Faculty of. Education, Kumamoto University, Kurokami 2-chome,. Kumamoto, Kumamoto 860. Many dye molecules which are distributed between the membrane.
J. Biochem. 89, 397-405 (1981)
Surface
Potential
Dye Molecules
Dependence
of the Distribution
onto Photosynthetic
Kazumori
MASAMOTO,'
and Mitsuo
Membranes
Katsumi
MATSUURA
, Shigeru
ITOH,
NISHIMURA
Department
of Biology,
Higashi-ku,
Fukuoka,
Faculty
of Science , Kyushu
Fukuoka
Received for publication,
Partition
of Charged
University
33,
812
July 24, 1980
of merocyanine
dyes,
which
have a negative
charge,
onto
photosynthetic
membranes of chloroplasts and bacteria was analyzed by measuring the fluorescence intensity change, absorbance change, and amount of dye in the supernatant after centrifugation. of valence
The partition
and concentration
depended
on the surface
potential,
which is a function
of ions in the medium.
The distribution of dyes between the membrane and aqueous phase was deter mined after centrifugation. The logarithm of the ratio of distribution was linearly related
to the logarithm
theory
and
the
of salt concentration
Boltzmann
as predicted
distribution.
Plots
of
the
from
the Gouy-Chapman
logarithm
intensity against the logarithm of KCl and MgSO4 concentrations lines with a slope ratio of about two. The absorbance change was also explained The
by the Gouy-Chapman
use of these
dyes
as probes
of fluorescence gave two straight upon salt addition
theory. of the surface
potential
of membranes
is dis
cussed.
Biological membranes have fixed charges on their surfaces due to lipid and protein molecules, which have net negative charge at neutral pH values (1-9). These charges produce a negative potential at the membrane surface with respect to the potential in the bulk aqueous phase and form a diffuse electrical double layer adjacent to the membranes (the Gouy-Chapman theory) (10-12). The application and usefulness of the theory have been demonstrated (11, 12). The effects of surface
potential have been shown with bilayer lipid membranes and liposomes (11, 13-18) and bio membranes (19, 20). In photosynthetic mem branes the surface potential is related to the changes in chlorophyll fluorescence (12), reactivity of ionic redox reagents (21-23), membrane stacking (24) and intramembrane electrical field change (7). A possible role of surface potential in the process of energy transduction of photosynthetic mem branes has also been suggested (23, 25, 26). Many dye molecules which between the membrane and the
1 Present address: Biological Laboratory, Faculty of Education, Kumamoto University, Kurokami 2-chome, Kumamoto, Kumamoto 860.
Vol.
89, No.
2, 1981
have
been
distribution
397
used should
as
membrane depend
are distributed aqueous phase probes.
on the
Such
concentration
a
398
K. MASAMOTO,
of the probe dye near the membrane surface. If the probe dye has a net charge, the distribution of the dye should be dependent on surface potential, as well as on the partition coefficient or binding constant of the dye, since in the presence of nega tive surface potential, cation concentrations near the membrane surface are higher and those of anions are lower compared with the concentrations in the bulk aqueous phase. Surface potential dependence has been reported with liposomes and biomembranes for dyes such as 9-aminoacridine, anilinonaphthalenesulfonate, etc. (13-15, 27-29). In this study we analyzed the surface-potential dependent changes in fluorescence and absorbance of dyes with photosynthetic membranes of chlo roplasts and chromatophores. The dyes mainly used were merocyanines, which have been devel oped as membrane potential probes in bilayer lipid membranes and axons (30-32). The surface potential was changed by adding salts to suspen sions of the photosynthetic membranes.
K. MATSUURA,
S. ITOH,
and M. NISHIMURA
where cm and cs are the concentrations of the ion in the surface layer within the membrane where the amphipathic molecules are partitioned and in the aqueous phase adjacent to the membrane. From Eqs. 1 and 4, the apparent partition coeffi cient (P) is given as,
The distribution ratio (D), which is the ratio of the amount of dye in the membrane to that in the aqueous phase, can be determined experimentally. It contains a volume factor depending on the membrane concentration (Eq. 6).
Here
Vm
dye
is
the
molecules
volume. and
By by
volume
are
of
the
partitioned
eliminating ƒÓ0
taking
the
membrane
and
where
V is the
from
logarithms,
medium
Eqs. we
3
and
5
obtain
THEORY
The concentration of an ionic species i at the surface (cs) is expressed by the Boltzmann equation as follows,
where ƒÓ 0,
cb the
valence have
is
the
bulk
electrical of their
the
ionic
potential
density as
(e,
follows
(C
elementary after
symbols to
relationship
M)
numerical
surface can
of dyes on salt salt-concentration ratio is given by
negative
approximated
surface
7 gives
coefficient
concentration. Similarly, the dependence of the distribution
the
be
a
z-z
When ƒÐ
as follows,
is
mainly
or
in
the
to
the
in
logarithm distribution not
to
the
hence
the
dye
either
of
salt of
become
to
phase, to
be
too
dye
The
total
the
the
loga
membrane
both), the
and dye
the
the
concen
fluorescence related
the
to that
two
the the
phases
one-sided.
