On the Behaviour of the so-called Components of the Complement at the Complement Fixation

(1)

53

On the Behaviour of the so-called Components of the Complement at the Complement Fixation

By

Fujimura, Shiro|Itibakase, Makoto

^{藤村,紫郎￨} ^{一番ケ瀬,眞}

(From the Biochemical Institute, Nagasaki Medical College, Japan)

Introduction

The complement, which is an important element both in lytic and opsonic action, was considered originally as an inseparable f ragment of the f resh serum. At an early time it was found out by Buchner that when the serum containing complement was dialysed against -water or diluted with distilled water it loses the complemental action. The separation of the complement into its components was attempted by many investigators, and the principle of their several methods was to apply a chemical procedure to the complement serum and to precipitate the globulin and separate it from the albumin which remains in the supernatant liquid. Namely Gerrata adopted for separation the dialysis of the complement serum against running water for twenty-two hours ; the precipitate thereby formed is collected by centrif uge as globulin. Sachs and Altmann separated the globulin f rac- tion from the complement serum by adding of diluted hydrochloric acid.

Lief m a n n's method depends an the precipitation of a portion of the globulin from the serum containing the complement by the action of a current of carbondioxide gas. By these procedures the complement serum is separated into two constituents, the so-called globulin fraction (mid-piece) and the albumin fraction (end-piece}, each of which is inactive by itself, but when mixed they acquire the property to cause haemolysis of ox red blood cor- puscles sensitized with the corresponding immune bodies as the original complement serum. The components of the complement, which were separated from each other up to the present, are the globulin component, the albumin component, both of which are heat labile, the third component and the fourth component. The third component, which is attacked by cobra venom or yeast, is usually associated almost entirely with the globulin component, but small quantities of it are found in the albumin fraction ; and the fourth component, which is made inactive by adding of ammonium hydroxide, is included in the albumin fraction. On the behaviour of these complement components at the complement fixation many authors have investigated.

Michaelis and Skwirsky have observed that at the complement fixation

not all the component of the complement are used entirely, but only the

globulin fraction is fixed. Skwirsky has cleared that at the luetic reaction,

the tuberculin-antituberculin reaction and the specific complement deviation,

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54 s. Fujimura and M. Itibakase

the globulin fraction is only used and the albumin fraction remains free in the solution. Gengon has shown that at the complement fixation besides the globulin fraction of the complement, one part of the albumin fraction is fixed by antibodies. These results about the behaviour of the complement do not' entirely coincide. After the experiment reported by D e i s sler it is evident that at the complement fixation the so‑called fourth component is fixed at first. Also M i sawa reported that at the various complement fixation reactions the fourth component is at first fixed, but the third component receives no marked influence. In a number of such experiments about the complement components, for the method of separation of the components first of all the splitting by the current of the carbondioxide gas according to Liefma,nn's procedure was adopted, which consists as follows : one volume of serum containing complement diluted with nine volumes of ice‑cold distilled water is splitted by passing carb"ondioxide gas through the mixture for a period of ten minutes, and then the thus formed precipitate is removed by centrifuging. To the clear supernatant fluid which consists of the albumin fraction, sodium chloride is added and made up its content to 0.85 per cent. The precipitate which consists of the globulin fraction, is washed twice with distilled water and then dissolved in an amount of 0.85 per cent.

salt solution equivalent to ten volumes of the original serum. Then from these fractions the third or the fourth components are inactivated by the action of yeast or the addition of arnmonium hydroxide respectively. It was found that the globulin fraction and the albumin fraction obtained according to the above procedure are not completely separated from each other. The authors fractioned the globulin and albumin fractions thus obtained by I /3, Ij2 or 1/1 saturation of ammonium sulphate into three portions, the euglobulin, the pseudoglobulin and the albumin fraction, and measured their contents. The results indicate that the globulin fraction contains all the euglobulin and a small amount of the pseudoglobulin in the complement serum, while the albumin fraction includes almost entirely all the pseudoglobulin besides all of the albumin in the complement serum.

