野口英世著

Journal of Experimental Medicine

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A BIOCHEMICAL STUDY OF T H E P H E N O M E N A
K N O W N AS C O M P L E M E N T - S P L I T T I N G .
FIRST PAPER: SPLITTING OF THE COI~PLEMENT ASSOCIATED WITHGLOBULIN PRECIPITATION.*

BY JACOB BRONFENBRENNER AND HIDEYO NOGUCHI.
(From the Laboratories of The Rockefeller Institute for Medical Research,and the Laboratory of Biolo#ical Chemistry of Columbia University,
New York.)
INTRODUCTION.

During the last few years much attention has been directed
toward a phenomenon known as the splittingof complement. The
conception is that complement, when treated according to certain
procedures, can be decomposed into two definite portions each ofwhich is by itselfinactive but can act in combination with the otheras complement. The two components of "the complement differ intheir behavior in hemolytic processes; one has the property of beingbound by the sensitized erythrocytes, while the other possesses alytic action upon sensitized cells that have been acted upon by the
firstcomponent. Thus, according to certain investigators,complement is composed of an intermediary portion, called the mid-piece,
and a lyric portion, called the end-piece. The terms "mid" and"end" are employed because the conception is that the mid-piecestands between the sensitizedcellsand the lyticportion of the complement, while the lytic portion when viewed graphically, becomesthe end-piece.
In view of the high labilityof complement it is natural to ask
if the phenomenon of splittingbrought about by certain procedures
may not be due to some modifications of the complement, such asa reversible inactivation through changes in the reaction of themedium in which the complement is present. It appeared to us ofinterest to approach this question from a biochemical standpoint.
In our firstpaper we deal with the splittingphenomena which are* Received for publication, April I, 1912.

598

Jacob Bronfenbrenner and Hideyo Noguchi.

599

associated with the precipitation of the globulin fraction, and in thesecond with those which occur without visible change in the physicalconditions of the serum proteids.
HISTORICAL.
In fresh blood serum Landois found a hemolytic action upon alien corpuscles,
and Fodor, NuttaU, and others, a bactericidal property, but none of the early
investigators made exhaustive studies upon the nature of the active principles.
It was Hans Buchner w h o firstpursued the study in a systematic manner andthrew much light on this interesting phenomenon. Buchner (189o) establishedthe fact that the active principle causing bacteriolysis or hemolysis is very labileand can be made inactive by a temperature of 55° C., by dialysis, or by dilution
with distilled water. The active principle was designated by him alexin.
Bordet (1899) subsequently found that the alexin of Buchner is composedof two distinct principles, one a sensitizing substance and the other a thermolabile alexin. Somewhat later Ehrlieh and Morgenroth (1899) made a similar
observation, but by Ehrlich the sensitizing portion was called the amboceptor,
and the alexin the complement. In this study Ehrlich and Morgenroth mention
that the complement becomes inactive when mixed with water. Markl (19o2)
observed that sodium chlorid in concentration above 3 per cent. suppresses theactivity of the complement.
While the disappearance of the complement activity after heating the serum
to 55° C. was recognized by all as due to the destruction of the active principle
at this temperature, no one was able to explain why the complement should lose
its action in distilled water. This question was difficult to approach inasmuch
as in a hypotonlc salt solution a hemolytic experiment could not be carried out.
Ferrata (I9O7), however, utilized the well known fact (noted by Hamburger,
Madsen, and Arrhenius), that the corpuscles can be kept intact when suspended
in an isotonic solution of sugar, and he studied the action of the salt-free complement (dialysed) in a salt-free isotonic medium, and found the complement to beinactive in this medium. Ferrata found also that during dialysis the serum
separates into two portions, one the serum globulin (precipitate) and the otherthe albumin fraction (in solution). When tested separately in an isotonic salt
solution neither of them caused hemolysis, while when united the action of complement was restored. He affirms that the component of complement remaining
in the albumin portion is inactivated at 55° C.
The finding of Ferrata was confirmed by Brand, who showed further thatthe component carried down with the globulin fraction attaches itself to thesensitized corpuscles and that the component in the albumin fraction will afterwards act upon the cells so altered, He introduced, therefore, the terms midpiece (Mittelstiick) for the former and end-piece (Endstiick) for the latter.
According to Brand the mid-piece of complement remains active when kept
as precipitate (globulin) in distilled water, but loses its property on standing insalt solution. Brand and Tsurusaki (19o8) found the mid-piece as well as theend-piece inactivated at 55° C.
Hecker (19o7) and Sachs and Bolkowska state that the binding of the mid-

600

Biochemical Study of Complement-Splitting.

