Evidence for the localization of chlorophyll in lipid vesicles: A spin label study

Vol. 71, No. 2,1976

BIOCHEMICAL

EVIDENCE

FOR THE LOCALIZATION VESICLES:

Walter

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

A SPIN

**

Oettmeier,

James

Received

National

IN LIPID

LABEL STUDY*

R. Norris

Chemistry Argonne

OF CHLOROPHYLL

and Joseph

J.

Katz

Division,

Laboratory,

Argonne,

Illinois

60439

May lo,1976

SUMMARY Fatty acid spin labels containing nitroxide groups at different positions in the fatty acid chain have been incorporated into lipid vesicles. Changes in esr parameters of the spin labels in the presence in the membrane of phytol, propionic acid phytol ester or chlorophyll a and the kinetics of chlorophyll a mediated photodestruction of the spin labels suggest a localization of the macrocyclic ring of the chlorophyll molecule in the polar head group region of the membrane. INTRODUCTION Lipid

vesicles

liposomes)

can serve

localization in

the

phyll been the

containing as model

of chlorophyll

photosynthetic within

the

membrane

systems

membrane

surface

of

of the

exist

for

macrocycle:

a) the

ring

the

is

between

*

Work performed under the auspices and Development Administration. ** Resident Research Associate.

Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.

mechanisms of

445

generally

accepted

is tilted

towards

of the into

of

the

the

chloro-

has not

45 and 54O (3-8).

position

protrudes

and

transfer

The location

chlorophyll

the

properties

chlorophyll-liposomes It

at an angle

possibilities

of

(1,2).

established. ring

5 (Chlorophyll-

and to electron

membrane

conclusively porphyrin

chlorophyll

yet that

the Four

chlorophyll water

phase

U. S. Energy

outside

Research

Vol. 71, No. 2, 1976

BIOCHEMICAL

the membrane and the chain

inside

is

located

the

ring

hydrophobic

within

the polar

d) the

ring

carbon

chains

bent is

acid levels

that

into

here

of the

fatty

layers

on the micro-environment

chain

probe

for

the fatty

ring

lipid:

c) or,

the hydro-

positions

location acid

spin

in the fatty

the environment

point

b) the

the bilayer.

membrane and yield

at that

phytol

region;

space between

from nitroxide

bilayer

by its

of the

acid

in

at different

labels

symmetric

region

in the

vesicles

Such spin

anchored

some new evidence

are substituted

chain.

RESEARCH COMMUNICATIONS

of the membrane;

the

the two lipid

in lipid

is

head group

completely

to report

of chlorophyll

itself region

backwards

buried of

We wish

labels

chlorophyll

the

is

AND BIOPHYSICAL

at different information

(9,lO).

MATERIALS AND METHODS Dl-a-dipalmitoyl-phosphatidylcholine was purchased from Sigma. Spin labels 5-Doxyl (2-(3-Carboxypropyl)-2-tridecyl-4,4dimethyl-3-oxazolidinyloxyl), 12-Doxyl (2-(lo-Carboxydecyl)-2hexyl-4,4-dimethyl-3-oxazolidinyloxyl) and 16-Doxyl (2-(14Carboxytetradecyl)-2-ethyl-4,4-dimethyl-3-oxazolidinyloxyl) were obtained from Syva Corp. Phytol (Aldrich)was freshly distilled in vacuum before use. Chlorophyll a was prepared according to Strain and Svec (11) and propionic acid phytol ester according to Katz et al. (12). Liposomes (lipid, Dl-a-dipalmitoylphosphatidyl-choline) in 0.1 M sodium phosphate, 0.1 M sodium pyrophosphate and 1 mM sodium EDTA were prepared as described recently (2). All components were incorporated into the starting lipid solution prior to evaporation. Esr measurements were made with a Varian E 9 X-band spectrometer connected to a Nicolet Model 1072 time-averaging computer. For photodestruction experiments, illumination of the cavity was provided by a 300 W Eimac xenon arc lamp (Model X6263). Light was passed through a 5 cm water filter and a 3850 Corning filter (cut off Q330 ml).

RESULTS AND'DISCUSSION Differences to 16-Doxyl immobilization

between

the

in liposomes of the

membrane and the

long

spin

esr spectra

(Fig.

of

the

spin

labels

5-

1A) are due to the partial

labels

known fact

at different of

446

increasing

levels fluidity

of

the of

the

Vol. 71, No. 2,1976

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Figure 1. Esr spectra of Doxyl spin labels incorporated into liposomes with various additives at 20'. The concentration of lipid was 45 mM, that of the spin labels 0.15 mM and that of the additives 1.5 mM. A. no addition, B. phytol, C. propionic acid phytol ester, D. chlorophyll a. Esr spectra were recorded at a frequency of 9.09 GHz, modulation frequency 100 kHz, modulation amplitude 2.0 gauss, power setting 10 mW. 8 30 set scans were collected.

membrane

towards

eters

spin

of

erties

and permit

which

directly

region,

where where

of

5-Doxyl

16-Doxyl

probes

only

at the

middle

probing

of

12-Doxyl

(Fig.

of

of propionic the

7-position

acid

within

nor at (Fig.

1B).

level

of the

1B)

(ATI 1 ' 2.5;

phytol

of porphyrin

alcohol

changes

probes,

ester, in

447

the in

chlorophyll,

prop-

the

phytol,

membrane

the

polar

into head

hydrophobic

Changes

membrane,

which

param-

of components

isoprenoid

molecule

spectral

in membrane

location

no significant

served

tion

the

in

changes

on the

chlorophyll

causes

Changes

reflect

conclusions

the

liposomes,

interface,

(9,lO).