The amount of pseudoglobulin included in the albumin fraction varies with the conditions of the experiments, sometimes it reaches 90 per cent. of the total pseudoglobulin in the serum. The authors attempted to separate the complement serum in the three fractions of albumin, pseudoglobulin and euglobulin as completely as possible and to demonstrate how each fraction reacts against antibodies at the complement fixation.

Experilnental

I. Separation of the complement.

The method adopted to separate the complement into its components

is as follows. Guinea‑pig serum was always used as complement. One

volume of fresh guinea‑pig serum was diluted wit. h nine volumes of 0.85

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On the Behaviour of the so‑caued Components ot the Complement etc. 55 per cent. sodium chloride solution and then was added to it neutralized saturated ammonium sulphate solution to 1/3 per cent. saturation of it.

The precipitate thus obtained is collected by centrifuging and washed twice with 1/3 per cent. saturated ammonium sulphate solution, then it is dissolved in a small amount of water and dialysed in an ice‑chest against distilled water in a collodium membrane to remove the ammonium sulphate as

thoroughly as possible, until the inner solution indicates no precipitation of barium sulphate with barium chloride solution, since it has a marked inhibiting effect on the complemental action. For this purpose it requires from 7 to 8 hours under application of suitable pressure on the internal solution and occasional refreshing of the outside water. When the ammo‑

nium sulphate is entirely removed, the precipitation of the euglobulin occurs again, then both the precipitate and the supernatant liquid are diluted toge‑

ther to ten volumes of the original serum and made up to 0.85 per cent.

concentration by adding of sodium chloride. This fraction is the euglobulin.

The diluted serum after removal of the euglobulin as precipitate is made up to I /2 per cent. saturation of ammonium sulphate and the precipitate thus obtained is collected by centrifuge as before and washed twice with 1 /2 saturated ammonium sulphate solution, then dissolved in a small amount of water and dialysed against distilled water in a collodium membrane in an ice‑chest as in the case of the euglobulin. Also in this case at the end of the dialysis the precipitation of the pseudoglobulin occurs. The content of the membrane is diluted to ten volumes of the original complement serum and made up to 0.85 per cent. concentration by adding of sodium chloride. This fraction is the pseudoglobulin. The remaining fluid freed of euglobulin arid pseudoglobulin is saturated with ammonium sulphate and the thus formed precipitate of albumin is collected by filtration on hardened filter paper on the sucktion filter and dissolved in a small amount of water and the dialysis in the ice‑chest, dilution with water to ten volumes of the original serum and adding of sodium chloride to 0.85 per cent. are performed as in the case of both globulin fractions. This fraction is the albumin.

About each of these three fractions of the complement serum and all their possible combinations the complemental action was examined for ox red blood corpuscles sensitized with the corresponding antibodies from rabbit serum. For this experiment to the series of test, tubes 0.5 cc of 3 per cent.

suspension of sensitized ox red blood corpuscles and an increasing amount of each fraction of the complement were added at simple or multiple com‑

binations and incubated for a period of 1.5 hours in 37'C and then the degree of haemolysis was observed.

The result is as follows :

Amount ot each fraction o.05 cc 0.1 0.2 0.3 0.5 0.7 1.0 1.2 1.5

Albumin fraction ‑ ‑ ‑ ‑ ‑ ‑ ‑ ‑

Euglobulin fraction

Pseudoglobuun fraction ‑ ‑ ‑

‑ ‑ ‑ ‑ ‑ ⁺

Alb. + Eug.