piece takes place at o ° C. as well as at higher temperatures, while that of theend-piece occurs only at higher temperatures. Thus they claim that the selective
absorption of the natural hemolysin by means of corpuscles at o ° C. effects not
only the fixation of the amboceptor, but also the mid-piece of complement.
Sachs and Teruuchi (19o7) observed that the complement loses its activity
in distilled water or isotonic sugar solution when the degree of dilution with distilled water is about I : IO, but not in much higher dilution. They were inclined
to believe that the inactivation of the complement was caused by a certain ferment of the serum capable of exerting an injurious action only in a salt-free
medium. They state that the inactivation of complement in a salt-free medium
does not take place at a low temperature or in the presence of an excess ofamboceptor. Complement a few days old does not undergo similar alteration(inactivation with water, etc.).
Tsurusaki found that the so-called splitting of complement is quite inconstant and seems to depend upon the quality of the dialysing membranes, upon
the variations in different individuals of the same species of animal, and upon
the temperature. For instance, the unaltered complement of rabbit was foundin the supernatant fluid when dialysed at o ° C.
Sachs, and Sachs and Altmann (19o8) then introduced a new method of
splitting the complement. They used a weak solution of hydrochloric acid by
which a certain portion of serum is precipitated (globulin). The precipitate
corresponds with the mid-piece, and the snpernatant fluid with the end-pieceobtained by dialysis.
Later Michaelis and Skwirsky found that when corpuscles, amboceptor, andcomplement were put together in a medium containing a certain quantity of
NaH2PO, and Na2HPO4 in proportions resulting in isotonicity, there was nohemolysis. But on further analysis, they found that in this mixture the corpuscles had fixed not only the amboceptor, but also the mld-piece of complement. The corpuscles so modified were called persensitized, because theyreadily underwent hemolysis when the neutralized supernatant fluid (containing
the end-piece) was added. In other words, persensitization can take place in a
medium containing an excess of NaH2PO, which suppresses the action of theend-piece, while the activity of the end-piece can be restored by neutralizing theexcess of the acid phosphate with Na~HPO4.
Liefmann and Cohn introduced carbon dioxid for splitting the complement,
whereupon the mid-piece is precipitated and the end-piece remains in solution.
This phenomenon occurs only in a serum diluted with a sufficient quantity ofwater. They studied the influence of certain acids and alkalis and concluded
that carbon dioxid exerts a far more inactivating effect than the acids or
alkalis studied by them. Cholesterin inactivates the mid-piece in a salt-free
medium. From the fact that the persensitization of corpuscles is affected witha minute amount of the mid-piece, they were inclined to think that this component acted like a ferment, an assumption held also by Ruszny~k.
Jacoby and Schutze state that the complement inactivated by shaking andalso the precipitate that is formed may be reactivated either by the mid-pieceor the end-piece. On the other hand, the supernatant fluid of the shaken complement becomes active only with the end-piece.
Complement is known to be carried down by certain colloidal substances

Jacob Bronfenbrenner and Hideyo Noguchi.

60I

through an adsorption (Landsteiner). Skwirsky confirmed this and points out avery interesting difference.existing between the colloidal adsorption and the specificcomplement fixation as it occurs in the Bordet-Gengou or the Wassermann
reaction. According to this author, later confirmed by Amako, in the former
instance the end-piece as well as the mid-piece is carried down from the solution, while in the complement fixation the mid-piece alone is absorbed, leaving
the end-piece free in the supernatant fluid.
Landsteiner, Liefmann and Cohn, and Fr~inkel state that the mid-piece prepared by the hydrochloric acid or carbon dioxid method from sera of different
animals can persensitize the corpuscles ~[or the end-piece of guinea pig serum,
but not vice versa. This phenomenon is ascribed by them to the presence of
natural amboceptor in the fraction containing the told-piece. Fr~inkel points
out certain differences in the behaviors of mid-pieces derived from the seraof different animals when these are acted upon by cholesterin, or sodium.
chlorid, or inactivated by the temperature of 57 ° C.
Marks observed that the reactivation of the end-piece by the mid-piece occurs
when the latter is employed in a smaller quantity than the ratio of I : I . He.
found that the heated as well as the fresh serum can furnish an active midpiece, but a prolonged heating of the mid-piece at 55 ° C. finally destroys its:
function.
Liefmann studied the relation of certain lipolds and soaps to the mid- andend-pieces of complement and found that they have no effect upon the end-piece,.
but that they destroy the mid-piece. He brought out also the fact that p e r sensitization can not take place unless the amount of amboceptor reaches nearly
fifty units.
That the active principle of complement undergoes an inactive modificationin the presence of various acids, alkalis, and salts has been carefully studied b y
Noguchi, yon Liebermann, Hektoen, and others. According to the experimentsof Noguchi, the complement is permanently injured by a strong solution o fthese substances, especially by the acids and alkalis, but when used within acertain limit, the activity can be restored with but slight loss of power.
EXPERIMENTAL PART.
T h e r e a r e a t l e a s t five d i f f e r e n t p r o c e d u r e s b y w h i c h it is c l a i m e d ~t h a t c o m p l e m e n t m a y be s p l i t ~ i n t o t w o c o m p o n e n t s . S a c h s e m p l o y shydrochloric acid; Liefmann, carbon dioxid gas; Ferrata, dialyzes
a g a i n s t d i s t i l l e d w a t e r ; M i c h a e l i s a n d S k w i r s k y u s e a m i x t u r e ofa c i d a n d a l k a l i n e p h o s p h a t e s ; t h e f i f t h m e t h o d is t h a t o f s p l i t t i n g
by complement fixation.

"Complement-splitting" is the term generally adopted by English speaking
bacteriologists. This is the reason for the employment of the term in thispaper, although a better word might be substituted, and the following work:
almost necessitates its replacement by the term "inactivation," since this is what:
has really been proved to take place in almost all cases of so-called splitting.

~,602Biochemical Study of Complement-Splitting.

In our first paper we shall consider only the first three methods.
In these the globulin fraction of the serum is precipitated duringt h e process of splitting.
COMPLEMENT-SPLITTINGWITH HYDROCHLORIC

ACID.