Introduction

anchors

group

center

labels

incorporated.

the

its

the

Table

I).

is the

are obsite

of

Incorporasubstituent

causes

changes

Vol. 71, No. 2,1976

TABLE I.

BIOCHEMICAL

Experimental

Doxyl

spin

labels

those

of Fig.

AND BIOPHYSKAL

values

in

of nuclear

liposomes.

RESEARCH COMMUNICATIONS

hyperfine

Experimental

tensors

of

conditions

are

1.

Additives

Spin 5-Doxyl

Labels

12-Doxyl

16-Doxyl

TII'

'I'

TII'

'I'

TII’

‘J-’

none

27.9

8.4

22.0

9.4

20.3

9.6

phytol

27.8

8.5

24.5

9.0

20.3

9.7

propionic acid phytol ester

28.0

8.5

24.2

9.1

20.4

9.6

chlorophyll

29.4

8.6

27.2

8.6

20.5

9.5

like the

5

those esr

(Fig. are

of phytol

signal

(Fig.

are

1C).

observed

As compared

increased

by 1.4

(5-Doxyl)

parameters

of

(Fig.

1D).

rin

ring

the

lipid.

These must

in

the

ring

protrudes

observed

as compared

to those

be expected.

buried

between

rotational

liposomes

in

cannot

the

behavior

near

the

into spectral

that

head

can be ruled water

group

with

Furthermore, hydrocarbon 16-D0xy1

if chains

the

the

the

porphy-

region

the

of of porphyrin

5- and 12-Doxyl

porphyrin lipid

has to be affected,

448

I).

pronounced

or phytol

of the

'

II

altered

site If

the

of

phytol

the

out.

phase,

parameters

observed

of

polar

T

(Table

are not

a) and d) for

the

ester,

(12-Doxyl)

however,

in

incorporated

phytol

us to conclude

possibilities

chlorophyll

changes

16-Doxyl,

changes

5 is

acid

and 3.0

lead

be located

Thus,

indeed

to propionic

results

pronounced

when chlorophyll

1D).

The spectral

More

propionate ring

is

bilayer, and it

the is

Vol. 71, No. 2, 1976

not.

BIOCHEMICAL

The results

distinguish

presented

between

This

to the

destruction

of

phyll

is

possibilities

spin

the

2.

observed

if

As can be seen rate

than

slowly.

12- and 16-Doxyl Spin

rate

where

are

closest

phyll region

label

the

the

labels

in in

donor

the

of the

presence

intensity

of

spin

-a is not

present

2, 5-Doxyl

is

, which

undergo

is most

the

in

(possibility

b)).

label

the

signal

at a faster

at

nitroxyl

polar

only the

fastest

radical

that

Should

shown

liposome.

occur

therefore, in

are

decomposition

a) and the

likely

photo-

of chloro-

the

destroyed

should

We conclude,

liposome

us to

photodestruction

height)

(chlorophyll

ring

allow

signal

photodestruction

together.

macrocyclic of

Fig.

the

The kinetics

decrease in

chlorophyll from

(13).

spin

No reduction

do not

b) and c).

labels

by the

RESEARCH COMMUNICATIONS

however,

by applying

three

2 (as judged

in Fig.

here,

can be achieved

technique

AND BIOPHYSICAL

the

chloro-

head group the

macrocylic

I Imid

Figure 2. Kinetics of the photodestruction of Doxyl spin labels incorporated into liposomes in the presence of chlorophyll a at 20'. Conditions as in Fig. 1, except that the concentraEion of spin labels was 2 mM. The magnetic field was adjusted to the center peak of the spin label resonance in traces D, Fig. 1. a - 13 5-Doxyl, O-O 12-Doxyl, A-A 16-Doxyl.

449

BIOCHEMICAL

Vol.71,No.2,1976

ring

be bent

12-Doxyl not

backwards

as compared

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(possibility

c)),

to 16-Doxyl

is

faster

destruction

of

to be expected,

and this

is

--in

to occur

in

observed. The main

amount

an oligomeric liposomes

form

(14),

--in vitro

reasonable cyclic

ring

of the

lipid

of chlorophyll

is

whereas

essentially

of chlorophyll bilayer

are associated

--in vivo

with

the

appears

state

of

monomeric

to assume

however,

the

vivo

similar

(2).

It

locations

within

the

for

those

lipid

chlorophyll

region

polar

the

the

seems to be

of the

macro-

head group

region

chlorophyll of

in

molecules

thylakoid

that

membrane.

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Lguger, P., Pohl, G. W., (1974) in Perspectives in S ., and Gitler, C., eds.) New York, San Francisco,

2.

Oettmeier, Naturforsch.

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Cherry, Biophys.

R. J., Hsu, K., and Chapman, Acta 267, 512-522.

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Vol. 71, No. 2,1976

BIOCHEMICAL

AND BIOPHYSICAL

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11.

Strain, H. H., and Svec, W. A. (1966) in The Chlorophylls (Vernon, L. P., and Seely, G. R., eds.) pp. 22-61, Academic Press, New York, London.

12.

Katz, J. J., Strain, H. H., Harkness, A. L., Studier, M. H ., Svec, W. A., Janson, T. R., and Cope, B. T. (1972) J. Am. Chem. Sot. 94, 7938-7939.

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451

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