‑ ‑ * ‑ ‑ ‑ ‑ +h i+f

(4)

56 S．Fujimura an（i M．Itibakase

Alb．十Pseudog．十冊掛惜冊帯帯惜 Eug．十Pseudo9．一一十層什｛什

Alb。十 Eug．十Pseudo9，汁惜辮柵惜・冊什琳 Native serum（1：10）十冊柵惜榊惜枡惜柵

From these experiments it is found that fQr the occu皿ence of the complemental action the pseudoglobulin and albumin fractions in the com−

plement serum seem to be necessary components and together only these two fractions form a system practically as active as the native serum．The euglobulin fraction appears to have no marked effect on the complemental action and therefore each fraction by itselfjs without any complemental action． This fraction can be neglected in our purpose．

II．Experiments about the behaviour of the components of the complement at the complement fixation．

In this experiment we wished to determine，in a positive case of Was−

sermann，s reaction and other complement fixation reactions，which fraction of the complement serum，albumin or pseudoglobulin，is fixed by antibodies or otherwise both fractions are fixed．

The complement fixation test was performed according to Browning，s method．The antiserum was inactivated by heating at56。C for30minutes，

ahd diluted10times with physiological salt solution，and O．5cc of it was taken． In selecting the luetic serums those were adopted which fix the complement over6units at the existence of antibodies and乱ntibody detector

（Antigen in vitro）．As an antibody detector in Wasserm我nn，s reaction to an alcoholic extract of beef，s heart，which was prepared by extraction of l part of minced beefys heart muscle with9parts of absolute alcohol for several days at room temperat皿e with occasional shakings，was added l per cent．cholesterin alcoholic solution in the ratio3：2and．diluted with physiological salt solution to30times volume of it as quickly as possible．

And in an interval of20minutes to2ho皿s after the dilution with salt solution it was used駄s antibody detector．The amount of this emulsion which Just fails to prevent the haemolysis of sensitized ox red blood cor。

puscles containing two units of complement，was found to be1．O cc．

In Wassermann，s test we took half of this amount of the emulsion．

At the other complement nxation reaction an antialbumin serum and anti−

dog，s heart extract serum were used．The antialbumin serum was prepared by immUniZing rabqbitS three timeS With1．O CC Of l per Cent．eggalbUmin

（M：erck）solution，with an interval of5to6days between each injection．

The antidog，s heart extract serum was prepared by immunizing rabbits

once with water extract of minced dog，s heartmuscle．As an antibody

detectαr in these cases l per cent．eggalbumin solution and an emulsion of

alcoholic Oxtract of dog，s heartmuscle in physiological salt solution which

was prepared by adding30volumes of physiological salt solutio血to l volume

of alcoholic extract of minced dog，s heartmuscle，were used，and they were

taken in half of the amount of these soluしions which Just fail to prevent

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On the Behaviour of the so 3alled Components of the Complement etc. ^'57 the haemolysis of a haemolytic system containing two units of complement.

In a series of test tubes, 0.5 cc of 10 per cent. antiserum, a fitting amount of antibody detector and inc. reasing amounts of both necessary components of the complement, albumin and pseudoglobulin, were put in and well mixed.

After a lapse of 1.5 hours in an incubator at 37'C with occasional shakings, 0.5 cc of sensitized ox red blood corpuscles and an increasing amount 'of one of the necessary components of the complement, albumin or pseudoglobulin, were added and the mixture was put into the incubator for I hour and l 5 minutes. The degree of haemolysis was then examined.

Results The

VI. In

complete and (+h)

(A)

results of the experiments stated above are shown in table the tables, cases where no haemolysis occurs is shown as (‑), haemolysis as ( }), while at intermediate cases the signs ( ), are used.

Luetic serum.