The procedure introduced by Sachs is as follows: One cubic~.centimeter of guinea pig serum (complement) is mixed with 8.2
cubic centimeters of N/25o hydrochloric acid solution (in distilledwater), and after standing at room temperature for one hour the
mixture is centrifugalized, whereupon the precipitate settles down
to the bottom leaving a clear supernatant fluid above. The supernatant fluid is carefully separated from the depositS and is mixed
with 0.8 of a cubic centimeter of N/25 sodium hydroxid solution (inIO per cent. sodium chlorid). The addition of alkali is intended toneutralize the acid introduced into the serum for splitting, while theresultant solution is made isotonic at the same time (about o. 9 per
cent. sodium chlorid). The deposit is washed With water andfinally collected by centrifugalization, and then dissolved in I O cubic.~entimeters of o. 9 per cent. sodium chlorid solution. The supernatant fluid and the precipitate, both now in clear solution, repre:sent the end- and mid-piece, respectively. Thus, the supernatantfluid has no action upon the sensitized corpuscles unless the precipitate portion is added, and vice versa. Apparently the complement
is separated into two components.
Experimental evidence was also brought forward to show thatt h e end-piece or supernatant portion carries the labile zyrnotoxic
component, and the mid-piece, or precipitate portion, possesses theproperty of enabling the end-piece to act upon corpuscles sensitizedby amboceptor, and that it is not destroyed by the temperature of
5 6o C.
The above experiments have been confirmed by various workers,
but we have been able to corroborate the results in part only, inasmuch as the supernatant fluid was found to be active upon sensitized
corpuscles in most of our experiments (table I, experiment II) andwas very seldom inactive.
Finding that the supernatant fluid was often active without the addition of
mid-piece, we took the precaution of filtering the end-piece through a hardened
filter paper in order to be certain of the absence of the mid-piece.

Javob Bronfenbrenner and Hideyo Noguchi.
TABLE

603

I.

Complement-Splittlng by Hydrochloric ,'lcid.
Experiment I (Ideal Case). s
Guinea pig serum x c.c. + N/25o H C I 8.2 c.c. (in distilled water).
Centrifugaliz~d after z hr. at room t e m p e r a t u r e ,Amounts taken.

Supernatant fluid I Deposit dissolved
LT ~÷ ' .o 8 c "c " N/z5 1
In ro c.c. ol~....
~ : ~ . e r c e n t . [ 0. 9 per cent. NaCI.

Guinea pig serumx c.c. "[9~9c-~ NaG[~•
, I After standing z hr.
t~qualbo " 'namounts
e ' " n oI i at room temoeraturetUl g r me ts. [(control).

2 units of amboceptor and o.z c.c. of a xo ~ suspension of corpuscles and the total
volume made up to z5 c.c. Results taken after x hr.
I.O

C.C.

0.7 c,c.
o.5
0.4
0.3
0,2
o.I5
O.I

c.c.
c,c.

N o H. 4

N o H.

No H.
No H.

No H.
No H.

C,H.

C.H.
C.H.
SI.H.
Tr.H.
No H.

c.c.
C.C.

c.c.
C.C.

CoM.

F.C.H.
Tr.H.

Experiment II (Usual Splitting).
Guinea pig serum z c.c. -t- N/=5o HC1 8.2 c.c, (in distilled water).
Centrifugalized after x hr. at room temperature,

Amounts
taken,

Supernatant fluid-{- 0.8 c.c. N/25 N a O H
in xo~ N a t l .

Deposit dissolved inzo c.c. of 0. 9 '$ NaCl.

Equal volumes of bothingredients,

Guinea pig serumx c.c. + 0. 9
NaC19 c.c.
After standing atroom temperaturefor x hr. (control).

2 units of amboceptor and o.z c.c. of a xo ~ suspension of corpuscles added to each tube and thetotal volume made up to x.5 c.c. Results taken after 3° and 6o min.
3° rain.

I.O C.C.
0.7 c.c.
o.sc.c.

0.4 c.c.
0.3 c.c.
0.2

C.C.

o.I5 c . c .
o.I c.c.
o.o7 c.c.

C.H.
C.H.
F.C.H.
Mch.H.
S1.H.
No H.

5o min.

3° 1rain.
ain.

No H.
No H.
C.H.

C.H.
C.H.

6o
5o rain. I[

No H.
No H.

3o rain.

6o min.

6o rain.
3° rain. I

C.H°

C.H.
C.H.
C.H.
F.C.H.
No H.

C.H.
C.H.
C.H.
C.H.
C.H.

C.H, [ C . H °C.H. C.H.
SI.H. Mch.H.
No H. No H.

a T h r o u g h o u t this work 0.9 per cent. sodium chlorid solution was used instead of o.85 per cent. usually employed by o t h e r workers.
• I n this a n d the following tables, f o r the sake of brevity, certain symbols
are employed to indicate the degree of hemolysis; thus C.H. = complete hemolysis; F.C.H. = fairly complete hemolysis ; SI.H. = slight hemolysis; Tr.H. =trace of hemolysis; No H. = no hemolysis; Mch.H, = much hemolysis.

604

Biochemical Study of Complement-Splitting.
TABLE

II.

The Effect upon Components of Guinea Pig Serum of Heating for 30 Minutes
at 56o C.
Experiment I?
Experiment II.
Guinea pig serum heated at 560 C.
Fresh guinea pig serum * c.c.+8.2 c.c. HC1 N/25o (in distilled water), for 30 minutes + 8.2 c.c.HCI N/25o(in distilled water).

Kept at room temperature for 6o
Kept at room temperature for 6o minutes, centrifugalized and supernatant minutes, centrifugalized and sufluid separated, neutralized, and made isotonic by o.8 c.c. of N / a 5
peruatant fluid separated, neutralNaOH in zo~ NaC1.
ized, and made isotonic by o.8 c.c,of N/25 NaOH in *o~ NaCI.
o.z5 c.c. of superuatant fluid, u units of amboceptor, and o.x c.c. ofxo~ suspension of corpuscles in each tube.

o.2 c.c. of supernatant fluid, 2units of amboceptor, and o.z c.c.
of zo~ suspension of corpuscles
in each tube.

