The dissociation properties of native C1

Vol. 107, No. 2, 1982

BIOCHEMICAL

RESEARCH COMMUNICATIONS

AND BIOPHYSICAL

July 30, 1982

Pages

DISSOCIATION

THE

J.

Rohert

Ziccardia

Department Research La

Received

June

PROPERTIES

and

of

Molecular

Institute

of

Jolla,

OF NATIVE

Jurg

618-623

Cl

Tschopp

Immunology Scripps

California

Clinic 92037

17, 1982

The first component of human complement (Cl) readily dissociates under physiologic conditions into two subunits - Clq and Clr2Cls2. The equilibrium constant for this reaction has been determined for native Cl in fresh normal human serum by hemolytic titration. Standard technology was modified to simulate physiologic conditions. Furthermore, assays were carried out at numerous concentrations of sensitized erythrocytes, thereby allowing the calculation of the total Cl concentrations. percent of associated Cl at different Increased Cl dissociation was observed with dilution. From these data, an association constant of 4.5 x 108 M-I was calculated for native Cl. Thus in normal human serum approximately ten percent of the Cl is present as free Clq and Clr2Cls2.

INTRODUCTION The

first

EOTA

into

component three

physiologic The

Cl

(Cls)

equilibrium

reconstituted

understanding

analysis

of

Cl

of

complement

subunits the

reincorporated An

human

distinct

conditions,

Clr2Cls2. for

of

-

from

purified

after

having

activation

a This is publication Clinic. This work was Ziccardi is the recipient Heart Association. Dr. Foundation.

Clq,

Clr

dissociation

constant

the

(Cl)b

kinetics

the

proteins

dissociation

and

latter (3,4)

purified properties

(4,5).

Furthermore,

are

for

Cl

Cl

is

presence However

primarily has having

radiolabeled of

the

(1,Z).

reaction and

and

in

Cls

products

for

been

dissociates

of under

Clq been had

and

calculated one

subunit

(4). important

increased

for

the

concentrations

proper of

number 2746 from the Research Institute of Scripps supported by USPHS grants AI 14502 and AI 17354. Dr. of Established Investigatorship 79-139 from the American Tschopp is supported by the Swiss National Science

b Abbreviations: AVB, veronal-buffered saline at physiologic ionic strength (0.15 M); Cl, first component of human complement; EAC4, antibody sensitized sheep erythrocytes bearing functionally active human C4; EAC14, antibody sensitizea sheep erythrocytes bearing functionally and C4; EDTA, active human Cl ethylenediaminetetraacetic acid; association constant; NHS, normal human Kassoc. serum; SAVB, veronal-buffered saline at reduced ionic strength (0.065 M). 0006-291X/82/140618-06$01.00/0 Copvrqht .4 II rrphlx

C) 1982 b.v .A tadem~ Pwss, o/ reproductron rn any .form

Inc. reserved.

61X

Vol. 107, No. 2, 1982 free

Clr2Cls2

Therefore human

we

have

serum.

native

Cl

is

and

of

the

as

been

detected

the

association

constantof

that into purified

a model

for of

4.5 10

components study

is of

the

RESEARCH COMMUNICATIONS

certain

pathological

properties x

of

lo8

percent

Clr2Cls2.

and

the

in

dissociation

approximately

Clq

from

activation

have

AND BIOPHYSICAL

investigated

indicating

reassembled used

Clq

An

dissociated

be

BIOCHEMICAL

of

M-l

has

the

Cl

Furthermore, almost

we

as

stable

biochemical

and

as

sera

native

Cl

been

calculated

in

normal

can

in

biophysical

normal for

human

conclude

native

(6).

Cl and

serum

that

Cl

thus

can

characterization

Cl.

MATERIALS

AND

METHODS

Normal Human Serum. Blood was drawn from healthy human donors and clotted 1 hour at room temperature. The clot was allowed to retract in ice for 30 minutes after which serum was obtained by centrifugation and kept on ice until used on the same day. Cl Hemolytic Titration. The method of Borsos and Rapp (7) for the hemolytic titrat'on of Cl was used with the following changes: 1. In order to determine the association constant of Cl under physiologic conditions, Cl was diluted and incubated with indicator cells in a buffer of physiologic ionic strength (0.15 M) instead of reduced ionic strength (0.065 M). 2. Buffers contained 0.1 percent bovine serum albumin to prevent nonspecific protein loss at the high dilutions utilized (gelatin was avoided due to its potential to interact with the structurally homoloqous CIq). In 'brief, hemolytic titration of Cl was carried out as follows: 1. Normal human serum, NHS, was diluted in AVB (i.e. veronal-buffered saline containing 0.1 percent albumin, 0.00015 M calcium, 0.001 M magnesium, pH 7.5, u = 0.15M) and incubated at 300C for 5 min. 2. 0.2 ml of diluted NHS was mixed with 0.1 ml EAC4 (i.e. sensitized erythrocytes bearing active C4) in AVB and incubated at 300C for 30 min. 3. Th'ese EAC14 were washed once with the cells taken up in 0.3 ml of a low ionic strength buffer - SAVB (i.e., isotonic veronal-buffered saline containing 4 percent sucrose, 0.1 percent albumin, 0.00015 M calcium, 0.001 M magnesium, pH 7.5, The low ionic strength prevents “Cl transfer" in the next reaction p = 0.065M). step (7). 4. An excess of oxidized C2 in 0.1 ml SAVB was next added and the mixture incubated at 300C for 10 minutes. 5. Finally 1.1 ml of guinea pig serum (diluted 1:50 in Verona1 buffer containing 0.04 M EDTA) was added and the mixture was incubated at 370C for 60 minutes (note that for the highest cell concentration utilized (Talble I), guinea pig serum was diluted 1:20 in order to be in excess). Hemoglobin released from lysed cells was measured by absorbance at 412 mu after spinning out unlysed cells. The reciprocal of serum dilution was plotted against In (1 - percent lysis). The dilution at which - In (1 - percent lysis) equalled unity corresponded to that quantity of Cl required to initiate the lysis of 63 percent of the offered cells. RESULTS That

