate with thevenom solution.
Since in this series of experiments the globulins and haemoglobin were associated and it was desirable to know whether thereaction takes place between b2emoglobin and venom, as well
as between the globulins and venom, separation of the hmmoglobin in a pure state was necessary.
Oxyhmmoglobin from horse corpuscles was prepared by Zinoffsky's ,z method, while that from dog's corpuscles was prepared by216

Protective Action of Snake Venom

laking the washed corpuscles with ether and removing the stroma
in the separatory funnel. The d e a r solution of haemoglobin was
shaken with a large volume of ether and allowed to stand fortwenty-four hours at o ° C. A large quantity of crystalline oxyhaemoglobin, which was purified by being twice recrystallized,
was thus obtained.
For testing against venom the haemoglobin was used in o.2
to o.i per cent solutions in water or 0.9 per cent sodium chloride.
To i.o cubic centimeter of the solutions of oxyhaemoglobin o.~
cubic centimeter 20 per cent venom was added. The results
were as follows: in the presence of sodium chloride no reaction
occurs ; in aqueous solution there is complete precipitation of thehaemoglobin of the horse, but no reaction with that of the dog.
That pure haemoglobin m a y react with venom is shown by theseexperiments ; t h a t the reaction is similar to that with the globulins in respect to solubility in salt is also shown; and t h a t adifference in composition exists between dog's and horse's oxyhaemoglobin the experiments also indicate.
In view of these results it seemed desirable to test venom
directly upon globin, which was prepared from the oxyhaemoglobin of the horse and dog by Schulz's ~s method. The solutionsof globin were neutral to litmus and gave the reactions describedby Schulz. To i.o cubic centimeter of globin solution o.i cubic
centimeter of 2o per cent venom was added. Dog's globin gave
no precipitate; horse's globin gave a voluminous precipitate
which was easily soluble in acids and alkalis, but not in salt solution. Indeed, a small quantity of salt hastens the appearanceof the precipitate with horse's globin.
This series of experiments would tend to show that the precipitability of oxyhaemoglobin by venom depends on the globin
constituent, and that the globins of horse and dog corpuscles are
distinct substances as regards, at least, their reaction with venom.
The question has already been mooted as to what the natureof the protective process described in this paper really is. Thatthe phenomenon is independent of the haemolytic reaction has
been conclusively proven. Moreover, the immediately preceding
experiments show that venom is a precipitant for several proteids

Hideyo Noguehi

217of the blood. It is doubtless upon the reaction which takes
place between the venom and these precipitable proteids that thephenomenon of protection depends. One of the striking appearances of the reaction is the carrying down of the hmmoglobin by
the venom; and it seems not improbable that this property
might be utilized as a measure of the protection of the corpuscles,
for when it is absent it is found that venom does not shield the
corpuscles from water laking. The following table shows that
this relation actually exists:
TABLE XII.
cobra-venom solution 2 . 0 e . c .
Washed blood corpuscles o . 3 c . c .

20 %

e

Origin oi"
orpuscles.

Horse

i

Behavior of the cor- Behavior of the cor- Precipitation of Solubility of the prepuscles whenpuscles left in the•the haemoglobin. ! cipitate* ino. 9 % sod.
[ chloride solution.
above mixture.
brought into water.

No

heemolysis

Nohsemolysis

RabbitRat
Guinea-pig
Dog
PigeonCrotalusi % intracorpuseular contents * . o c. c .
2 o % cobra-venom solution o.t c. c.

Ii¢,

Complete h~em,olysL, Com,p,lete h~emolysis
SlightIncomplete
"
No
"

I
!Dissolves but reprecipitates on standing.
Almost complete Partially soluble ; large
fluid retains
precipitate on standfaint color.
rag.
Complete
Slightly soluble
None
Complete

SlightEas!lysoluble.

* The precipitate was easily soluble in different acids and alkalis.

Yet, since it is not only the hmmoglobin which is precipitatedby venom, but the intracorpuscular globulins as well, it is notpossible to describe the phenomenon of protection as if it depended wholly on a reaction with the former proteid. It is
probable that all the precipitable proteids are acted upon andaffect the result, although it seems not unlikely that the fixationof the hmmoglobin in an insoluble form is what directly prevents
its diffusion out of the corpuscles into the surrounding medium.
In the course of this paper allusions to a change in the permeability of the corpuscles have been made. The question as towhether the so-called protected corpuscles are altered in their
physical condition so as to exclude certain substances, which
under normal conditions easily enter them, is pertinent. It is218