Results taken after :Results taken after :3° rain. 6o rain. 9° min. 30 min. 6o min. 9° min.

+o
No H
+ A l a n i n o.3 e.e. of 7.5% solution in
o.9 % NaC1.
C.H.
+ D e p o s i t from splitting i n ( o.I5 c . c . C . H .
experiment I dissolved.~
in Io e.c. 0.9% s a l t [solution.
~. o.07 c . e . C . H .
+ D e p o s i t from splitting in [" o.2 e.e. No H
experiment I I dissolved.)
in Io e . c . o . 9 % salt s o - ]ution.
Lo.I c.e.C.H.'ocontrol,

S1.H.

C.H.

NoH.

NoH. NoH.

C.H.

C.t~.

C.H.
C.H.

N o H . No H.i No H. N o H.
N o H . N o H . N o H . No H.

C.H.

C.H.

S1.H.

C.H.

NoH. NoH. NoH.
N o H . N o H . N o H . No H.

C.H.

C.H.

NoH. No.H NoH.

While the phenomenon suggests a double constitution of the complement, yet it does not exclude the possibility of its being causedby an excess of acid or alkali. Guinea pig serum, when mixed withhydrochloric acid in the proportion prescribed in this procedure,
becomes almost neutral or but faintly alkaline. This causes theprecipitation of certain proteids, mainly the globulins. Thus theacid introduced is quickly taken up by certain alkaline salts of theserum and there is none left free. The precipitated globulins alsobind a certain amount of the acid. The supernatant fluid which isalmost neutral and contains chiefly the albumin fraction of theserum, is then mixed with sufficient free alkali to neutralize exactlys T h e t o t a l v o l u m e w a s b r o u g h t up t o x c.c. by 0.9 p e r cent. s a l t s o l u t i o n inboth experiments.
" T h i s phenomenon does not occur constantly. W i t h some specimens ofc o m p l e m e n t ( v e r y f e w ) t h e m i d - p i e c e is u n d o u b t e d l y d e s t r o y e d by h e a t i n g t h e
w h o l e s e r u m p r e v i o u s to t h e s p l i t t i n g , but i t is e a s i e r t o d e s t r o y t h e m i d - p i e c e
a f t e r its s e p a r a t i o n .

605

Jacob Bronfenbrenner and ttideyo Noguchi.

the amount of acid added, but this amount of alkali is in excess
when added to the serum mixture from which the globulin fraction
was removed after its precipitation by hydrochloric acid. Thus it
happens that in the serum treated with the acid and then neutralizedwith the alkali in the manner prescribed, in the presence of theglobulin fraction, the complement action is restored. On the other
hand, if the globulin fraction is first removed and the same amountof the alkali is added to the remaining supernatant fluid, the complement activity of the supernatant fluid is paralyzed. The examiTABLE

III.

Demonstration of Complement Inactivation by Sodium Hydroxid in theProcess of Complement-Splitting.
Tubes.
.

2

x

3

4

I
]

5

6

t

Guinea pig serum . . . . . '1.oc.c.
1 . 0 C.C.
I.O C.C.
I.O C.C.
I.O C.C.
I.O C.C.
N/25o HCI . . . . . . . . . . .
[ 8.2 c.e.
8.2 c.c.
o.9 % NaC1 . . . . . . . . . . .
I
Distilled water . . . . . . :. [8.2 c . c .
After I hour at room temperature all the tubes were centrifugalized. F r o m tubes 1
and 2 only supernatant fluid was used; from the others the entire contents of the tubes wereused.
N/25 N a O H in xo%
L
NaC! . . . . . . . . . . . . . .
0 . 8 C.C.
o,8 c.c.
[
I o % NaCI . . . . . . . . . . .
-I 0.8 c.c.
0.8 c.c. I
I[ 0.8 c.c.

i

ISupernatant fluid
T h e entire contents of the tubes
Reaction of resulting
fluids to alizarin-in- 5.6 c.c.
2.5c.c. 2 . 5 C.C.
6 . 2 C.C.
digo 7 (calculated for HCI
NaOH
HCI
NaOH
the whole volume).
N/~So
N/~5o N/25o
N/=5o
Nl25
N125o
Complement activity of
the resulting mixtures(with o.z c.c. of xo%
suspension of corpuscles and 2 units ofamboceptor.
C.H.
.kf I.o c.c.
No H.
S1.H.
[ 0.7 c.c.
Tr.H.
| 0.5 c.c.
C.H.
/ 0 . 3 C.C.
No H.
,H.
F.C.H.
C.H.
1 0.2
c.c.
~.C.I-I.
F.C.H.
Mch.H.
C.H.
~ ~ | o.~5 c.c.
¢Ich.H.
Tr.H.
F.C.H.
~ |o.i c.e.
qo H.
<
( 0.07 c.c.

H tcc H :cc

~

NoH

o"H

v T h e u s e o f this indicator, first i n t r o d u c e d by B e r n h a r d t , w a s s u g g e s t e d tou s by P r o f . W . J. Gies. T h e indicator is n o t noticeably influenced by t h e
p r e s e n c e o f p r o t e i d s o r c a r b o n dloxid.

