Raman studies of lipid interactions at the bilayer interface: Phosphatidyl choline-cholesterol

Vol.

94, No.

May

30,

2, 1980

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Pages 638-645

1980

Raman Studies

Department

of Lipid Interactions Phosphatidyl choline

of Chemistry,

-

at the Bilayer Cholesterol

Ellen Bicknell-Brown Wayne State University,

Interface:

Detroit,

Mich.

48202

and Department Received

Kenneth University

of Chemistry,

April

G. Brown of Detroit,

Detroit,

Mich.

48212

8,198O

SUMMARY: The Raman C=O stretch region of phospholipids is shown to be a sensitive probe of molecular interactions in the interface region. We report here the effect of cholesterol on the Raman C=O stretch and Raman symmetric C4N+ stretch of aqueous dispersions of dipalmitoyl phosphatidyl choline Addition of cholesterol causes a shift of some intensity from the ZflPpClh.strong 1737 cm-l C-O - stretch to lower frequency - producing a second strong C=O stretch band at 1720 cm-'. Cholesterol methyl ether does not produce this splitting. Evidence is presented which suggests that the C=O frequency shift is due to a conformation change in the acyl region of DPPC induced by the presence of cholesterol. Addition of cholesterol causes no change in the Raman symmetric C4N+ stretch band of DPPC - suggesting that the effect of cholesterol does not extend to the head group region. Introduction:

Raman spectroscopic

interactions

have

(2800-3000 which

cm-')

provide

until

studies

now concentrated

and the skeletal

information

on phospholipid

side

about

chain

side

fluidity

chain over

by Raman spectroscopic

carbon

region

It

is

important

phospholipids and polar stretch

frequency dispersion explored

(1000-1150

to extend

group

frequencies

(2)

in dipalmitoyl of anhydrous the possibility

cm-l)

by Lippert

as well.

phosphatidyl crystalline of using

choline

these

0006-291X/80/100638-08$01.00/0 Copyrrghr NC1 1980 b-v Academic Press, Inc. All righrs of reproduction in any form reserved.

side

in

638

that

(4).

(1).

studies

of

interface

the Raman C=O (3)

dramatically

We have,

bands

has carbon-

stretch

(DPPC) change

vibrational

chain

the acyl

'i+ c-r;r-c C

DPPC in water

range

and Peticolas

We have observed

and the Raman symmetric

cm-l),

of cholesterol

transition of the

and structure

region

The effect

entire

and

(1000-1150

of Raman spectroscopic

interactions

region

region

order. the

structure

C-H stretching

studies

the utility

to include head

on the

C-C stretching

been demonstrated stretching

of phospholipid

upon

therefore,

as probes

to study

Vol.

94,

No.

2, 1980

phospholipid

BIOCHEMICAL

interactions

a Raman study

BIOPHYSICAL

the interface

region

of molecular

RESEARCH

interactions

can indeed

interactions

now results

which

demonstrate

be used as an important,

in

Materials and Methods: Cholesterol were purchased from Sigma.

COMMUNICATIONS

We report

region.

of phospholipid-cholesterol

the Raman C=O stretch new probe

in

AND

the phospholipid

methyl

ether

of that

sensitive

interface

region.

and cholesterol

(99+% pure)

Samples were prepared by dissolving the phosphatidyl choline and cholesterol (or cholesterol methyl ether) in a 1:l molar ratio in chloroform. The chloroform was removed under vacuum and the dried mixture was dispersed in excess water. Samples were incubated at temperatures above the lipid phase transition until "swelling" was complete. Samples were centrifuged for ten minutes in a clinical centrifuge to remove some excess water. The spectra State University Spex 14018 double photomultiplier A spectral band Results

were measured equipped with monochromator tube. Laser pass of 4 cm- f

and Discussion:

dipalmitoyl

stretch ture

and choline of both

gel-to-liquid

The Raman spectra

phosphatidyl

of DPPC and cholesterol

samples crystal

with a laser Raman spectrometer at Wayne a Coherent Radiation CR8 argon ion laser, with spatial filter and a cooled RCA ~3104 ower at the sample was approximately 2OOmw. and pen period of 10 sec. were used.

choline

of the aqueous

(DPPC/H20)

was 19'C, phase

regions which

are in figures

is

below

cc

"

frequency

1:

compared

dispersion

in the

la and lb.

the mid-temperature

of

carbonyl

The temperaof the

transition.

