Interaction of the polyene antibiotic amphotericin B with phospholipid bilayer membranes: A circular dichroism study

Vol. 88, No. 3, 1979

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

June 13, 1979

Pages 998-1002

INTERACTION B WITH

OF

THE

POLYENE

PHOSPHOLIPJD

ANTIBIOTIC

BILAYER

MEMBRANES

DICHROISM G.

BOUDET

Departement

4,

April

A CIRCULAR

BOLARD

Recherches

place

Physiques

au

C.N.R.S.

et

Marie

Pierre

75230

Received

J.

associa

Universite

:

STUDY and

da

Laboratoire

AMPHCITERICIN

nP

71

Curie

Jussiau

PARIS

Cedex

05,

FRANCE

4, 1979

SUMMARY

circular

The study

dichroism

its

interaction

dylcholine

vesicles

dichroism tion.

As

monitors a result

signs crystalline is

that

of

be

without

other

than

association,

rejected

on

by the

Hsu

basis

are

in

the

dependance

state

Chen of

It monitored appear

spectra

vesicles

“Fungizone”

data

of

of

and

has

and

those

new the

proposed

B

dimyristoyl cholesterol.

; the cholesterol according to the

different model

or

wether

state the

not

with

to

amphotericin

lecithin,

species

according

also

of

with

the

obtained

in

used

to

phosphadi-

appears that circular by electronic absorpwhich are of opposite gel state or in the liquid the rate of these changes

vesicles.

Feingold

been

dipalmitoyl

It

for

the

the

liquid

is

gel

concluded

state

should

crystalline

state. The model of

polyene

and

antibiotic

biological

membranes.

interaction

may

a star01

and

on

stereochemically

B

specific

(9)

even

At

higher

in

the

presence

in

easy

object

several

well

as

having

concentration

(10

NMR

studies

of

study

in have

has

@ 1979

by Academic Press, Inc. in any form reserved.

of reproduction

it

M)

a the been

-5

may

confirmed

system

of

7

performed

998

its

in

a

in

lipid

pores

destruction

(IO)

using these B,

double

gel

per-

results. due

bonds, vesicles.

electronic

that

cholesterol

vesicle

amphotericin

of

aqueous with

extended

mode

cholesterol,

of

create

phospholipid on

;

presence

study

conjugated

of

shown

(1,2,3)

M can

of

absence

solution

cause

and of

presence

in

the

been

interacted

A recent

0006-291X/79/110998-05$01.00/0 Copyright All rights

10

properties

optical molecule

-4

cholesterol.

and

of

(51,

after

membrane,

has

manner

bilayers

variety the

or

aqueous

defined

the

of

It in

than

absence

the

cholesterol

a

antibiotics

presence

ratio.

lower

characteristic

The

tance

in

as

the

concentration

chromatography

meation

an

a

size

(6,7,8).

(4)

at

with

polyene

on

stoichiometrically

layers

amphotericin

depending

with

interact

other

with

: amphotericin

interact and

monomolecular

As

complex,

sterol

B can

amphotericin

of

be

the

B can

amphotericin

to

the make

For spectra

it ins-

and

Vol. 88, No. 3, 1979

recently ding

the

absorption

dimyristoyl

with

results t.D.

is

very

much

more

bean

used

by

with

lecithin

be

-

B was

388

been

et

used

to

(12)

not

did

here

B

“Fungizone”. since

; it the

it

is

already

has

interaction

of

out

a detailed

carry

bin-

report

absorption

investigate

authors

these

We

modifications

t o

its

amphotericin

electronic

than

monitor

(11).

on

environmental

al. but

studies

tool

filipin study.

METHODS

from

phosphatidylcholine (IJPPC)

from

and

Sigma

obtained

sodium

has

vesicles

better to

Bittman

purchased

ning

at

(C.D.)

sensitive

dipalmitoyl

were

nm

a

vesicles

L-IX

tin

to

AND

choline

band

dichroism

expected

MATERIALS

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

phosphatidylcholine

circular

of

BlOCHEMiCAL

and

-

used

Serva

from

egg

L-a

type

yolk,

the

Squibb

L-CX

-

phosphatidyl-

dimyristoyl purification.

further preparation,

without

as

V-E,

phosphatidylcholine

(DMPC) Amphoteri-

contai-

Fungizone,

deoxycholate.

dispersions

were prepared by dissolving weighed amounts of phoscholesterol in chloroform and than removing the solvent The appropriate buffer (tris - HCl pH 7.5) was added to give the in vacua. desired concentration and the sample was quickly vortexed, then sonicated to clearness for about 5 minutes in a MSE 150 W Mk 2 sonicator, the sonication bath being maintained in each case above the transition temperature of the phospholipid. Amphotericin was added either directly from the aqueous stocksolution or after having added dimethyl formamide to the stock-solution (the final proportion in the sample being lower than I’$%) in order to compare our results with those of other authors. The only differences between these two preparations occur in the intensity of the dichroic doublet at 330 nm. All the experiments described below have been done with a final amphotericin B concentration of 1.25 x TO-5M. We have chosen this concentration because it is of the same order of magnitude as that used in the NMR and permeability measurements and is well adapted to our C.D. experiments. Circular dichroism was measured with a Jobin-Yvon dichrograph Mark III, carefully calibrated with a low-pressure Hg lamp.