606

Biochemical Study of Complement-Splitting.

nation of the so-called neutralized supernatant fluid for alkalinity
indicates a slight excess of alkali over that present in the same
dilution of the untreated serum.
That this slight excess of alkalinity is not solely responsible forthe loss of action of the untreated complement, but that the natureof the alkali employed has more to do in paralyzing the complement
seems very probable. Thus, the free alkali in the form of sodiumhydroxid fails to restore the original alkaline salts, and exerts aninjurious effect upon the active complement constituents. That
this is the case is easily shown by the experiments in table IIL
These experiments show that the supernatant fluid from which
the native alkalinity has been almost completely removed byhydrochloric acid still has nearly two thirds of the original complement power of the serum. But by adding gradually increasing
amounts of sodium hydroxid, the activity is steadily reduced (tableV) until in some cases it disappears completely (table V I ) , when
the amount of sodium hydroxid reaches the point supposed to benecessary to restore the original alkalinity of the treated serum.
The completeness of the inactivation of the complement action of
the supernatant fluid is also enhanced by a slight excess of the freesodium hydroxid toward which the complement shows a great susceptibility. It may be assumed that the salts that cause the native
alkalinity, principally the carbonates and phosphates, are split byhydrochloric acid and form sodium chlorid and the acids, 'but thegreater part of carbon dioxid leaves the fluid, thus causing the freealkali introduced to act directly upon the complement.
It is interesting, however, that, when the serum was acted on byhydrochloric acid and sodium hydroxid was subsequently added,
without removing the globulin fraction, much if not all of thecomplement activity was restored (table IV, A).
The reason for sodium hydroxid not causing the same injurious
effect upon the complement in this instance (table IV, A) must be
due to the presence of the globulin fraction which seems to have agreater affinity for the free sodium hydroxid than the albumin
fraction in which the active principle of the complement seems toreside.
That the globulin fraction has a greater affinity for sodium

Jacob Bronfenbrenner and Hideyo Noguchi.
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608

Biochemical S t u d y of C o m p l e m e n t - S p l i t t i n g .
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Jacob Bronfenbrenner and Hideyo Noguchi.

609hydroxid than the albumin fraction is easily demonstrated by experiments described later.
In order to ascertain whether the loss of complement action in
the albumin fraction, the so-called end-piece, is really due to areversible inactivation through the action of sodium hydroxid, suchas had been observed by Noguchi in his earlier experiments, andnot due to the removal of one of the interdependent components,
the mid-piece, it was necessary to make a series of reverse experiments with an acid.
The following experiments show that the complement activity of
the inactive end-piece can be restored to a great extent by means ofneutralization of the alkali with adequate amounts of hydrochloric
acid (table V I I ) .
We now come to consider the mechanism of the restoration of
the complement activity of the end-piece by the globulin fraction
known as the mid-piece. From the reactivation experiments withhydrochloric acid, it is not improbable that the combination of thecomplement and sodium hydroxid can also be split by means of anamphoteric compound. Among the amphoteric substances we findvarious proteids, proteoses, and many non-coagulable synlhetic
amino acids. The serum globulins are undoubtedly amphoteric.
In the present experiments we have also used egg-white, alanin,
deuteroalbumose, and peptone. 9
These experiments (table V I I ) demonstrate that the complement action of the so-called end-piece can be readily restoredby adding adequate quantities of the amphoteric substances herein
employed. One may wonder how such a weak acid as the aminoacid, which does not form a salt at ordinary temperature, canreverse the combination of the alkali and the complement fraction.
But, it must be remembered that the affinity between the complement and alkali cannot be stronger than that which exists between
any other amphoteric bodies. Moreover, the direction of the reversion is dependent upon the total number of acid radicals possessedby a given substance. Thus, a greater amount of the substance is*We are indebted to Dr. F. J. Birehard, of The Rockefeller Institute for
Medical Research, for the albumose and peptone preparations. The albumose
was prepared from fibrin according to Pick's method, and the peptone fromWitte's peptone according to Sigfried's method.

Biochemical Study of Complement-Splitting.

610

0

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Y~ , ~ . ~oJacob Bronfenbrenner anc~ Hideyo Noguchi.

Ji

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e$

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v
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-

et2

Biochemical Study of Complement-Splitting.

required if it possesses fewer acid radicals. That the globulins
have a greater affinity for sodium hydroxid than the albumins may
be accounted for by the larger size of the molecules of the former.class of proteids. The solubility of the substances seems also to beimportant, as certain insoluble amino acids, such as asparagin,asparagin acid, and leucin failed to reactivate the end-piece.
COMPLEMENT-SPLITTING WITH

CARBON DIOXID.

Liefmann splits the complement in the following manner. One
half of a cubic centimeter of guinea pig serum (complement) ismixed with two cubic centimeters of distilled water, and carbon
dioxid gas is passed through until no more precipitate is formed.
The treated serum is then centrifugalized to separate the precipitatefrom the supernatant fluid. The clear supernatant fluid is carefully,decanted and filtered through hardened filter paper into anotherreceptacle and is used as the end-piece after being made isotonic
with sodium chlorid. The deposit, which represents the globulin
fraction of the serum, is then dissolved in 2. 5 cubic centimeters of,0.85 per cent. salt solution. This portion is used as the mid-piece.
In a certain number of instances the phenomenon of splitting isideally shown, the end- and mid-pieces separately having no action
upon the sensitized corpuscles (table X). The two components~btained by the carbon dioxid method behave in much the same way:as those prepared by the hydrochloric acid method of Sachs. It
may be stated, however, that the end-piece portion remains quite
active in many instances, as has been observed also by Liefmann
and others (tables IX and X).
In considering the processes that lead to the inactivation of the~end-piece by carbon d~oxid and the subsequent reactivation of thisportion by the mid-piece of the globulin fraction, one can conceivethat an essential difference exists between the inactivation by carbon
dioxid and that by the hydrochloric acid method. In case of thehydrochloric acid-splittlng, the carbonates as well as other alkaline
salts of the serum are completely converted into chlorids. Small
amounts of free carbon dioxid or other weak acids remaining in thefluid cause a partial inactivation of the complement.
On the other hand, the passage of carbon dioxid through aJacob Bronfenbrenner and Hideyo Noguchi.

o

Z

q

0

~

0

z

°

u

~,~1~ ~" ~ ao~ :: ~~
"L~.

