Paramagnetic fluorescence quenching in chlorophyll a containing vesicles: Evidence for the localization of chlorophyll

Vol.

78, No.

2, 1977

PARAMAGNETIC

BIOCHEMICAL

FLUORESCENCE

VESICLES:

J.

EVIDENCE

Luisetti,

Ulm,

Received

QUENCHING

H.J.

Experimentelle

Universitat

11.

RESEARCH

COMMUNICATIONS

IN CHLOROPHYLL

A CONTAINING

OF CHLOROPHYLL

Galla

Physik

7900 Ulm

August

BIOPHYSICAL

FOR THE LOCALIZATION

H. Mijhwald,

Abteilung

AND

III

(Donau),

Oberer

Eselsberg

1977

SUMMARY Spinprobes (fatty acids, androstane, cholestane) that differ in the location of the paramagnetic centre relative to the polar membrane surface have been incorporated into lipid vesicles containing chlorophyll a. Quenching of the Chl a fluorescence is observed following the Stern-Vollmer-relationship. Quenching is more effective with spinprobes carrying the nitroxide group near the polar head groups of the lipid moiety. Quenching is also observed using a water soluble spinlabel. The porphyrin ring of the Chl a molecules is suggested to be localized in the polar head group region of the bilayer membrane. INTRODUCTION Lipid lized

b ilayer

as model

the

ves icles

systems

to

of

chlorophyll

localization The porphyrin

(1,2).

surface

at

bilities

for

position

a)

the

the

ring

membrane,

b)

region

the

of

hydrophobic lar

region

layers. different

the

ring

into

is

d)

between

the et

the

the

(4)

ring

is

is

the bent

measured in

the

Four

possi-

discussed

outside

polar

head

backwards

of

kinetics

the

the group

into

buried

spectral the

are

towards

phase

completely chains

and changes

Copyright 0 1977 by Acudernic Press, Inc. All rights of reproduction in any form reserved.

aqueous

within

hydrocarbon

al.

45 and 54O (3). ring

and

membrane

to be tilted

the

ring

properties

photosynthetic

porphyrin

located

c)

the

reported

the

a can be uti-

organization

between

of

protrudes

region,

spinlabels

is

chlorophyll

the in

an angle

bilayer,

Oettmeier

study

ring

membrane

(9):

containing

two

in

the

apo-

lipid

parameters of

the

the

of chloro-

754 ISSN

0006-291

X

BIOCHEMICAL

Vol. 78, No. 2, 1977

phyll

a mediated

clude

that

lar

calization

of

looking labels fatty

photodestruction

the porphyrin

head group

region

ring of

chlorophyll

carrying

(TEMPOL*,

chain

is

located

most likely

bilayer

group

in the po-

androstane

different

of the

quenching

profile.

probes

is

based on a reduction

of

fluorophore

The fluorescence

Fatty

at different

were used as quenchers.

regions

They con-

the

acid

position

Additional

spinin the

spinlabels

and cholestane)permitted membrane, thus quenching

of the

lo-

membranes upon

of Chl a fluorescence.

the nitroxide

spinlabelled

spinprobes.

the membrane. We have examined

to probe

the

of these

a in lipid

at the quenching

acid

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

excited

yielding

us a

by paramagnetic state

lifetime

(5,6).

MATERIAL AND METHODS D-L-d--dimyristoyllecithin from Fluka was checked by thin layer chromatographie and used without further purification. Spinlabels were purchased from Syva, Palo Alto. The pyrene derivatives were synthesized in our own laboratory. Chlorophyll a was obtained by isolating the plant pigments from fresh spinach. Purification was performed on a chromatographic column filled with a suspension of powdered sugar in petrolether following the method of Smith and Benitez (7). Vesicles were prepared from a chloroform solution containing lipids, chlorophyll and fatty acid spinlabels. After evaporation with nitrogen the probes were dispersed in a 2 mM solution of CsCl by sonication in the dark and under nitrogen atmosphere for about 10 minutes. The temperature was kept above the phase transition temperature of the lipid. Probes containing cholestane or androstane spinlabel were prepared similarly but the spinlabel was added from a IO-2 m methanolic solution according to Bieri et al. TEMPOL was added from an aqueous (7). solution. Fluorescence spectra were taken with a Schoeffel M 460 Photometer equipped with a cooled red sensitive photomultiplier. All measurements were performed at 320 C except the TEMPOL quenching experiments at 12O C. The quenching process is expected to follow the Stern-Vollmer relationship: (IO/I) l=k.?'. c, where I, is the fluorescence without quencher, I is the emission intensity in the presence of a quencher, k is the quenching constant, Z the lifetime of the excited state and c the concentration of the quencher. * Abbrevation used: dinol - 1 - oxid)

