Biological applications of fourier transform infrared spectroscopy: a study of phase transitions in biomembranes

201

3oumalofhfolecularSh-ucture.113 (1984)201-212 ElsevlerSclencePubhskrs BV, Amsterdam-_~dInTheNetherlands

BIOLOGICAL

APPLICATIONS

PHASE TRANSITIONS

IN

HENRY H.

MANTSCH

Divlslon

of

OF FOURIER

TRANSFORM INFRARED

SPECTROSCOPY.

A STUDY OF

BIOMEHBRANES

Matlonal

Chemistry,

Research

of

CouncJl

Canada.

Ottawa,

KIA

OR6

(Canada)

ABSTRACT The thermal response of aqueous lipid membranes has been investigated by Fourier transform infrared spectroscopy. Changes in infrared spectral parameters are applied to the analysis of the structural changes which occur within the lipid bllayer as the temperature is varied. Such stud1 es prove de the basis for the interpretation of phase transitions in complex bl omembranes.

INTRODUCTION When studying spectroscopy

complex

one

classical

spectroscopist

generally

small

the

of

field

transform The

of

of

UV or

biological

water

a fact

conventional the

impair both

which

advent , along

spectroscopy

has

with

the

has

opened examples

from

our

polymorphism infrared plasma

in

laboratory membranes of

spectroscopy, membrane

0022~2860/34/SO3

of

00

live

to

recent

The

taken

of

by the

of problems

in

Fourier

answers.

solvent

a range

of

require of

widely an aqueous

choice,

measurements

heavy

water

or

severely

the

study

interferometrlc

blol

systems.

and

precluded

hand11 ng techniques, ogl cal

kinds

particularly

invarlantly

the

onal

encountered

structure

many

and

useful

spectroscopic water

spectroscopy of

are

encompasses

only

vi brat1

that

molecular

infrared

almost

of

but

often

part

itself.

not

infrared

However,

not

the

provide

which

use from

there

which

can

is

structure does

of

appl lcatlons

water

Raman techniques,

absorbers,

to

structures

whereby

molecular

the

While

details

science

by the

different

Nevertheless,

spectroscopy

molecular

environment,

quite

solving

biological

domain

samples

a situation

molecules.

infrared

different

bl ologlcal

faces

Fourier

which

ln

Ottawa

to

the

and

I shall range

characterization,

temperature-induced

using strong

limited of

aqueous

of for

the

to the

bacteria.

0 1984 Glsevler Science E’ubh_sbers B V.

ESR,

of

systems. infrared

study

data of

aqueous

of

llpld

illustrate study

by Fourier changes

KMR,

appl lcation

sophisticated

use from

the Infrared

the

transform

availability

up new avenues

are

in

the

this

are

transform structure

of

the

EXPERIHENTAL Instrmentatr

ferr

on

field

The

of

infrared

llcludlng

sophisticated

llplds

and

hereon

were

transform

aqueous

low

with

instruments and

to refs.

1 and

Spectra

aqJisition

various

this

no:

and

models

lipids

conventional

below,

changes

at

in

and

band

above

experiments

are bath

selected

a spectrum,

This

been

response

to

in

live

covered

in

absorption

subtract

the

modern

should

the

masks

several

namely and

as that

determined Among the

of

greatly

routinely various

deconvolutlon

extremely useful

(whereby for

sample

changes

modern

in in

study

IS

Frequency

lineshape of

band

is

often

are

take

into

required

operation main

to for

precautions

recorded

they

with

cells

recorded

at

of

the

account

the

bandwidth than

task

of

values

+ 0.05

technique

removed

contours

to

spectrum.

better the

twc

digitized

and

of

methods,

to

water

range

As the

IS

a routine

are

that

order

the

uncertalntles

a known

spectra and

temperature

it

however.

spectrometers

reduction

rhe

water

of

simplified. 71th

data

the

spectra.

temperature

from deterioration.

bands IS

nhicn

alternative

preparations.

lipid

process

computer

recording

the

bacteria

lipid

6,7),

materials

the

live

Spectra increments

This

Ar

a wide

subtraction

window

spectral

allows

all

(refs.

that

3.4).

study

as

temperature

of

the

Absorbance

for

In it

in

study.