concentration

determined

Iby

section.

In

five

of

functional

hemolytic

ly

titration

separate

active as

experime

nts,

Cl

in

fresh

normal

described

in

the

the

number

of

added

107.

For

each

63

percent

human

Materials indicator

serum

was

and

Methods

cells

(i.e.,

7 EAC4) dilution

was

varied yielding

from

1.3

enough

Cl

x to

10' initiate

to

24 the

619

x

lysis

of

cell of

dose, the

the offered

serum cells

Vol. 107, No. 2, 1982

BIOCHEMICAL

AND BIOPHYSICAL TABLE

HEMOLYTIC

Number of EAC4

I

TITRATION

OF Cl

IN NHS

Associated Cl Mo?eculesb

Serum dilution for 63% lysisa

RESEARCH COMMUNICATIONS

Total Cl MoleculesC

Percent Associated

1.27

x lo7

6,900

1.27

x lo7

3.1

x 109

0.41%

2.80

x lo7

5,100

2.80

x lo7

4.2

x 10'

0.67

6.50

x lo7

4,000

6.05

x lo7

5.4

x 109

1.1

12.1

x lo7

3,550

12.1

x lo7

6.1

x 10'

2.0

23.9

x lo7

2,400

23.9

x lo7

9.0

x log

2.7

aSerum dilution of the offered

such cells

that there is (column 1).

enough

bEquals the number of EAC4 (column 1) molecules in 0.2 ml of serum diluted 'Total number of Cl molecules in Cl concentration (immunochemically 1.8 x 10-7 M (10,ll). dEquals

column

was

determined

there

is

follows

I column

(Table one

the

active one-hit

The

total

calculated of

from

1.8

x lo-'

calculated the

1).

to

determine

the

x = 100

2xK

x

following

X = percent

the

lysis

hemolytically 2.

is

of

active

63%

Cl

shown in column NHS was taken

2. as

Total

with

associated

greater

Cl

used

4 '

Kassoc and

to

[Cl]t

'

the

column

Cl

of

EAC4.

column

4)

was

concentration Cl

was

readily

5).

As

expected,

various the x

Kassoc

[Cl],

Cl

data

concentration

theoretical + 1

concentration.

from and

association

"l't

620

I,

experimental

total

generate

= total

number

Cl

associated

molecules

dilution.

assuming

+1Cl

versus

calculated

'

serum

I,

Cl

the

(Table

(Table

hemolysis

active

to

dilution

function,

initiated

of

equal

constant,

was

Cl

number

of

experiment

association

'

the

this

percent

distribution

since

determined

each

equation

Poisson

simply

at

the

curves

the

Thus 3)

decreased

aSS*Cx ml, associated

initiate

dilution

(7-g).

column

for

percent

! where

this

present

figures

theoretical The

lysis

Finally,

Cl

as

at

to

immunochemically

associated

to

the column

According

molecules

the

ml

ml of serum diluted as determined) in undiluted

cell

I,

of

to

(Fig.

Cl

and represents as shown in

2).

of

these

plotted

compared

per

from

order

were

Cl

M (10,ll).

percent In

column

(Table

of

0.2

x 100.

I,

Cl)

number

(4)

theory

associated

(i.e..

I

(3)

0.2

Cl in

Cld

Table then

constants

(1 1 curves:

Vol. 107, No. 2, 1982

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

G

F:gure

The 6 x 10' 1.8

of the association constant of Cl in NHS. The percent of Cl (from Table I, column 5) is plotted versus total Cl con(Table I, column 4 f 0.3 ml i 6 x 10z3). Open circles experimental points. Solid lines represent theoretical curves from equation (1) assuming numerous association constants.

experimental

data

M-l

a

.

x 1O-7

percent

Determination associated centration represent generated

1.