Vesicle phatidylcholine

and

RESULTS Amphotericin les

B

concentration 329

(CMC),

( AE

=

( A,

= -

ted

and

650

; + 700)

are

lipid

spectra vesicles.

of

In

all

to

409,

bands 1.25

the

at x

cases

the

exhibit bands

an

at

384

and

409

and

-7

described

999

at

( be

365

nm

;

384

nm. in

below

= -

in

under we

the

401, 329

absorponly

of

observed

388

aggrega-

CMC,

presence

and

nm

observed

bands

mical-

388

the

the

have

the

419,

at

coupling

i.e.

as

micellar

centered

M,

amphotericin

water

critical

420

excitonic

10

in

bands

signal

of

10m5M

present

be

above

bisignated

concentration at

i.e.

spectra

attributed a

D .C.

10m4M,

dichroic

intense

observed

positive

Absorption

very

At

(13).

bands weak

a

to

thought

absorption negative

72)

species

tion two

-

of

the

corresponding

with

is

a concentration

At

(13).

“Fungizone”

phospho-

around

nm

BIOCHEMICAL

Vol. 88, No. 3, 1979

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE

I

lecithin

DMPC

DPPC

100

T
c

220

T>T

c

of

409 nm

and

384

and of

hand

the

lated

a) above

(-

10)

(-

10)

375

(-

a)

374

(-

15)

374

(-

15)

nm

decreases

hand,

the

their

position

at

the

The

375 but

to

nm at

are

by

peaks vesicles

transition

corrected

to

the the

from

the

differential large

size

shifted

Bittman

and

crystalline

of

the

to

415

or

more

(11,

Chen

state.

less

but

12)

the

On

rapidly

and

at

388 in

the

other

a

rate

unre-

nm.

I)

mM lecithin

vesicles

room

at

temperature,

i.e.

temperature.

and

the

disappears the

slightly

near

shifted of

observed.

a much

slower

bisignated

totally

bands

disappearence

be

phase

immediately

mM cholesterol,

of is

phospholipid

the

to

liquid

415

C.D.

amphotericin.

observed

at

1.7

rapidly

respectively. band

in

the

of

due

are

been

of

indicated

aggregated

(Table

0.1

intensity

AE

above

corresponding

transition of

presence and

background

shift

presence

( AE)

the

The

nm disappears

phase

the

intensities

the

the

In

c

392

329

of

470

419

of

presence

4)

30)

at

the

(+

16)

that

B

377

(-

band

amphotericin

of In

DMPC

4)

(-

amphotericin

and

(+

392

the

has

377

419

scattering

as

10)

ia)

(Tc).

free

lecithin

to

fications

in

16)

(+

18)

positive

intensified

case

C.D.

nm

(+

394

36OC

below

of

421

10)

(-

El in

particles

18)

(+

(-

cholesterol,

dichroic

(+

394

392

amphotericin

continuous

c

421

c

and

temperature

220

419

Wavelength

with

c

Without

3 or

in

400 420

the

bisignated

and

cholesterol

rate.

1000

389

signal

4 hours.

On

almost

constant

remains

nm

from

intense

nm

signal we

to

419

observe

the

other

392 a

the

329

though

and

allows

at

nm

negative same

modi-

BIOCHEMICAL

Vol. 88, No. 3, 1979

b)

In

the

We

have

results and

by

presence

to

those

Hsu

Chen

case

of

Above

DMPC the

with

pears

421

the

the

and

Bittman

(14)

by

permeability

case

the

Without

cholesterol

1 hour.

The and

nm same

mixture

is

observes

and

type

a

new

raised

above

our absorption

electronic

These

at

377

In

addition,

DMPC)

:

spectra

as

with

with

DMPC,

bisignated

signal

signal

between are

nm.

phos-

23oC

in

the

with

or

without

With

the

lecithin.

at

room

at

329

350

replaced

nm

and

by

tempera-

that

disap-

450

nm

positive

if

is ones

mM cholesterol

Cl.1

we note

phase of

with

10°C

bands

at

reappearence

the

C.D.

C.D.

the

by

compare

transitions

(at

negative one

to

measurements.

36nC

of

the

modification.

(11)

(at

initial

the

order

DPPC.

temperature

modified 394

of

same

DPPC).

in

state

temperature

observes

the

diately

in

vesicles. C.D.

Chen

transition

and

DPPC

crystalline

41gC

in

or

by

phase

rapidly

observes

Feingold

transition

totally

also

by

gel-liquid

phase

the

ture

of

and

one

Below

mM DMPC interactions

obtained

and

cholesterol

1.7

these

present

pholipid

at

of

studied

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

one

temperature

the

transition

temperature,

one

imme-

bisignated

band

at

329

nm.