TEMPOL (2,2,6,6

- tetramethyl

- 4 - piperi-

Vol. 78, No. 2, 1977

BIOCHEMICAL

a01 molar

Figure with TEMPOL chlorophyll according to

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

a' of TEMPOL

concentration

1. Stern-Vollmer plots of fluorescence quenching at 120 C in dimyristoyl lecithin bilayers. The concentrations are a) 1 Mole %, b) 3,3 Mole % the lecithin. Identical curves are obtained.

RESULTS AND DISCUSSION a) Quenching Stern-Vollmer

plots

the chlorophyll fig.

Straight

1.

indicating

lines

like

relative

minimize periments transition attached

the label

concentration at

temperature. to internally

quenching

probes

to the

within C that

12O

According accessable

and the given

lipid

shown in

lipid.

It

between

the

reduction chlorophyll

of

the

the equeous In order

a fluorescence

is below

the

lipid

phase

to the EPR-spectra

no TEMPOL

sites

at this

was observed

by addition

was immediately

756

to

the membrane our ex-

concentration

spinprobes

1

is known

of

10

-3

molar.

The emission quenching was effective in a concentration -2 -1 between 10 and IO molar concerning the paramagnetic After

of

containing

phase of the membrane (8).

were performed

temperature

for

TEMPOL can distribute

phase and the hydrophic

by TEMPOL:

caused by TEMPOL are

are observed

% chlorophyll

molecules

a fluorescence the paramagnetic

fluorescence

a

and 3.3 Mole that

of chlorophyll

range probes.

of ascorbic reestablished

acid

BIOCHEMICAL

Vol. 78, No. 2, 1977

AND BIOPHYSICAL

I)

F)

d)

RESEARCH COMMUNICATIONS

h)

9)

Figure 2. Lipoids used in this study and the relative location of the nitroxide group as well as the chromophore according to the membrane surface indicated by the lecithin molecules. a)dimyristoyl lecithin, b) TEMPOL, c) 5-nitroxide-stearic acid, d) 12-nitroxide-stearic acid, e) 16-nitroxide-stearic acid, f) androstane label, g) cholestane label, h) (rJ-pyrene butyric acid, i) w -pyrene-decanoic acid.

to

the original

value

b) Quenching Three

fatty

nitroxide

acid groups

in the

absence

caused by fatty spinlabels along

acid

differing

the

fatty

in the membrane profile

At

temperature

sition)

the

randomly of the rate, in fig. using at the

spinlabels

distributed labels.

of are

is

shown in fig.

the

lipid

into

for

a small

(the fatty

acid

757

chain).

2.

phase tran-

the membrane and the EPR- spectra

5-nitroxide-

quenching

paramaqnetic

the

Their

stea-

and 16-nitroxide-stearate

d and e. Only

end of the

plots

of

were used.

as can be shown by taking

16-nitroxide-stearate apolar

chain (9)

incorporated

12-nitroxide-stearate 3, c,

spinlabels:

32O C (above

The Stern-Vollmer

molecule.

in the position

acid

localization the given

of any quencher

effect group

are

shown

is observed is

localized

The quenching

effi-

Vol. 78, No. 2, 1977

BIOCHEMICAL

AND BIOPHYSICAL

molar

ratio

RESEARCH COMMUNICATIONS

of spinlabel

Figure 3. Stern-Vollmer plots of paramagnetic quenching in dimyristoyl lecithin bilayers at 320 C. The spinlabels incorporated into the lipid matrix are c) 5-nitroxide-stearate, d) 12-nitroxide-stearate, e) 16-nitroxide-stearate, f) androstane-label, g) cholestane spinlabel. The chlorophyll a concentration is 1 Mole % according to the lipid.

ciency chain is

increases versus

situated

than

near

polar the

carbonyl

not

of the standing

position

porphyrin

out

nitroxide

group

group

the that

is a much better

12 is

ring

along

5-nitroxide-stearate,

in a medium position.

from carbon

localization

the

head group.