1750 1700 Figure

and of the aqueous

(DPPC/cholesterol/H20) C-N + stretch

dispersion

,

.

750 700 km-l)

The Raman C=O stretch and C4NC stretch bands of dipalmitoyl phosphatidyl choline/H20 dispersions: a) without cholesterol b) with cholesterol at 19' C.

639

Vol.

94, No.

2, 1980

The most

BIOCHEMICAL

striking

AND

difference

between

DPPC/H20 and DPPC/cholesterol/H20 region

(1720-1740

stretch with

band

over

to that

is

found

the

if

at 1737 cm present.

two bands

the

C=O stretch cholesterol

ether

The Raman spectrum

ether/H20

bandwidth, -OH group C=O stretch There from

has only

as in

band,

are

-1

interpretations

C=O stretch

band

2:

Raman carbonyl ether/H DPPC.

sample

required

for

the

is

splitting

cholesterol

methyl

DPPCfcholesterol

at 1737 cm it

the effect

stretch

0 and c) 2 11 spectra

of the

shift

-1

with

appears

a 30 cm that

observed

-1

the in

the

of some intensity

to a new band appearing

frequency Fi gure

(measured

of the Raman spectrum.

two possible

the 1737 cm

band is

intensity

Therefore, for

frequency

frequency

of the mixture

may be required

region.

the DPPC/H20 Raman spectrum. is

band

C=O

prepared

C=O stretch

containing

of DPPC/H20.

on the cholesterol region

a sample

one C=O stretch

the spectrum

has one strong

integrated

-OH group

stretch

in the C=O stretch

, the

band in

phase

carbonyl

in the DPPC/cholesterol/H20

stretch

methyl

of gel

in the

The lower

The total

of the DPPC carbonyl was measured.

-1

COMMUNICATIONS

The samples

Raman bands

1720 cm-1 .

of the single

To determine

1 occurs

a 30 cm-1 band width.

with

no cholesterol

at approximately

by planimeter) equal

-1

band

with

the Raman spectra

figure

have -two overlapping

frequency

of the sample

in

RESEARCH

The DPPC/H20 Raman spectrum

at 1737 cm

cholesterol

The higher

found

cm-l).

BIOPHYSICAL

region of DPPC/cholesterol/H20 were measured

640

at approximately

km-‘) a) at

DPPC/H20 gispersions 19 C.

b)

DPPC/cholesterol and of d)crystalline

methyl

Vol.

94, No.

BIOCHEMICAL

2. 1980

1720 cm-l

when cholesterol

1) One is

that

which

in

some acyl

the C=O stretch

change

beginning

is

with

linkages

occurs

frequency

for

shift

change

significantly

lesterol

is

hydrogen

bond

possible.

to the

to us that

effect

hydrogen

carbonyl

stretch

at the

possibilities

to cause

from

in a pure

it

C=O group

that

a

of DPPC below,

that

DPPC/H20

system

would

However,

possibility

is

which

offers

bonding

and that

the have to no

with

cho-

groups

if

not

be so different Thus,

cholesterol

causes

we are

investigating

would

cholesterol

the DPPC C=O groups,

the C=O frequency.

region.

would

the DPPC carbonyl

of cholesterol

with

seem that

groups

hydrogen

bond with

bonding

is

for

-1 17 cm downward

would

an environment

to hydrogen

in

induced

are discussed

the observed

stretch,

seems likely

-1 a 20 cm shift

is

2) The other

occurs

to one in which

system were

dispersion:

change

of some DPPC carbonyl

It

to water

hydrogen-bonding as to cause

bonding

possibility

COMMUNICATIONS

model.