while

the

such

temperature

continuously

since

solvent

spectrometers

(refs.

at

transitIons

mount.

spectrometer

waits

spectrum

5)

and

under

the

temperature,

preventing

water

of

of

system

apparatus cell

range

same

temperature.

on auxiliary

sample

useful

the

bands.

output is

of

well

behavior

the phase

of

a thermostated the

IS of

temperature-induced

and

temperature

(ref.

corresponding

dependent

minutes

D,D)

water

same temperature

Since

studies

Fourier

are

of

a function

control

another

bacteria

fnfrared

the

particularly

pathlength

processing

last

discussed

of

phase

spectrum

as

particular

the

the

be considered,

identical

the

under

records

a few or

infrared

all

to

increase found

Water (H,C

advantages

thermotroplc

the

temperatures,

lncrerents

then IS

has

many

completely

equlllbratlon.

be

at

according

brought

approach

for

results

instruments

infrared

parameter-s

circulating

recorded

the

the

temperature

records

dispersive

studying

collecting

temperature

be

suitable

the

available

handling

Variable

being

quite

experimental The

in

now widely

here, tne interested reader is referred

variable

can

are

instrumentation.

towards

involve

temperatures

usually

which All

be discussed

data

directed

monitoring

are

dramatlcally are

2.

Experiments memb,-ane

cost

changed

spectrometers

preparations.

osler

shall

has

infrared

membrane

obtained

documented

and

lnstrumentatlon

Fourier-transfor;n

years.

of

cm-1

band

from

the

comprised

of

data

now can (ref.

be 8).

narrowing

or

spectra),

IS

more

one

than

a

203 component

; the

integrated of

this

LIPID

method

PHASES-

The of

deconvoluted

lntensjtles to

the

primary

book

level

introduction

to

by Ueissmann

are

as in

to

an

hydrophobic interface

yield

a rather

aqueous

of

especially

polar

their

temperatures

are

into groups.

ability direct

IS

to

in

correct

refs.

dl scussl

on

9-11.

the

the

the

take

up a variety

An

be

found

in

the

of

separated

of

the from

in

parts. e

a hydro-

conmfon to

hydrophobic other

properties

of

at of

the

bllayer

sufficiently

each

structures

segregation

of

bonds

A property

characteristic

this

1.

can

same molecule

by chemical

segregation

of

of

embedded

behavior.

Many

an arrangement

12).

proterns (bottom).

in

together

IS Figure

membranes

(ref.

regions,

consequences

the

membranes

blologlcal

independent

distinct

found

as shown in

presence

linked

environment

moieties

of

Claiborne

by the part,

be

but

An appropriate

biological

membrane with lipid membrane

characterized

a hydrophlllc

can

structure

subJect and

bands

retained.

TRANSITIONS

of

a bilayer the

narrower

are bands

organization

into

and

flexible

apolar

of

lipids

edited

Lipids

lipids

overlapped

Model of a biological (top), and typical

Fig. 1. of lipids

phobic

locate

has

rrequencies

STRUCTURE AND STRUCTURAL

amphlphlllc

excellent

spectrum

and

and by lipids

different polar

all

and

an and

With regard to studies of the polymorphism

in membrane lipids, the infrared

spectrum can be separated to great advantage into spectral regions nhlch inate from possible For

to

the

trum

vlbratlons refer

purpose

of

an

of to

of

aqueous

different

molecular

“head-group’

and/or

“hydrocarbon

tnis,

illustrating membrane

In

moleties.

Figure

preparation

tail”

2 shows

obtained

this

from

manner

spectral

a typlcal human

orrgit

IS

regions.

Infrared

spec-

erythrocytes.

t i 2-

.-w.

C-V

WAVENlJMBER,cti’

Infrared spectrum of aqueous dispersions of human erythrocyte phosFIJ. 2. phatidylethanolamine ‘n water (DaO) recorded in a 6um thick BaF, cell with a Digllab FTS-11 Fourier transform spectrometer equipped with a mercury cadmium tellurlde detector (ref. 13).