410

and

388

nm

the

vesicles

DISCUSSION

very

The (11) and

arise

to

from

not It

at

C.D.

peaks

clude

that

absorption

changes

from

adsorption

of

amphotericin important

is

occur

fast

the are

not

at

for

since

: at

a 10m6M

represents ricin)

as

absorption

surprising water

rate

the

the

same

and

C.D.

in

do

concentration

the

C.D.

spectrum

our

results

the

changes

;

in

for

909

the

species. free

said

nm)

represents

not new

We This

conis

not

amphotericin (409,

of

do

peaks.

spectrum

(about

388

changes

addition,

same

case

absorption

species

(420,

the

the

C.D.

absorption

the

monitor also

onto

are

configuration.

as

not

is the

monomeric

a channel

wavelength

it

the

monomers

absorption

instance

mainly while

in

that

note

near

amphotericin

molecules to

same

observed

the

in

384

total

only

nm) amphote-

the

aggregated

species. The

or

lecithin ditions

of

consists the

spectrum

C.D.

DMPC the

of

adsorbed

vesicles kinetic

three

appearance

in

species amphotericin.

from the

(111, negative

of

monomeric

resulting

the

interaction

crystalline

liquid

permeability bands which

at

are With

of

(8) 419,

neither DMPC

1001

state and

392

and

free and

amphotericin

NMR 375

in

the

measurements nm

micellar DPPC

(i.e.

and

vesicles

and

(lo)),

monitors

thus

nor

free in

or the

gel

con-

Vol. 88, No. 3, 1979

state

in

(i.e.

and

the

Feingold)

and

these

phase

In compare ships

in

meric

amphotaricin/phospholipid

it

gel

between

the

appears

high

rate,

obtained

we

relate

are

not

clearly

Hsu

even

by

HSU

the

same

Chen

without Chan

and

dichroic

the

on

with

the

that

state.

Thus,

proposed

by

channels

it

obtained

species

below

and

latter Chen

constituted

not

above

the

cannot

Feingold

and

of

amphotericin-cholesterol

Kruyff,

B.,

to

relation

fluidity

-

relation

results and

possible

permeability on

association

the Hsu

is

-

fluidity

conclusions vesicles

model

transmembrane

if

species

obtained

state,

crystalline

liquid

a

of

temperature.

conclusions

the

at those

that, these

conditions

the

measurements

appear

parallel

changes,

these

permeability

bands

indicate

transition

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the

results

furthermore

permeability

the

of dichroic

Thus

Feingold

the

conditions

positive

cholesterol.

to

BIOCHEMICAL

mono-

ships be

in

used

the

to

model

the choose

with complexes.

REFERENCES

1.

Norman,

A.W.,

and

Deenen,

Van

2.

Schroeder,

3.

Bittman, 379

4.

Demel,

F., R.,

R.A., (19721,

Holland,

J.F.,

Fischkoff,

Van

6.

Finkelstein,

A.,

antibiotics,

Eisenman

Zutphen

(19711,

L.L.,

(1972)

Van

(1974)

F.J.L.,

Van

Kessel,

Acta, (1972)

Proc.

Natl.

W.S.M.

290,

l-14.

11,

Biochem. Acad.

Sci.

3105.

USA,

12,

R.

(1973)

ed.

p.

Kinsky,

(1968)

S.C.

Acta in

241,

310.

Membranes,

bilayers

Lipid

and

377.

Oerlemans,

W.J., Biophys.

De

L.L.M.,

Biophys.

Gerritsen,

P.,

Deenen,

1.

Biochim.

Biochim.

Hoogevest,

Van

150,

Holz,

B.,

Kruijff,

L.L.M.

8.

Geurts Biophys.

Bieber,

S.A.

Crombag, R.A., Biophys. Acta,

Demel, Biochim.

Da

Biochim.

5-3799.

5.

7.

De

L.L.M.

Acta

Kruijff,

B.

A.,

339,

(1978)

Demel,

R.A.,

Van

Deenen,

30-43.

Biochim.

Biophys.

Acta,

511,

397-407. 9.

Sassa,

G.,

10.

Gent,

M.P.N.,

11.

Chen,

W.C.,

12.

Bittman,

R.,

Chen,

13.

Rinnert,

H.,

Thirion,

14.

Hsu

then,

Weissmann,

G.

(1967)

Prestegard, Bittman,

C.,

Feingold,

Biochim.

J.H. R.

W.C.

(1977) and

C.,

Biochem.

Biochem.

16,

Anderson DuPont,

D.S.

Biophys.

(1976)

(1973)

O.R. G.

(1977) Biochem.

972-970.

1002

Acta,

135,

Biophys.

416.

Acta,

426,

17-30.

Ul45-4149. (1974)

Biochem.

13,

Biopolymers Biophys.

16, Res.

1364-1370. 2419-2427.

Comm.

51,