- probably

hydrogen-bonding

added

Both

environment

DPPC aqueous

frequency.

environment

in the DPPC carbonyl

hydrogen-bonding

RESEARCH

a conformational

at a lower

introduced.

hydrogen

BIOPHYSICAL

in the

the hydrogen-bonding

In order

it

the from water

seems unlikely

the change this

in the DPPC possibility

in

in progress. The second

ational

change

Evidence is

present

in the hydrogen-bonding

when cholesterol

work

is

AND

in the C=O region

has been presented

dependent

on geometry

have proposed the acyl

that

that

C2-Cl

which

cholesterol

which

lowers

bond and that

the

the

demonstrates

in phospholipids

the C=O stretch

allows

frequency gauche

C=O stretch

that

(2).

or induces

Brown

sensitive

c -c//O 1 3 \

frequency.

the C=O stretch

Brown, is

a conform-

frequency

and Person

to rotation

conformer

(2) about

C=O stretch

is

cl72

about

lo-20

-1

cm

higher

frequency

than

the

trans

0 c3 'c2-Cl

C=O stretch. H20 samples

If are

the two C=O stretching compared

to those

frequencies

of crystalline

641

4 \

0

conformer

0

of the DPPC/cholesterol/ DPPC (see Table

l),

it

can

Vol.

94,

No.

Table

2, 1980

1.

BIOCHEMICAL

AND

BIOPHYSICAL

Frequencies (v) and bandwidths (BW) of choline C=O stretch and C N s etric 4;"" of various DPPC mixtures tt 19 C C,N+

RESEARCH

COMMUNICATIONS

the dipalmitoyl stretch in the

symmetric v(cm

-1

phosphatidyl Raman spectra

stretch

of

choline

BW(cm-I)

)

DPPC/H20

718

14

DPPC/cholesterol/H20

718

14

718

14

710

11

DPPC/cholesterol

crystalline

methyl

ether/H20

DPPC

acyl

C=O stretch

(higher

frequency)

BW(cm-I)

"(cm-l) DPPC/H20

1737

30

DPPC/cholesterol/H20

1737

30

1737

30

1737

12

DPPC/cholesterol

crystalline

methyl

ether/H20

DPPC

acyl C=O stretch

(lower

v(cm-I) DPPC/H20

_----

DPPC/cholesterol/H20

1720

DPPC/cholesterol

crystalline

be seen that crystalline (a gauche and 2) the

methyl

conformer frequencies

-SO

12

1720

are very

DPPC is

BW(cm-l)

-----

ether/H20

DPPC

they

frequency)

Since

similar.

due to the presence in the B chain

1) we believe

of two acyl

and a trans

of the GO "doublet"

642

the doublet

linkage

conformer

in

conformers in

the y chain),

in DPPC/cholesterolh20

are

the

Vol.

94, No.

2, 1980

BIOCHEMICAL

crystalline

AND

same as those

of the

C=O stretches

in the Raman spectrum

to a conformational

change

bonds

bringing

has occurred,

conformation reference

may cause

of the

In figure samples

with

C=O stretch

in which

ratio

to the 1737 cm

3:

to rotate chains

is -1

(figure

band appears

of cholesterol is

a gauche

about from

its

the

thereby

stronger to the

interchain

less

strong

two DPPC/cholesterol/H20

ratios

are

compared.

ratio 3b).

cm-'

1720

is

1.0

is

(figure

The intensity that

The frequency-

greater

for

3a) than ratio

to be approximately

that

sample for

of the 1720 cm

proportional the

the

cholesterol

to the effect

one.

The Raman C=O stretch region for samples with ratios a) 1 to 1 cholesterol-DPPC ratio. DPPC ratio. C denotes a cholesterol band.

643

axis,

for

at

a local

in

(2,5).

to DPPC, indicating probably

due

C2-Cl

regions

appearing

0.5

some acyl

two

mid-transition

the

arrangement

cholesterol-DPPC molar

are

As discussed

below

chain-planes

the cholesterol-DPPC

the molar ratio

chain

chain-planes

intensity

from

conformation.

the side

"parallel"

different

on DPPC conformation

Figure

of

the

cholesterol about

conformers

that

c/2-cl=c; 3

3, the Raman C=O stretch

shifted

molar

array

with

rotation

at temperatures

of the "tilted"

interactions

acyl

COMMUNICATIONS

seems likely

of DPPC mixed

p CZ-cl~o

the entire

the packing

interaction

band

c3,

9, such a rotation

changing

which

these

RESEARCH

it

DPPC doublet,

in DPPC in which

to a trans

temperature

BIOPHYSICAL

varying cholesterol-DPPC b) 8.5 to 1 cholesterol-

-1

Vol.