The

structural

by monitoring Among

the

stretching red

spectra

temperatures. membranes.

changes various

vibrational vibrations of

egg

occurring

infrared modes of

of

the

maJor

acyl

yo 1 k lecithin

Lecl th i ns are

in

aqueous

absorption

the

diagnostic

chal in

bands

ns.

the

most

membranes

as

a function

value

Figure C-H

lIpId

are

the

3 displays

stretching

cwrsnon phosphollplds

can of

be

methylene a series

region In

studled

temperature.

at

C-H of

different

mammalian

infra-

205

3000

2900

2950

WAVENUMBER

Fig, 3. Temperature-induced lecithin in the region of with increasing temperature.

The

vibrations

infrared (refs. and

of

spectra 14-20).

symetric

2872

o-n-l

methyl

are

changes in the C-H stretching

acyl

chains

of

esters

of

The

strong

bands

fatty at

are

readily and

2920

sL.retching

modes.

asymnetric

and

symmetric

bands

exhibit

of

width

these

which

can

be

infrared bands;

acids

the All

and

CM’

CH,

group.

frequency

the

the

2800

2850

and

spectra of egg the peak height

assigned other

2850

are

the

CH,

comparison

methylene-chain cm-l

respectively;

by

the

weaker

stretching

to

antlsynnnetrlc bands

modes

structural

with

compounds

at

of

tenperature-dependent

related

yolk decreases

2955

the

and

temmlnal

variations

changes

at

the

in

molecular

1 eve1 . Illustrated frequency stearoyl lstlc

in of

those

C16:O

in

frequency

calorimetric and

The highly

whereas

found

band

lecithin.

tne

dlsordered

s.1 gmo~ da1

4 are cm-l

conformationally

hydrocarbons,

ing

Figure 292G

(Cl8:O) of

ationally as

the

54OC for

acyl llquld

the

in

values

above with

can

transition, C18:O

values

ordered

lecithin

acyl 2923 a high The

be taken 1-e.

24°C (ref.

profiles

(C14:0),

frequency

chains

Tm,

temperature

myristoyl

hydrocarbons.

curve, phase

detailed

below

chains m-1

are

midpoint

for 21).

the

2920 as

content

as

obtained

palmitoyl

the

cm-l

found

are

in

characteristic of

gauche

of

the

midpoint C14:0,

for

(C16:O)

the and

character-

solid-like of

confom-

conformers

such

steep of

the

41.5”C

correspondfor

the

10

20

3.0

40

50

60

TEMPEPATURE

Fig. 4. Temperature-dependence stretching bands In the sp ectra and stearoyl lecithin (squares). computing the centre of gravity (for details see ref. 83.

There

IS

stretching

and In

such

effect The

in

the

gel

and

spllttlng

due

helm of

the

eratures chains

only

rocking

In

CH,

an

acyl

scissoring

between behave

like

chain and

from

at in

At

1470

and

of

CH,

trans/gauche the

at

higher

the

similar

to

Tm IS

for

modes

at

chain

independently

Cl6:O

structure

720

show modes

phase

temperatures packing of

and

subcell

IS each

of

above

field

17,

26-29).

has

been

at

temperatures

Tp. where (ref.

and CH,

interchaIn

hexagonal other

chains Tp the

[refs.

50°C

bllayers

correlation

change

lack

at

the

acyl below

an-l

by

and of

saturated

orthorhombic-like

rccklng

rotors

35°C the

vlbratlonal

characterized

acyl

the

conformational

frequency

temperatures

a solid-solid an

of

average

frequency

with

Tp.

packing

Tp and Tm the rigid

is

Cl4:0,

lecithins

at

of

the

this

in

a change

coupling

leclthlns

as a transition

Tp to

in

modes

lnterchaln

pretransition

characterized

of

degree

monitor

higher

frequencies

n-alkanes.

14OC for

temperature

“prerransltlon”,

CH, to

3 (at

of

the

on the to

increase

melting

observed

as the

used the

steep

that

depend

been

22-25);

the

Figure the

phase,

to

scissoring

The

In

literature

chains

The

at

90

the frequency of tne antlsynnnetrlc CH, myrlstoyl (triangles), palmitoyl (circles) Infrared frequencies were obtained by wide seqment of the bands of the topmost 2 an-l

have

(rafs.

disorder.