::'~'~~ ::'~.
~ :: ~' ~

613

614

Biochemical S t u d y of Complement.Splitting.
6

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~ ~.~.~'~'~'~.~1~ ~ ~ ~1 . . "~.
8"~'~'~.

~oJacob Bronfenbrenner and Hideyo Noguchi.

615

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616

Biochemical Study

o

o

o

of Complement-Splitting.

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Jacob Bronfenbrenner and Hideyo Noguchi.

617

TABLE XII.
Reactivation of Complement Inactivated by Carbon Dioxid.
Amounts of reactivating
substances.

' I.O

I0.3 c.c.
0.2

C.C.

~.~ o.I5 c.c.
= ~0 0 . I
I::I I o.oS

c.c.
c.c.
c.c.
c.c.
C.C.

, o.o3
66 "~~ 0.02
0.01
~.
0
~ 0.005 C.C.
o -,-,

Mid-piece

Egg-white{CO2),

I: IO,

Sheep
serum, 3°rain. at56°C. z:5.

Protoalbumoses 5 ~.

Deuteroalbumoses 2

C.C.

~,~ i o.5
0.7 c.c.
c.c.
~ 0.4 c.c.
.~ ~~Alanin 3 ~.

C.H,
C.H.
C.H.
F.C.H.
Mch.H.

SI.H.
Mch.H.
Mch.H.
C.H.
Mch.H.
SI.H.

C.H.
C.H.
C.H.
C.H.

S1.H.
S1.H.
F.C.H.

No. H,S1.H.
Mch.H.
SI.H.

No. H.
Tr.H.
Mch.H.
SI.H.

0

mixture of guinea pig serum and water precipitates the globulin
fraction just as in the case of the hydrochloric acid treatment, but
the carbonates as well as phosphates remain unaffected by this treatment. The inactivation of the complement in this instance is doubtless due to the overcharging of the serum water with carbon dioxid
gas, which by virtue of its acid nature combines with the amphoteric molecules o f the serum albumin fraction in which the activityof the complement is residing. In a medium, whether in the undiluted native serum or in a serum diluted with sodium chlorid solution of isotonic concentration, in which the sodium chlorid content
reaches a sufficient concentration (table X I ) , a weak acid like carbon dioxid fails to enter into even a loose combination with thealbumin molecules, and hence leaves the complement comparatively
uninjured. The reason that carbon dioxid acts upon the complement molecules in serum sufficiently diluted with water is that thestrong electrolyte, sodium chlorid, can in that dilution no longer
interfere with the action of a weak acid. In diluted serum carbon
dioxid causes an acid inactivation of complement and the activity o fcomplement may be restored by certain amphoteric substances (table
XII) and also by an alkali (table XV, C), but not by an acid.
The phenomenon of reactivation by means of adequate quantities of sodium hydroxid may raise a question as to why the result--,618Biochemical Study of Complement-Splitting.

6o

o

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d

o~~

~9

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°~

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6

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C
c

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:

Jacob Bronfenbrenner and Hideyo Noguchi.

619

ant N%CO~ or NaHCO~ no longer exerts the same effect as the freesodium hydroxid and carbon dioxid, since these are readily dissociable in the fluid. But one must not forget that in the stage ofreactivation the fluid is now made isotonic with sodium chlorid, andthis restrains the dissociation of carbonates or phosphates sufficiently to prevent the free action of the carbon dioxid or sodium
ions upon the complement fraction. It is important to notice thatan acid has no reactivating property for the end-piece prepared withcarbon dioxid, while exactly the opposite is the case with the endpiece made with hydrochloric acid, which is an inactivation byalkali.
In the experiments in table X I I I we show that amphoteric
substances capable of reactivating complement inactivated by carbon dioxid lose this reactivating property if treated with carbon
dioxid before being added to the inactive complement. The way
in which amphoteric substances reactivate complement is thus clearly
demonstrated.
C O M P L E M E N T - S P L I T T I N G BY M E A N S OF DIALYSIS.

Before Sachs, Liefmann, and others, introduced various methodsof splitting the complement, Ferrata discovered that by dialyzing
complement against water through a fish membrane or a celloidinsac, it could be separated into two inactive components, an albumin
fraction corresponding with the end-piece of the later investigators,
and a globulin fraction (which precipitates during dialysis) corresponding with the mid-piece. This result, although not always
obtained, was nevertheless confirmed by other workers.
On several occasions in the present series of experiments we havealso confirmed this finding. Our explanation for this phenomenon
is, however, entirely different from that given by previous workers.
As will be shown in the following protocols, the supernatant fluid(albumin fraction) when separated from the precipitate (globulin
fraction) and made isotonic with sodium chlorid, is quite inactive
upon the sensitized corpuscles, but can be reactivated by adding,
not only the globulin fraction of the same serum (isotonized withsodium chlorid), but also the globulin fraction prepared by thehydrochloric acid or carbon dioxid methods (table X I V ) . Indeed,Biochemical Study of Complement-Splitting.