I to

and (+)

Table I

Ist nuxture : a n t i ser u m

antibody detector

albumin and pseudoglobulin fracfions of the complement 2nd mixture: haemolytic system

albumin fraction of the complement

Unit of complement No. of

experiment 2 4 6 8 10 12

2

Table I I

Ist rmxture : a n t i s e r u m

antibody detector

albumin and pseudoglobulin fractions of the complement 2nd mixture: haemolytic system

pseudoglobulin fraction of the complement

Unit of complement No. of

experiment 2 4 6 8 10 12 1 i+ + i{+ i+f i+} +f}

2 i+ {+} + ii+ i+f i+}

3 + {+f #f +ff ftf ii+

4 +}* + +it i+} #} 4tf

(6)

58 S．Fujimura and M．Itibakase

（B） Antialbumin serum．

Table III 1st mixture＝

2nd mixture：

antiserum

antibody detector

albumin and pseudoglobulin fractions of the complement

haemolyticsystem

albuminfractionofthecomplement

Unit of complement

No。of

experiment

1 2 3 4

2 4 6

十

8 辮

10

十柵柵

12

惜惜柵惜

Table IV

1st mixture：

2nd mixture：

antiserum

antibody detector

albuminandpseudoglobulinfractionsofthecomplement haemolyticsystem

pseudoglobulinfractionofthecomplement

Unit of complement No．of

experiment

1 2 3 4

2

什枡十

4 報惜冊柵

6 世掛惜柵

8 惜惜柵惜

10

継冊柵冊

12

惜惜柵惜

』（C） Antidog，s heart alcoholic extract serum。

Table V

1st mixture＝

2nd mixture：

antiserum

antibody detector

albuminandpseudoglobulinfractionsofthecomplement haemolyticsystem

albumin fractionofthecomplement Unitofcomplement

No．of experiment

1 2 3

2 4 6 8

什惜

10

什惜冊

12

柵

惜

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On the Behaviour of the so‑called Components. of the Complement etc.

59

Table VI Ist nuxture: antiserum

antibody detector

albumin and pseudoglobulin fractions of the complement 2nd mixture: haemolytic system

pseudoglobulin fraction of the complem nt

Unit of complement No. of

experiment 2 6 ro

+}f ⁴^+f ^++f+ff ⁸

+f +f}

As can be seen from these experiments, at Wassermann's reaction and also at the other complement fixation reactions the albumin fraction of the complement remains free in the liquid without being fixed by the antibidy, therefore when haemolytic system and the pseudoglobulin fraction of the complement are added, the albumin fraction which remains free acquires complemental action and causes haemolysis, while the pseudoglobulin of the complement is fixed by the antibody strictly so that by a supplement of albumin fraction of the complement and ha molytic system haemolysis can not be caused.

Next we examined the relation between antibody and complement com‑

ponent when antibody, antibody detector and only one necessary component of the complement, albumin fraction or pseudoglobulin fraction, are coexistent.

In a series of test tubes antiserum and antibody detector were put in as in the above experiments and an increasing amount of one component of the complement, albumin or pseudoglobulin, was added and well mixed.

After incubating this mixture at 37'C for 1.5 hours with occasional shakings, haemolytic system and the another component of the complement, albumin or pseudoglobulin respectively, were put in and again at 37'C for 1.25 hours incubated and then the degree of haemolysis examined

The result is as follows :

Results (A) Luetic serum.

Ist mixture :

2nd mixture :

No. of ex periment

1 2 3 4

Table VII antiserum

antibody detector

albumin fraction of the complement haemolytic system

pseudoglobulin fraction of the complement

Unit of complement

+}} +1+ + ･ ii+ {+f

+h + ii+ i+f + i+ {+f i + +ff +w ii+ ii+ + ii+ ii+

(8)

