The detection of a conformational change in the antibody molecule upon interaction with hapten

Vol. 38, No. 1,197O

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

THE DETECTION OF A CONFORMATIONAL CHANGE IN THE ANTIBODY MOLECULE UPON INTERACTION Carol Contribution Institute, Microbiology,

Warner,

Verne

WITH RAPTEN

Schumaker

and Fred

Karush

No. 2467 from the Department of Chemistry and Molecular Biology University of California, Los Angeles, and from the Department of University of Pennsylvania.

ReceivedOctober

31, 1969

SUMMARY The technique of differential sedimentation is used to detect any change in sedimentation coefficient and in frictional coefficient of they:G immunoglobulin molecule upon interaction with hapten. Interaction of anti-lactoside antibody with the univalent haptens, lactose and p-(p-dimethylaminohenzeneazo) phenyl S-lactoside, results in a slight increase in sedimentation coefficient of the antibody molecule, which corresponds to a decrease in frictional coefficient. The interpretation is that the antibody molecule assumes a more compact configuration upon interaction with univalent hapten. The overall which

still

structure

eludes

support

an elongated

Noelken

et al.' CM

compact,

biochemists.

studied

that

y&G immunoglobulin,

by electron

asymmetric examined

studies

led

antigen

they

having that

structure.

been

also

Most

particle

have argued

Y-shaped

slightly

of the 7s immunoglobulin

the data

The shape

when it with

open,"

complexes that

exposing

a critical

has also

antigen,

reported

was only

Feinstein

the electron

and Rowe These

microscope.

molecules

disulfide

a more

and Rowe‘

of 105A'.

when antibody

and Green3

have made a most

understanding

of the shape

of the antibody

micrographs.

They have been able molecule

hapten-antibody

with

with

molecule

Feinstein

with

25OA" by 4OA'.

compatible

of the antibody In,l965,

seems to

combine

with

bond and perhaps

the

site.

Valentine

antibody

are

is a problem

data

of roughly

was not combined

them to hypothesize

complement-binding

also

a maximum dimension

antigen-antibody

"click

of the hydrodynamic dimensions

microscopy.

G molecule

in a Y-shaped

significant molecule

to photograph configuration,

mixture. 125

contribution with

regular

their

to the electron

complexes,

when studying

with

a bivalent

each

BIOCHEMICAL

Vol. 38, No. 1, 1970

Based on the results hypothesized antibody

a simple (see Figure

assumed

to have

of these

scheme for 1).

the legs

occur,

one would

flected

expect

by a decrease It

tional

is

If

of

this

and in

upon in terac tion

with

(Lac Ab)

compared

small with

same solvent, Spinco

to

of the moleturn,

be re-

to report

the change

in fric-

of the antibody

molecule

OF ANTIBODY WITH UNIVALENT HAPTEN

c

The antibody molecule is shown schematically to be Y shaped, with three globular regions. The hapten molecules are depicted by the solid squares. The “click open” hypothesis illustrated in this figure is inconsistent with the data presented in this communication.

system

used

from rabbits.

sedimentation

detect

were

to

hap ten.

for

the experiments

Two univalent

p-(p-dimethylaminobenzeneazo)-phenyl tial

in

hapten

AND METHODS

The antibody body

with

is

of the molecule.

coefficient

HAPTEN

MATERIALS

would,

and

hapten,

change

coefficient

communication

with

coefficient This

we

hapten

interaction

in frictional

sedimentation

THE INTERACTION

FIGURE 1 LEGEND:

interaction

and upon

in the sedimentation

the purpose

coefficient

before

calculations).

studies,

of monovalent

such a conformational

an increase

unpublished

microscopic

the interaction

of the Y closed

the open configuration.

(C. Warner,

two electron

The molecule,

assume

cule

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Model

changes

technique

with

or without

E ultracentrifuge

haptens

S-lactoside developed

in sedimentation

the reference

was anti-lactoside

protein, hapten. at 20°,

were (Lac

by Schumaker coefficient

runs

of

and

The differen-

the antibody

were

performed by Schumaker

to

molecule

?/-G immunoglobulin

as described 126

dye)4.

lactose

and Adams5 was used

a non-specific All

used:

anti-

in the

in a Beckmanand Adams 5 .

Vol. 38, No. 1, 1970

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

RESULTS AND DISCUSSION The basic difference

experiment

was the measurement

of the antibody

The results

are

molecule

shown in Table

before

1.

All

of the

sedimentation

and after

errors

treatment

coefficient with

in sedimentation

hapten.

coefficient

TABLE 1

Experiment

Description

aveibergs)

(6f/f

x 100)

Lac Ab plus lo-Sr lactose

Lac Ab

+0.056

-0.4%

2

Lac Ab plus 10m4M Lac dye

Lac Ab

+o.Mo

-2.05%

sign indicates it is compared.

differences,

sedimentation

coefficient,

account

the binding experiment is

complete

to note 0.3

y-Y-Gimmunoglobulin

molecule,

reference

in all

solution

The results Lac Ab molecule than

lactose

S less

change

of the

of all

than

in

to the observed

of hapten

obtained

interacts

4 and also

of the molecule.

coefficient

correspond the interaction

The percent

the Lac Ab

change in 6 of Schumaker taking

antibody was chosen

available

the sedimentation from Pentex,

molecule

due to

in each

binding

sites.

coefficient coefficient

Inc.,

and used

of the as the

runs.

coefficient

Thus,

weight

than

the Lac Ab had a sedimentation

show a small when it

faster

by the method

saturation

that

S.

corresponding

The concentration

of hapten. to ensure

sedimented

* 0.016

in molecular

was approximately

strongly

the molecule

was calculated

the increase

interesting

which

that

6Sio w, were less than 3 coefficient, 6f/f x 100,

frictional

It

ss”20 wa

to

1

aThe plus to which

into

Compared

increase

in sedimentation

with

hapten.

produces

to a decrease of antibody

in

increases frictional

and hapten 127

of the

The Lac dye binds

a greater

The observed

coefficient

change

more

in sedimentation

in sedimentation coefficient cannot

of the molecule.

be described

by the

Vol. 38, No. 1, 1970

simple

model

which

of the molecule closing

BIOCHEMICAL

we proposed.

portion

"click

open"

small

univalent

univalent

is

possible

is an open Y and that

of the molecule

hydrophobic

It

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

due to increased

only

haptens.

hapten

induces

than

It

applies

the normal

binding

results

hydrophobic

to large,

seems that in

protein

solution

in a slight

Feinstein

from

and not

to assume a more compact

REFERENCES Noelken, J. Biol.

M. E., Nelson, C. A., Chem. 240, 218 (1965).

2.

Feinstein,

A. and Rowe, A. J.,

3.

Valentine,

R. C. and Green,

4.

Karush,

5.

Schumaker,

V. and Adams,

6.

Schumaker,

V.,

F.,

Nature

N. M.,

J. Am. Chem. Sot.

Biochemistry

Buckley,

P.,

2,

C. E. and Tanford, 205,

I,

3427

128

Biol.

27,

615 (1967).

(1957).

Biochemistry (1968).

C.,

147 (1965).

J. Mol. 3380

to

of Lac Ab with

before.

1.

the

and Rowe's

antigens

the interaction

the molecule

configuration

interactions

Perhaps,

of the Lac dye hapten.

h ypothesis

configuration

hapten

that

I,

3422

(1968).