Protective Action of Snake Venom

to be considered whether the rendering of certain of the proteids
insoluble is not associated with an obstruction to osmotic p r o cesses taking place between the corpuscles and the surrounding
medium. If in the case of water and the protected corpuscles
this fluid was successfully excluded from the corpuscles by aninsoluble although superficial barrier, laking could not occur.
However, m a n y of the experiments described show t h a t theabsence of laking does not depend upon changed conditions of
osmosis, b u t upon the formation of insoluble compounds.
A set of experiments was undertaken to determine whether,at the same time, the permeability of the corpuscles may not havebeen altered. For this purpose saponin was employed, since, as
stated, Kobert has shown t h a t saponin causes hmmolysis by virtue of the fact t h a t it dissolves the lecithin of the corpuscles.
Now, the failure of saponin to act upon the protected corpuscles
could be due either to their impenetrability for saponin, or to theinsolubility of the venom-h~emoglobin compound in the mens t r u u m in spite of the change of the ceils caused by a solutionof the lecithin in saponin. The following experiment was devised with a view of determining the nature of the protection
from saponin.
Twenty per cent cobra venom in salt solution was treated with
washed horse corpuscles (5 cubic centimeters of venom plus 0. 5
cubic centimeters of corpuscles) for three hours at the room temperature. The corpuscles were rendered water-protected andalso non-hmmolyzable when treated with 2 per cent watery solution of saponin.
Two cubic centimeters of the venomized corpuscles were
dropped into 15° cubic centimeters of distilled water; sedimentation was complete in twenty hours. The corpuscles weretwice washed in the centrifuge with water. Examined microscopically they were entangled with venom granules. These corpuscles were protected perfectly both from water and o. 9 percent salt solution. The sediment was mixed with an equal volu m e of 2 per cent solution of saponin, and the mixture wasallowed to stand for ten minutes. The corpuscles were diluted
and washed in water until all the saponin (determined by testingItideyo Noguchi

219with unprotected corpuscles) was removed. A thick suspension
in water of the saponin-venomized corpuscles was now added
separately to water and to o.9 per cent saline solution (o.2 cubic
centimeter of suspension to 2 cubic centimeters). The salt solution quickly caused laking, while, as was to be predicted, thewater was without effect.
The interpretation of this experiment is probably as follows:
The saponin enters the venom-protected corpuscles in spite of
their fixation and takes up the lecithin, whereby the corpuscles
are seriously damaged. However, as the venom-hmmoglobin
compound is insoluble in water, no visible expression of thisdamage can occur. Since the venom-hmmoglobin compound issoluble in o. 9 per cent saline solution, it quickly dissolves in thelatter whether it is capable of leaving the corpuscles or not.
Unless the corpuscles have been previously injured, the dissolved
hmmoglobin compound does not diffuse into the medium; but
in view of the action of the saponin upon the corpuscles a sufficient injury has been inflicted to allow the h~emoglobin to escape.
Venom protection of blood corpuscles does not seriously affectthe permeability of the corpuscles, but it prevents, under many
conditions, the diffusion of the haemoglobin into the surrounding
medium.
SUMMARY.

Washed blood corpuscles of certain species of animals in aconcentration of about 5 per cent suspended in salt solution containing above 4 per cent of cobra venom undergo changes intheir resistance to certain physical and chemical agents. They
become non-haemolyzable by water, ether, saponin, and quite
strong solutions of lecithin, provided always that the excess ofvenom has not been entirely removed. On the other hand, certain acids and alkalis, excepting ammonia, lake the venomized
corpuscles more easily than they lake normal corpuscles. Venom
solutions of 2 per cent and less exert no protective property uponblood corpuscles, but they induce changes in the corpuscles
whereby they are rendered more easily laked by the same physical and chemical agents.

220

Protective Action of Snake Veno~,~The changes in the corpuscles upon which this protection
relies can be set aside by repeated washings of the venomized
corpuscles in 0. 9 per cent salt solution. When the protection isremoved the corpuscles are left in a state of diminished resistanceto injurious agencies.
The substance in venom upon which the protective action depends is not destroyed by the temperature of 95 ° C., although at
this temperature a part of the venom is coagulated. The protective body is found in the coagulated portion ; while the venom
haemolysin is contained, in full activity, in the clear filtrate.
The agglutinin for red corpuscles disappears at 75 ° C. ; hence,the protection of corpuscles by venom depends neither upon
the haemolysin nor the agglutinin.
Blood corpuscles absorb the haemolysin from concentrated
solutions of venom as readily as from weaker solutions. This
fixed haemolysin can be brought into action, under suitable conditions, by the addition of complements. But the haemolysin isunable, in the presence of complements in excess, to cause lakingof the corpuscles unless the protection produced by the venom
has first been set aside.
Cobra venom forms a precipitate with blood serum when comparatively free of salts or when diluted with water; it forms a
precipitate with a watery extract of blood corpuscles, and withthe separated globulins, haemoglobin and globin of the blood.
The precipitates are insoluble in water, b u t dissolve in weakacids and alkalis. The stroma of the red corpuscles causes nochange in venom solutions.
Protection of the corpuscles against haemolysis depends upon
the formation by v e n o m of a water-insoluble compound withcertain of the corpuscular constituents, chiefly probably withhaemoglobin. There is no reason to believe that the permeabilityof the corpuscles is greatly altered.
The restoration of the susceptibility of the corpuscles to theordinary laking influences is accomplished by dissolving thevenom-proteid compound. Since this compound is soluble inacids and alkalis and even in a large excess of salt solution,
restoration is easily effected. Corpuscles which have been pro-

Hideyo Noguchi

221

tected and again rendered hmmolyzable are less resistant to injurious influences than normal corpuscles, resembling in thisrespect corpuscles which have been treated with relatively weakersolutions of venom. Venom, therefore, always exerts a direct
injurious influence upon blood corpuscles; but strong solutionsof venom may obscure this injurious effect through the predominance of the protective action which they display.
Not all blood corpuscles are susceptible to the protective action of venom, and different corpuscles display varying degrees
of capacity for protection. Dog corpuscles are not protected bycobra venom, and watery extracts of dog corpuscles, as well as
pure oxyh~emoglobin and globin of this animal, are not precipitated by venom solutions.
Crotalus and moccasin venoms possess this protective property,
but in less degree than cobra venom.
I wish to express my gratitude to Professor Flexner for his
encouragement and for many valuable suggestions during theexecution of this work.
ADDENDA.