Table VIII

1st mixture：

2nd mixture：

antiserum

antibody detector

pseudoglobulinfractionofthecomplement haemolyticsystem

albuminfractionofthecomplement

Unit of eomplement

No．of

experiment

1 2 3 4

2 4 6 8

十

10

十什冊什

12

什汁柵柵

（B） Antialbumin serum．

Table IX

1st mixture：

2nd mixture：

antiserum

antibody detector

albuminfractionofthecomplement haemolyticsystem

pseudoglobulinfractionofthecomplement

Uniしof complement

No．of

experiment

1 2 3 4

2 十

什柵十

4 冊惜惜惜

6 惜辮柵冊

8 冊冊冊柵

10

柵柵柵珊

Table X

1st mixture：

2nd mixture：

antiserUm

antibody detector

pseudoglobulinfractionofthecomplement haemolyticsystem

albuminfractionofthecomplement

Unit of complement

No．of

exper玉ment

1 2 3 4

2 4 6

什

8 珊惜

10

冊冊冊帯

12

冊

枡

惜

(9)

On the Behaviour of the so‑called Components of the Complement etc.

₆₁

(O Antidog's ^heart alcoholic extract serum.

Ist mixture :

2nd mixture :

No. of

ex periment

l

2 3

Table XI antiserum

antibody detector

albumin fraction of the complement haemolytic system

pseudoglobulin fraction of the complement

Unit of. complement

6 8

+

i + ++} ii+

ii+

*+f

Ist mixture :

2nd mixture :

No. of

ex periment

1 2 3

Table XII antiserum

antibody detector

pseudoglobulin fraction of the complement haemolytic system

albumin fraction c f the complement

Unit of complement

+f}

i+ +

+ +

From these results it is evident that the albumin fraction of the complement is not fixed directly by antibody, but the pseudoglobulin frac‑

tion also at the absence of albumin fraction is fixed directly by antibody under existence of antibody detector.

III. Separationof the antiserum.

In this experiment the antiserum was treated with ammonium sulphate as the complement serum and separated into the three fractions of albumin, euglobulin and pseudoglobulin as perfectly as possible, then tested in which fractions the antibody is transformed by complement fixation reaction accord‑

ing to Browning's method.

The result is as follows : (A) Luetic serum.

Antiserum Albumin Euglobulin Pseudoglobulin

2

+f}

Table XIII

Unit of complement

4 6

i+} ii+

8

+

10

+f}

+ +

12

+iv i+f

+

(10)

62 Antiserum

Alb. + Eug.

Alb. + Pseudog.

Eug. + Pseudog.

Alb. + Eug. + pseudog.

Native serum (1 : 10)

S. Fujimura and M.

2 4

Itibakase

6 8 10

+ +

12

+

(B) Antieggalbumin

Antiserum Albumin Euglobulin Pseudoglobulin

Alb. + Eug.

Alb. + pseudog.

Eug. + Pseudog.

Native serum (1 : 10)

serum.

Table XIV

Unit of complement

2

+

4

i+}

6

+

8

ii+

+ +

10

+fr

i+

+h +r

12

i+f

+h +

+++

+1+

++f

+

(O Antidog's heart

Antiserum Albumin Euglobulin Pseudoglobulin

Alb. + Eug.

Alb. + pseudog.

Eug. + pseudog.

Native serum (r: 10)

From these ex eri

entirely included in the bulin fraction somewhat together both of these antiserum. The authors fraction of the an i of the complement serum tubes first 0.5cc of the instead of native

increasing amount of the

alcoholic extract

Table XV

Unit ot complement

2

i+f

4

+

l

serum.

6

+

8

+ +h

i+f

i+

*+}

i+

+ +

10

+ + + +h +

f+f

i+f

i+

12

+

+t+

+++

+

4+}

+}+

++}

+

experiments it is decided that the antibody is almost globulin fraction of the antiserum and in the euglo‑

richer than in the pseudoglobulin fraction, and

fractions gain antibody action as strong as native

then experimented to clear the question by which

antiserum, euglobulin or pseudoglobulin, the pseudoglobulin

is fixed. For this purpose in a series of test

euglobulin or pseudoglobulin fraction of antiserum

antiserum a antibody, 0.5 cc of antibody detector and an

pseudoglobulin fraction of the complement were

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On the Behaviour of the so‑called Components of the Complement etc.