94, No.

2, 1980

BIOCHEMICAL

Considerable

broadening

the presence

of cholesterol

to increased

freedom

stretch that

frequency

of the (see

of motion

is very

and that

motion

in the

The 720 cm preciably with

and is about

sample,

Thus,

observed

is

result

suggests

freedom

of

freedom

of

(S ee Table

that

the

C4N+ stretch

that

the most

cholesterol

is

not

ap-

stretch

the DPPC/H20 samples + Since we observe that the C4N -1 higher sample is at 10 cm

is

of the crystalline

sensitive

likely

in DPPC/H20

for

of lipid

or to

the similarity

samples

affect

in agreement

DPPC

to environment

explanation

has little

of the head group. This conclusion 2H nmr (6) and 31 P nmr (7) studies

is

the same for

1.)

Raman band

head group

The C4N+ symmetric

of the DPPC/H20 -1 10 cm broader than that

the

the C=O

enhances

of the choline

we feel that

This

in is due

since

to monitor

of cholesterol.

are approximately

in the C4N+ stretch

cholesterol

region

greatly

Raman band

we believe

geometry.

the broadening

linkage

may be used

stretch

cholesterol.

frequency

Raman band occurs

We believe

COMMUNICATIONS

region.

and bandwidths stretch

C=O stretch 1).

to conformation.

by the presence

and without

RESEARCH

of cholesterol

C4Nf symmetric

symmetric

BIOPHYSICAL

the acyl

bandw-idth

linkage

altered

frequencies

in

the presence acyl -1

lower

Table

sensitive

the Raman C=O stretch

motion

AND

with

and without

on the amine

with

region

interpretations

of

head group-cholesterol

interactions. Thus, bandwidth

the

results

DPPC in aqueous

mixture lipid

dispersion.

at the acyl

C=O stretch

Raman carbonyl probe

suggests in about

bands

not that

half

and DPPC. This

can be monitored

the frequency

region

freedom

stretch

of a second

strong

C=O stretch

cholesterol

induces

of the side

conformation

by measuring

in phospholipid of motion

and

the C4N+ symmetric

chains change

and molecular

a conformational

and the cholesterol-

the relative samples

of

in a 1 to 1 molar intensities

-1 and 1720 cm-1 . The results at 1740 cm

stretch

of geometry,

interface

linkage

of cholesterol

but

affects

The appearance

of cholesterol

interaction

the

cholesterol

of the Raman C=O stretch

upon addition change

show that

of

show that

affords

the

a sensitive

interactions

in the

region.

References 1.

Lippert, J. L., 68, 1572-1576.

and Peticolas,

2.

Brown, E. B., Brown, J. Am. Chem. Sot.

3.

Brown, K. G., Peticolas, Commun. 54, 358-364.

K. G.,

W. L.

(197 1) Proc.

and Person,

W. L. and Brown,

644

Nat.

W. B. (1979). E. B. (1973)

Acad.

Sci.

Submitted Biochem.

U.S.A. to Biophys.

Res

Vol.

94, No.

BIOCHEMICAL

2. 1980

4.

Brown,

K. G.,

5.

McAlister, 1189-1195.

6.

Stockton, Smith, I.

7.

Yeagle, P. L., Hutton, W. C., Acad. Sci. USA 72, 3477-3481.

J.,

Brown,

E. B.,

Yathindra,

AND

and Person, N.,

BIOPHYSICAL

RESEARCH

W. B. (1980)

and Sundaralingam,

COMMUNICATIONS

Manuscript

M. (1973)

Biochemistry

G. W., Polnaszek, C. F., Leitch, L. C., Talloch, A. P., C. P. (1974) Biochem. Biophys. Res. Comm. 60, 844-850. Huang,

645

C. and Martin,

in Preparation.

R. B. (1975)

12,

and Proc.

Nat.