After the completion of this study the paper of Sacharoff andSachs (Mi~nchener reed. Wochenschrift, i9o5, lii., 297) on thehmmolytic action of photodynamic substances came to my attention. I therefore tested the protected corpuscles of the
rabbit by exposing them directly to sunlight in the presenceof eosin. It was found that the protected corpuscles are notsubject to the h~emolytic action of this florescent substance;
while the corpuscles which have had the protection removed bymeans of salt solution are readily h~emolyzed by eosin when exposed to the sun's rays. In keeping with the other experiments
upon the injurious effects of smaller and non-protective dosesof venom, it was also found that venomized but unprotected
corpuscles are more readily hmmolyzed by eosin in the lightthan the normal corpuscles.
Another fact may be added. The supervenomized corpuscles
have become insusceptible to tetanolysin.

222

Protective Action of Snake Venom
BIBLIOGRAPHY.

1. Mitchell a n d Reiehert.--Smithsonian Contribution to Knowledge, 1886,
No. 547.
2. C . J . M a r t i n a n d McG. Smith.--Journal and Proceedings of Royal Societyo] New South Wales, ~892 , xxvi, 240.
3. Phisalix.--Compt. rend. de la Soc. de Biologic, 1899, 834, 865.
4. Kanthack.--Rep. of Medical Officers o] Loc. Gov. Board, L o n d o n ,1895-1896, 235.
5. Mitchell a n d Stewart.--Trans. College of Physicians of Philadelphia,~:897, xix, io 5. Memoirs of the National Academy of Sciences, 1898, viii.
6. S t e p h e n s a n d Myers.--Brit. Med. Journal, 1898, P a r t I, 621. M y e r s . - Journal of Pathology and Bacteriology, 1899-i9oo, vi, 415, Stephens.--Journalof Pathology and Bacteriology, i899-19oo, vi, 273.
7. C . J . M a r t i n . - - ( 2 ) .
8. George Lamb.--lndian 2~edical Gazette, 19Ol, x x x v i , 443.
9. Flexner.--American Journal of the Medical Sciences, 19o3, exxvi, 202.
lO. George L a m b . - - ( 8 ) .

1I. Cunningham.--Scientific .~iemoirs by the Medical Officers of the Army o]India, 1895 , P a r t I X , a n d 1898, P a r t X I .
12. F l e x n e r a n d Noguchi.--Journal of Experimental ~/Iedicine, x9o2 , vi, 277.
x 3. Calmette.---Compt. rend. d. l'Acad6mie de Sciences, 19o2 , cxxxiv, 1446.
14. Kyes.--Berliner klin. Wochenschrift, i9o2, x x x i x , 886, 918; i9o3, xl,
956 , 982.
15. F l e x n e r a n d NoguchL--Journal o~ Pathology and Bacteriology, I9o3, viii,
379. University of Pennsylvania Medical Bulletin, 19o3, xvi, 163,
16. Mitchell a n d S t e w a r t . - - ( 5 ) .
7. S t e p h e n s a n d M y e r s . - - ( 6 ) .
18. F l e x n e r a n d N o g u c h i . - - ( i 2 ) .
~9. Kyes.--Berliner klin. Wochenschri~t, 19o2, xxxix, 886, 918.
2o. Kyes a n d Saehs.--Berliner klin. Wochenschrift, 19o3, xl, 21, 57, 82.
2i. Kyes a n d S a c h s . - - ( 2 o ) .
22. F l e x n e r a n d N o g u c h i . - - ( i 2 ) .
23. Neisser and Wechsberg.--:~Ii~nchener reed. Wochenschrift, 19Ol ' xiv, 697.
24. D e t r e a n d Sellei.--W$ener klin. Wochenschri]t, i9o4 , xvii, 1311.
25. Kobert.--Beitrage z~tr Kenntnis der Saponinsubstanzen. S t u t t g a r t ,19o4, 48.
26. F r e u n d a n d Joachim.--Zeitschr. f. physiologische Chemic, 19o2 , x x x v i ,4o7.
27. Zinoffsky.--Zeitschr. ]. physiologische Chemic, i886, x, 16.
28. Sehulz.--Zeitschr. ]. physiologische Chemie, 1898, xxiv, 449.

29. Lamb.--Scientific ~Vlemoirs by Officers of the Medical and Sanitary Departments o~ the Government o] India, 19o3, No. 4, 8.