620

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Jacob Bronfenbrenner and Hideyo Noguchi.

621

the reactivation may be even better and more constant with midpieces prepared by these other methods than with its own mid-piece.
Further, it is found that an acid (table XV, B) or certain amphoteric substances which bear no relation to the serum (table X I V )
TABLE

XV.

Reactivation of Complement Inactivated by Acid (Carbon Dioxid) Compared
to Reactivation of Complement by Alkali (Dialysis or Distilled Water).
Complement + distilled water (i:to) at room temperature for x hr.; the whole
made isotonic ; o.5 c.c. used in each tube.
Incubationtime.

+ Alanin 7.5 5$.

02C.C.
~ hrs.

x hr.

2 hrs.

x hr.

2 hrs.

N o H.
No H.
No H.

No. H.
Tr.H.
S1.H.

Tr.H.
S1.H.
Mch.H.

No. H .
S1.H.
S1.H.

S1.H.
F.C.H.
F.C.H.

C.HY-

N o H.

No H.(?)

No H.
N o H.

No H.
No H.

x hr.

O.I
o.~25
o.I5

4"

No alanin.

O,3C.C.

Control

F.C.H.

F.C.H.

0.I
0.2
o.3
0. 4
o.5

N o H.
No H.
Tr.H.

NoH.
NoH.
Tr.H.(?) i No H.
Tr.H.
i S1.H.(?)

z

No. H.
Tr.H.
Sl.H.

End-piece of complement dialyzed for z2 hrs. against running water ; madeisotonic ; final dilution x:xo; 0.3 c.c. in each tube.
Incubation time.

+ Alanin 7.5 ~.
0,2C.C.

x hr.

°:L 5

[oi5

2 hrs.

x hr.

No H.
No H.
No H.

Control~ I 0"I
0.2
0.3
0.4
Z

0.5

No alanin.
O.3C.C.

No H
No H
Tr.H.

I

2hrs.

xhr.

2 hrs.

N o H.
SLH.
SI.H.

No H.
Mch.H.
Mch.H.

No H.
S1.H.
SI.H.

No H.
Mch.H.
F,C.H.

C.H.

C.H.

No H.

Tr.H.

N o H.
No H.
Tr.H.

NO H .
No H.
No H.

Biochemical Study of Complement-Splitting.

622

End-piece (CO2) for xo min. ; made isotonic; final dilution x:io; 0. 3 c.c.
in each tube.
Incubation time.

+ Alanin 7.5 ~.

No alanin.
0 . 3 C C.

z hr.

~
-

fO.I

N o H.

~

N o H.
N o H.

o.I2S~ 0"I5hr.

2 hrs.

z hrs.

z hr.

2 hrs.

N o H.
N o H.
No H

No H.
No H.
N o H.

Tr.H.
Tr.H.
Tr.H.

Tr.H.

C.H.

N o H.

S1.H.

N o H.
Tr.H.
Mch.H.

Tr.H.
C.H.
C.H.

C.H.
C.H.

M

Control

S1.H.

~!0.I
0.2
0.3
0.4
o.5

No H.
Tr.H.
Mch.H.

Tr.H.
F.C.H.
C.H.

reactivate this dialyzed end-piece. On the other hand, alkalis do
not reactivate it (table XV, B).
From the experiments given above it becomes highly probablethat dialyzed serum loses its complement activity chiefly by analkali inactivation of a reversible character. The cause of this
TABLE

XVI.

Rate of Dialysis of Sodium Chlorid and NaHCO~ from the Mixture of Fifty
Cubic Centimeters of I per cent. Sodium Chlorid + Fifty Cubic
Centimeters N/5o NaHCO~.
NaCI
AgNOs ~H4SCN

CombineqAg

4.o c,c. 2.0C.C.
3.o c.c. 2°0C.C.
2.0 c.c. 1,2C.C,i hr.
i . o c . c . o.6c.c.
2 hrs.
o.5 c.c. o.4c.c,
3 hrs.
o.5 c.c. o.5c.c.
4 hrs.
o.5 c.c. o.5c.e.
R u n n i n g w a t e r i . o e.c. I.OC.C.

2.0C.C,
I.OC.Co

Afterleo2o min.
4o min.

o.8c.c,o.4c.c.
O.IC.C.
0
0
0

NaHC03
NaCi

H~SO4 I NaOH Combined
N/zoo ( N/zoo
acid0.02 gin.16.0 c.c .o c.c. 5.0 C.C.
o.0I gin.14.0 c.c.lI.o c.c. 3.0 c.c.
o.008 gin.12.5 c.c.lo.5 c.c. 2 . 0 C.C.
0.004 gin.12.5 c.c.lI.3 c.c. 1 . 2 C.C.
o.oor gm./2.o c.e.IL3 c . c . 0.7 c.c.
Trace(?) 11.5 c.c.io.9 c.e. 0.6 c.c.

NaHC03
N/so

2.5

C.C.

L5

C.C.

o

1.5 c.cqi.oc . c .

o.5 c.c.

I.O C.C.
o.6 c.e.
0.35 c.c.
0.3 c,c.
o.~5 c . c .

oi . 5 c . c . i . o c.c.

0.5 c.c.

0.2 5 c.c.

~6At t h e t i m e i n d i c a t e d , 5 c.c. o f t h e c o n t e n t s o f t h e c o l l o d i o n s a c w e r e r e moved and examined for hydrochloric acid and NaHCO,.