80

of of

the

acyl

and

systems

indicate

referred

in

long can

observed

arrows

ClS:O),

of

hence

conformatlonal the

evidence

bands

disorder ratio

ample

73

’ C

coupling At

tempindividual

29).

for

207 Figure of

5 shows

temperature

cl

in

10

the

change

the

spectra

30

20

JO

in

the

of

50

60

TEtPESA-uRE

There

IS

a large

the

lipid

phase

observed acyl chain the

with

Tm and The

length

and

greater

The the

at

chal ns.

the

temperature

amounts

change

in

bandwidth.

incubation

proteins

of by

the

In

the

gel

chain

GII-l

band

and

stearoyl

as

a function lecithin.

phase the

at

the

the

or

occurs.

(ref.

32-34).

packing

has

relative

(ref.

annealed The

at

effects

through The changed

22,

30,

this

another

infrared resulting

of

with

at

Tm),

affects llpld

CH1

31). near

4°C

a

transformation

phase

spectra In

the

the

acyl

increasing

temperatures of

in

membranes

bandwidth

of

bandwith

introduced

leclthln

0

clearly

nature

increasing

Tm decreases

protein

the Cl6

in

introduced

disorder

and

the change

with

into

of

but

correlate

disorder

greater

at

a smaller,

to

Tm increases

frequency

are

Tm and

the

cholesterol

temperature

at 3/4 peak height of C14:O (triangles),

dlfflcult

to

at

frequency

membranes

increasing

acyl

both

cholesterol

subtransition

the

of

width spectra

values,

AHm (i.e.

or

change

lecithin

IS

related

the

The

It

bandwldth

greater

of

2920

53

bandwidth

bandwidth

in

incorporated

reversed

the

Uhlle

directly

dependence

transformat?on

as

IS

60

70

the

When hydrated

known

Tp

change

modes.

of

in

Tp.

absolute

incorporation

stretchlng

be

at

the

palmitoyl

of the full bands in the (squares).

increase

increase

of

‘C

Fig. 5. Temperature-dependence antisytnnetric CH2 stretching (circles) and C18.0 1ecTthlns

identifiable

width

myristoyl,

can

transition, show that

a more

rlgldly

after

208 packed

gel

reduced; in

phase

this

hydration.

Thus

dehydration In

and

certain

obtalned

1 ine

phase been

as

that

the

that

the

of

indicated

established

cr

from

converts

by

head

X-r*y

and

contain

to

macroscopic

freeze

fracture

1s greatly are

in

respectively

acyl

typlcal

a ml ccl

structure

It

has

been

concentration of

the

phase

shown

of

transition

gauche

amine

further to

the

in

frequency

of

such

F gure the

gel

shift phase

force

in

the

acyl

this

(ref.

frequency

phase

of

the

a frequency crystal

of

about

1 cm-l Both

CH,

gauche

for

egg

shl ft

of

about

50°C

transitions

transition

the

formation

Since

population mode

yolk

wth

reversible.

this of

is

the

an

phosphatidyl

2 an-l (A

associated are

Induced

18).

stretching

plot

ohase

at

the

solld-like (part C). hexagonal

increasing

triggers 13,

has

36).

thermally 1s the

which

ln

The

llqu’d

5).

this

(refs.

Increase

to

(C to

chains

temperatures

a traasltlon.

7 shows

behind

rearrangement,

the

higher

a further

phase,

rrith

miceliar

at

involves

monitor

driving structural

bonds

phase

llquld-crystallIne

associateo

the

a major

non-bllayer

adequate ethanol

that

requiring

liquid.

non-lamellar

of

techr;iques

chains)

lamellar

lar

Fig. 6. Illustration of the lamellar bllayer structures as ln the gel phase (part A) and ln the liquid-like liquid-crystalline phase Fart 8 shws the non-bilayer structure referred to as the Inverted phase Lonsistrng of tubular inverted mlcelles.

transition

changes

23,35).

unsaturated the

heating

group

result (ref.

erythrocytes,

The

head

transformation

group

(which

further

Fl gure 6.

in

the

the

subtransition

lipid

human

upon

of

causing

and

the

membranes

egg yolk

shell

forces

lncubarlon

hydratlan

phase

hydration

the

phosphollpld

from

crystal

and

suggests

to

at C)

the

18°C and

a

transition

209

.