63

put in and well mixed. After incubating at 37'C for 1.5 hours, haemolytic system and the other necessary component of the complement, i. e. albumin fraction or pseudoglobulin fraction respectively, were added and again in‑

cubated at 37'C for I .25 hours with occasional shakings and then the degree of haemolysis was examined. . The experiments indicate following results.

(A) Luetic serum.

Ist mixture :

2nd mixture :

No. of experiment

1

2 3

Table .XVI

euglobulin fraction of the antiserum antibody detector

pseudoglobulin fraction of the complement haemolytic system

albumin fraction of the complement

Unit of complement

2 4 6 8

+,

10

+

12

i+

+

tt+

Ist mixture :

2nd mixture :

No. of

ex periment

1 2 3

Table XVII

pseudoglobulin fraction of the antiserum antibody detector

pseudoglobulin fraction of the complement haemolytic system

albumin fraction of the complement

Unit of complement

2 4 6 8

i+

+ +

10

i+}

i+

12

il+

+

++f

(B) Antieggalbumin _serum.

Ist mixture :

2nd mixture :

No. of experiment

1

2 3 4

Table XVIII

euglobulin fraction of the antiserum antibody detector

pseudoglobulin fraction of the complement haemolytic system

albumin fraction of the complement

Unit of complement

2 4 6

+

8

+

i+

10

ii+

+

12

#f

+lr

f'

*i+

(12)

Table XIX

1st mixture：

2nd mixture：

：No．of

experiment

l 2 3 4

pseudo910bulin fraction of the antiserum antibody detector『

pseudoglobulin fraction of the complement haemolytic system

albuminfractionofthecomplement

Unit of complement

2 4 6

什

8

・什

什惜

10

惜惜柵珊

12

柵柵惜柵

（C） Antidog，s heart alcoholic extract serum．

Table XX

1st mixture：

2nd mixture：

No．ノof

experiment

1 2 3

雪

euglobulinfractionoftheantiserum antibody detector

pseudoglobulinfract40nofthecomplement haemolyticsystem

albuminfractionofthecomplement

Unit of complement

2 4 6 8

十

惜柵

10．

冊柵柵

12

珊柵珊

Table XXI

1st即ixture：

2nd mixture：

No．of experiment

1 2 3

pseudoglobulinfractionoftheantiserum antibody detector

pseudoglobulinfractionofthecomplement haemolyticsystem

albuminfractionofthecomplement

Unit of complement

2 4 6

十什

8 冊惜冊

10

珊惜惜

12

柵柑柵

From these results it is evident that in the complement五xation the pseudoglobulin fraction of the complement is fixed by both fractions−

pseudoglobulin and euglobulin−of the antiserum and the albumin fraction

of the antiserum seems to stay without any marked reaction．

(13)

On the Behaviour of the so−called Components of the Complement etc． 65

Summary

1． The separation of the complement into its components according to the carbondioxide gas method is imperfect and the albumin and globulin ca，n not be exactly separated by this method．The bulk of the pseudoglo−

bulin remains in the albumin fraction．

II．The method of separation of the complement into albumin，pseudo−

globulin and euglobulin is described．The thus separated fractions in them−

selves have no complemental effect，but the mixture of the albumin and the pseudoglobulin fractions represent an almost equal complemental action as the native complement serum．The euglobulin fraction seems to have no marked r61e on the complemental action．

III．In antiserum the euglobulin and pseudoglobulin ca皿y an antibody action and their mixture acts as strongly as native antiserum．The，albumin fraction has no action as antibody．

IV．At complement fixation among the essential components of the complement serum for complemental action only the pseudoglobulin is fixed by the corresponding antibody，and the fixation of the albumin fraction of the complement is very slight．This fixation is performed by both the euglobulin and pseudoglobulin of the antiserum．

On the Behaviour of the so-called Components of the Complement at the Complement Fixation