Jacob Bronfenbrenner and Hideyo Noguchi.

623

inactivation may be found in the gradual changes taking place inthe salt content of the serum within the sac during dialysis. As
may be expected, sodium chlorid passes out of the celloidin sac
with a greater velocity than the remaining salts, such as carbonatesor phosphates, and this together with an increase in volume (byendosmosis of water) results in removing the restraining power ofthe chlorids upon the dissociation of the carbonates. These salts
then inactivate the complement.
A study of the comparative velocity of osmosis of sodium chlorid
and Na2CO3 through a celloidin membrane is given in table XVI.
At the time indicated, five cubic centimeters of the contents of thecollodion sac were removed and examined for sodium chlorid andsodium bicarbonate.
SUMMARY.

It is generally accepted that complement may be split into a midpiece and an end-piece. The mid-piece is thought to be in the• globulin fraction, and the end-piece in the albumin fraction. The
restoration of complement activity by putting together the albumin
and globulin fractions does not prove, however, that each fraction
contained a part of the complement, for the albumin fraction can bereactivated in the absence of the globulin fraction.
Complement-splitting as brought about by hydrochloric acid,carbon dioxid, and dialysis, is really an inactivation of the wholecomplement by certain acids or alkalis; either added in the freestate to the serum, or liberated as a result of the dissociation ofcertain electrolytes.
That the whole complement, and not a part only, is present in thealbumin fraction of the serum can be demonstrated by the removalof the inhibitory action of the acid or alkali. This can be effectedby the addition, not only of alkali or acid, but also of any amphoteric
substance. When hydrochloric acid, carbon dioxid, or dialysis are
employed to produce the phenomenon known as complement-splltting, the complement is merely inactivated, not split.
BIBLIOGRAPHY.
Amako, T., Ztschr. f. Immunit~tsforsch., 191o, viii, 168.
Bernhardt, IT~ien. klin. H/chnschr., 1911, xxiv, 6o6.

624

Biochemical S t u d y of C o m p l e m e n t - S p l i t t i n g .

Border, J., Ann. de l'Inst. Pasteur, 1896, x, 193; 1898, xii, 688.
Brand, E., Berl. klin. Wchnschr., 19o7, xliv, xo75.
Buchner, H., Arch. f. Hyg., 1893, xvii, 138.
Buchner, H., and Orthenberger, M., Arch. ~. Hyg., 1890, x, 149.
Ehrlich, P., and Morgenroth, J., Berl. klin. Wchnschr., 1899, xxxvi, 481.
Ferrata, A., Berl. klin. Wchnschr., 19o7, xliv, 366.
Fr~inkel, E., Ztschr. ~. Immunitiitsforsch., 1911, viii, 781; 1911, x, 388.
Hecker, R., Arb. a. d. k. Inst. ~. exper. Therap., 19o7, No. 3, 39.
Guggenheimer, H., Ztschr. ~. Immunitiitsforsch., 1911, viii, 295.
Kiss, J., Ztschr. ~. Immuniti~tsforsch., 19o9-1o, iv, 703; 19o9, iii, 558.
Landsteiner, Centralbl. ~. Bakteriol., Re~., I9IO, xlvii, Suppl., 88.
von Liebermann, L., Arch. ~. Hyg., 19o7, lxii, 3o6; Biochem. Ztschr., 19o7, iv, 25.
Liefmann, H., Centralbl. ~. Bakteriol., Ref., 191o, xlvii, Suppl., 87; Mi~nchen.
reed. Wchnschr., 19o9, lvi, 2o97.
Liefmann, H., and Cohn, M., Ztschr. ~. Immunittitsforsch., I9IO, vi, 562; I9IO,
vii, 669; I9IO, viii, 58.
Markl, Ztschr. f. Hyg., 19o2, xxxix, 86.
Marks, H. K., Ztschr. ~. Immunitiits[orsch., 191o-11, viii, 508; 1911, xi, 18;]our. Exper. Med., 1911, xiii, 59o.
Meyer, K., Arch. ~. Hyg., 19o8, lxvii, 114.
Michaelis, L., and Skwirsky, P., Ztschr. ~. Immunittitsforsch., 19o9-1o, iv, 357,
629; Berl. klin. Wchnschr., I9IO, xlvii, 139.
Mutermilch, S., Compt. rend. Soc. de biol., 1911, Ixx, 577.
Noguchi, H., Proc. Soc. Exper. Biol. and Med., 19o7, iv, 45, 157; Biochem.
Ztschr., 19o7, vi, 327.
Noguchi, H., and Bronfenbrenner, J., ]our. Exper. Med., 1911, xiii, 43Ruszny~k, S., Ztschr. ~. Immunitiitsforsch., 1910-11, viii, 421.
Sachs, H., Centralbl. ~. Bakteriol., Re[., 19o8, xlii, Suppl., 174.
Sachs, H., and Altmann, K., Kraus and Levaditi's Handbuch der Technik und
Methodik der Immunit~itsforschung, Jena, 19o9, ii, 965; Berl. kiln.
Wchnschr., 19o8, xlv, 699.
Sachs, H., and Bolkowska, G., Ztschr. ~. Immunitiitsforsch., I9IO, vii, 778.
Sachs, H., and Teruuchi, Y., Berl. klin. Vdchnschr., 19o7, xliv, 467, 520, 6o2.
Skwirsky, P., Ztschr. f. Immunitiitsforsch., 191o, v, 538.
Tsurusaki, H., Biochem. Ztschr., 19o8, x, 345.