-

2852

. I -

2851

. I

I. -_v VW 2850.

0

I

_v Tm

10

20

30

40

TEMPERATURE,

The

complexity to

due

provides

to

of

an

which

The

of

In

grown

shows on

a

studies

involving

precludes such

circumventing

8 which

was

spectrum as

fatty

bands. these

monitor

In

acid

bands

acyl the

diet

the

cases

these the

the

the

A frequency

8 contains the

the

use

problems

infrared

addition

observation of

containing

of

deuterium

of of

specifl

oeuterated

(refs.

spectrum

the

frequencies of

trans-gauche

2090

cm-1

of

a hlgl,ly the

of

of

for

upper

of

bands

c 1 lplds

37-41). whole

This

IS

membranes

of

labelled

ordered llmlt

cm-l,

fatty

the

membrane.

in

gel being

the CD,

phase about

lipid

of

cm-l,

are

chain, CH,

acyl

(refs.

a low gauche

2096

the

chains

be used

band

of

Interfering acyl

to

can

ensemble

stretching with

from

bands hence

and bands

a lipld

Similar

CD2 stretching

syrinnetrlc

free as

and

llplds

stretching

a region

incorporated

disorder

ratjo the

from

C-D

IS

the

conformatlonal

average

frequencies,

2200

acid

probe

of

characterisltic

near

fatty

a specific

degree

a variety

temperature-dependent

occur

chains deuterated

provide modes,

on

Figure However

well.

Since

stretching depend

Higher

of

.

proteins the

Figure

and

generally

vibrations.

means

rn

membranes

membranes

well-known

a useful

organism

natural

lipid

exemplified

acids

centre of llpld

BIOMEMBRANES

substances bands

60

Temperature-dependence of the frequency (measured as of the symmetric CH, stretching bands in the spectra obtained from egg yolk phosphatidylethanolamlne.

Fig. 7. gravity) membranes

NATURAL

50

“C

to 5,

42,

43).

IS population. indicative

of

an

increased

populat>on

frequency

and

of

a simple

gel

and

4c

a

of

gauche

conformational

two-component

liquid

crystal

However,

rotamers.

disorder

is

overlapping phases

not

band

(ref.

the

linear.

model

relation

This

to

between

necessitates

obtain

the

the

use

proportions

of

41).

1000

2000

3000 WAVEMJh~BER,cm-l

Fig. 8. laldlan:i

iT-IR spectrum of the bacterial El grown at 30°C in the presence

Figure

9 shows

in

the

spectra

of

live

cells

transition the

gel

The

to phase

isolated

live

are

in

liquid

with

the to

an

between

at

CD,

symmetric

20 and

34OC they

On cooling

stretching

Be1 ow 20°C,

membranes.

a gel

widths

of

bacteria,

the

Thus,

crystalline in

the

phase.

undergo

the

live

aisorder IS

also

4OC higher.

membranes

conformational

phase;

of

Isolated

Acholeplasma acid.

undergo

the

band

membranes a

system

reverts

to

hysteresis.

while

and

liquid

and

crystalline

50 percent

content,

gel

membranes

about

30-C,

blologicai

the

profiles

cells

a slight

membranes

compared

of

temperature

the

However,

temperature

phase

the

tne

isolated

transition.

1 .e.

of

plasma membrane of of perdeuteromyrlstic

the

a given in

important

at

phase live

tl-e

which

bilayer, in

membrane

phase

are the

the

forme-

temperature

is

only

membranes.

lipid

factor

crystal

of

growth

content

cell

liquid

transitions

transition

temperature, the

to

the

about

The IS I.e.,

of

dependent the

for

occurs the

liquid

at

bacteria,

20 percent,

“fluidity”

regulation.

same

as

of on the

amount

crystalline

a

Temperature dependence of the frequency of rhe symmetric CD, Fig. 9. stretching band of the lipids of Acholeplasma laidlaw B grown at 30°C on perdeuteromyr7stIc acid. Shown are frequencies from spectra of live cells with the temperature ascending from 20 to 40°C (+) and descending from 40 to 15°C (x)

and

frequencies

ascendjng

from

5 to

from

spectra

45OC

(a).

of

isolated

membranes

with

the

temperature

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