12-nitroxide-stearate

quenching

but

the

upon shifting

quencher

Nevertheless

effective.

Therefore

a

in the polar

headgroup

region

of the membrane surface

is

favoured

by

our experiments.

Cholestane with

its

fore

conceivable

pared fig.

nitroxide

to the

spinlabel ring that

androstane

(fig. into

its

2 g) is

to protrude

the membrane surface.

quenching label.

suggested

This

3.

758

efficiency is revealed

It

is

is there-

smaller

by curve

comg in

BIOCHEMICAL

Vol. 78, No. 2, 1977

Our results porhyrin

ring

is

confirm

a chlorophyll

located

near the

membrane. The macrocyclic the water 1.

phase but

Quenching xide

ring

to be bent

lipid

positioned fatty

androstane

bedded deeply

chains

3. Also

small

label

with

part

of the

into

nitroxide

stearic

part

using

of

and

that

are em-

of the membrane (12-

the cholestane

positioned

(IO -1 molar

is

spin-

in the hydrophilic

observed

at a lipid

our method of finding quenching

above given the

lipid

centre chains

chains) acid,

ce of these

the

acid

spinlabels of the

is

only

at high

concentration

of

order

Using pyrene is positioned

of efficiency

carbon

acids

pyrene

(fig.

is

759

decanoic

2 i)

Pyrene

at carbon

following

also

thirteen

acid

quenched

(the

and

cen-

seven of the

by:

acid.

of effiacid

at carbon is given

decanoic

order

butyric

acid

as fluoropho-

5-nitroxide-stearic

12- and 16-nitroxide-stearic pyrene

w-

ring

in the

acid,

ring

a fluorophore.posi-

as quencher.

aromatic

quenched

acid.

aromatic

using

butyric

12-nitroxide-stearic

of the

lipid

and eight acid

spinprobes

TEMPOL quenching

checked

16-nitroxide-stearic tre

atoms five

the nitro-

molar).

with

ciency:

using

group

2 h) and w - pyrene

the

with

membrane.

concentrations

re and the

of

into

because:

carbon

is observed

by stereospecific

acid

of the

acid).

quenching the

We also

(fig.

region

to protrude

spinlabels

the hydrophobic

4. Using watersoluble

tion

head group

(S-nitroxide-stearic

is observed

and 16-nitroxide-stearic

1o-3

using

where the

label).

2. Small quenching

label

polar

backwards

between

acid

conformation

is assumed not

is most effective

group

the

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

5-nitroxide-

The fluorescenby androstane

Vol. 78, No. 2, 1977

and cholestane quencher will

for

be given

BIOCHEMICAL

label, both.

whereas

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

androstane

A detailed

label

description

is

the better

of these

experiments

helpful

discussions

elsewhere.

ACKNOWLEDGEMENT We are indepted and to U. Theilen Luisetti

is

to Prof.

for

his

supported

Sackmann for

excellent

technical

by DAAD (Deutscher

assistance.

Akademischer

J.

Austausch-

dienst).

References: 1.

Lauger,

P., Pohl, G.W., Steinemann, A., Trissel, H.-W. in Perspectives in Membrane Biology pp. 645 - 659 Academic Press, New York, San Francisco, London. (1974)

2. Oettmeier, W., Norris, (1976) Z. Naturforsch. 3. Podo, (1976)

J.R.,

Katz, -

31c,

163

F., Chain, J.E., Blasie, Biochim. Biophys. Acta

J-J. 168.

J.K. 419,

19 -

41.

4. Oettmeier, W., Norris, J.R., Katz, J.J. (1976) Biochem. Biophys. Res. Commun 71, 5.

Birks, (1970)

6. Bieri, (1975)

J.B. Photophysics

of aromatic

V-G., Wallach, D.F.H. Biochim. Biophys. Acta

molecules, 389,

7. Smith, J.H.C., Benitez, A. (1955) In Modern Methods of Plant Springer Verlag. 8. Shimshick, E.J., (1973) Biochem.

McConnell, 12,

9. Schreier-Mucillo, S., (1976) Arch. Biochem.

2351

413

-

-

415

Whiley

Analys is

IV,

2360.

Marsh, D., Smith, I.C.P. Biophys. 172, 1 - 11.

760

Intersience.

427.

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451.

pp.

142

-

195