Using

M for

of

tine

and

in

consistent

mean

[Cljt Cl

are

NHS

is

value

of

equation present

with

a

4.5

x

KassOc lo8

(l),

we

can

conclude

free

Clq

and

Clr2Cls2.

as

of

3

M-l

x

lo8

for

that

M-'

to and

Kassoc

approximately

ten

DISCUSSION An under of

association

constant

physiologic the

(7)

conditions.

is

present

as

utilized

for

these

Cl

was

Materials

and

Methods

macromolecular

or

Clr2Cls2

were

includes was

not It

percent

when

assayed

is of

at

hemolytic

titration

associated

Cl,

the

still

titrateable.

normal and

gel

with

Cl

is

the

less

than This

few

modifications it

in

native 10

hemolytic

was

technology in

assumed while

favor

activateable

that free

of

Cl

percent

described

active,

was

for

approximtely

Standard

Evidence

data

dilutions

in by

Table

for

reduced

ionic

six

percent

of

percent

was

at

621

I

hemolytic

required

out

serum,

hemolytically

Cl

calculated

this

while

the all

Clq

and

assumption dissociated

Cl

(5).

detected

carried

human

the

associated

from

been

calculations

was

analysis

has

Clr2Cls2.

For

all

high is

in

Clq

M-l

inactive.

by

serum

Thus

Cl

apparent

the

lo8

section.

that

readily

x

studies

hemolytically finding

4.5

free

associated

the

dissociation

therefore

of

calculated

(column

5)

that This

titration. this

assay.

strength the

While (7),

serum from

only

is the

is the

thereby

Cl

a small due stanaard increasing

associated total

to

and serum

Cl

Vol. 107, No. 2, 1982

BIOCHEMICAL

TABLE ASSOCIATION

Cl

Cl

SO”PCB

K

x lo*

Reconstituted

Cl

6.7

x lo7

Exchanged

Cl

aReported

as

2.0 a lower

bCalculated

from

Cl

well is

than

that

activation

Siegel

ultracentrifugation

60%

Kilchherr

with

was at

the of

is

study

= 1.8

and

Cl/ml

Cl

study

--et

al.

(3)

et --

al.

(4)

of

the

10m7

x lo-*

one

although should From 89

II)

biochemical

. and

Cl

subunit

Thus

(10,ll)

and

reconstituted

(Cls) Cl)

Cl,

are

native

Cl

be

noted

these

percent

M

reincorporated

(4).

These

listed

is

in

somewhat that

native

reconstituted

biophysical

values

Table more

the

association of

from

II. stable

KaSSOc

for

constants,

we

Cl

and

75

percent

Cl

can

be

used

characterization

of

Cl.

ACKNOWLEDGEMENTS We wish assistance

to and

thank Ellye

Deborah Casbero Lukaschewsky for

and Julie secretarial

Harper Ziccardi services.

for

expert

REFERENCES 1.

2. 3. 4.

5.

Lepow, I.H., Naff, G.B., Todd, E.W., Pensky, J. amd Hinz, C.F. (1963) J. Exp. Med. 117, 983-1007. Ziccardi, R.J. and Cooper, N.R. (1978) Science 199, 1080-1082. Siegel, R.C., Strang, C.J., Phillips, M.L., Poon, P.H. and Schumaker, V.N. (1981) Fed. Proc. 40, 963 (Abstract). Kilchherr, E., Fuchs, H., Tschopp, J. and Engel, J. (1982) Mol. Immunol. 19, 683-691. Ziccardi, R.J. (1982) J. Immunol. 128, 2500-2504. 622

the

M (10)). for

native

limit).

(Table

x

exchanged

for

(it

a lower

had

concentrations,

associated

1.0

(i.e.,

KaSSOc Cl

1.8

calculated

study

exchanged

M.

be

or

having

this in

as

to

radiolabeled

in

x 10e7

previously

for

physiologic Cl

for

been

and

reported

[Cl],

x lOI

have

or

reconstituted model

75%

agreement

reconstituted

calculate

a

ultracentrifugation

(1)

calculated

reasonable

Cl

Reference

present

(6

purified

that

exchanged

Cl ssociated NHS 6

titration

determined

(3,4),

been

as

of in

89%

equation

constants

having

There

of

and

proteins

after

7a

concentration

Association purified

Percent

hemolytic

(immunochemically

titrateable

CONSTANTS

limit.

Kassoc

concentration

as

x 10

r4-'

RESEARCH COMMUNICATIONS

II

Methodology

assoc

4.5

Native Cl in NHS

AND BIOPHYSICAL

technical

as the

Vol. 107, No. 2, 1982 6. 7. 8.

9. 10. 11

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Laurell, A.B., Martensson, U., and Sjoholm, A.G. (1976) Acta Path. Microbol. Stand. Scet. C. 84, 455-464. Borsos, T. and Rapp, H.J. (1963) J. Immunol. 91, 851-858. Rapp, H.J. (1955) Mathematical Theory and Analysis of Immune Hemolysis. Dissertation. The Johns Hopkins University School of Hygiene and Public Health. Colten, H.R., Borsos, T. and Rapp, H.J. (1?67) Science 158, 1590-1592. Ziccardi, R.J. and Cooper, N.R. (1977) J. Immunol. 118, 2047-2052. Ziccardi, R. J. (1981) J. Immurol. 126, 1769-1773.

623