The surface potential dependence ance change of a dye will be a little plicated.
be
fluorescence
provided
between
the to
apparent
to
the
linearly
concentration, the
in not
respect
fluorescent
expected
the
also If
(but
with
constant,
expected of
ratio.
phase
linear
is
as is
logarithm and
bulk is
tration
regarded
distribution
due the
intensity
does
be
concentration
coefficient, of
intensity
The partition coefficient (Po) of amphipathic ionic species between the membrane and the aqueous phase adjacent to the membrane is,
can
salt
related
partition
be
Po
of
linearly
expressed
Eq. 2 can
and
logarithm
charge
substitution,
potentials,
Eq.
partition
the
rithm
At high
concentration,
of the apparent
among of
and
charge/A2)
some
other
and
the dependence
ion, zi
concentration in
the
surface,
According the
(mV), salt
of
the i,
meanings. theory,
symmetrical
at
species
usual
Gouy-Chapman surface
concentration
potential
At a fixed membrane
amount
of absorb more com
of dye added
J.
to the
Biochem.
SURFACE medium
POTENTIAL can be expressed
where
CT is
in
medium.
the
Eq.
6
level
the
of
CT
where ƒÃm cients
where
the can
be
are
the
absorbance
At
dye
in
a
cT
obtained
molar
(A)
at
of
dimer
for
a given dye
absorption
coeffi
and
Eqs.
5,
in
9,
as
membrane
a
the
and
bulk
10
function
the of ƒÓ0
concentration,
dependence
values
is
of
Po
of
(Vm/V)
can
be
are
addition
METHODS
were
emission
and
490nm
(ANS,
measured
VY-49
on
the
for
auramine ƒfor
for
tively.
Those
anions)
were 575
run
430
Corning and
9782 emitting
(monovalent
acriflavine
590nm
by
was
with
safranin ƒfor
for
the on
Hitachi
follows:
and
505nm
filters
were
sides,
mixed
cation),
merocyanine
dyes
and
for
NK2272
Vol. 89, No. 2, 1981
610nm
(merocyanine
(To placed
mixing,
515
520
and
was
measured
supernatant
dye
530 and
a
the
ratio of
in
of
Hitachi
used
parameter this
to
express
changes
tra of dyes were measured SM-401 spectrophotometer Safranion ƒ-
Industries
(Okayama,
from
model and
acriflavine and Nippon Japan).
amount
distribu the
volume
(D=P(Vm/V)).
absorbance
Chroma,
the without
the
includes
measured in a dual-wavelength Hitachi 356 spectrophotometer.
from
buffer
chromatophores.
used
experiment
membranes
spectropho
indicate the
80,000
supernatant
between
the
to
with
after at
the 124
in
KCl
minutes
centrifuged
dye
was
(pH and
difference
associated
Therefore, tion
was
the
chromatophores of
MgSO4
absorbance and
bacterio
0.01mM
absorbance
with
The
dyes
(monovalent
540),
The
were
(3.3 ƒÊM)
(10 ƒÊM Tricine-NaOH
Ten
suspension
3 h.
tometer.
ical
respec
NK2273,
for
dyes dye
2mM
concentrations. the
•~g
and
tral data processor,
and
dyes.
merocyanine Each
of
NaCl,
various
of
chromatophores 3ml
Salt-induced 380
of merocyanine
centrifugation.
in
1mM
factor
respectively) 460
cation),
(monovalent
at
Excitation
as
cation),
(monovalent
580
a
25•Ž.
were
anion),
and
exciting
in
8-anilino-l-naphthalenesulfonate
monovalent
shiba
dyes
using at
wavelengths for
of
suspensions
spectrofluorimeter
and
nm
changes
salt
of
MPF-2A
and
intensity chromatophore
formulae
ratios
determined
chlorophyll)
Chloroplasts were prepared as reported pre viously (33). Once-washed chloroplasts were sus pended in a mixture of 2mM Tricine-NaOH (pH 8.0), 1mM NaCl, and 0.01mM MgSO4. Chro matophores were prepared from cells of Rhodop seudomonas sphaeroides as described previously (7), washed once, and suspended in the same medium as for chloroplasts. and
Structural
Distribution
estimated.
8.0),
Fluorescence
1.
510 and 560 nm for NK2274. The indocyanine dye NK2612 (monovalent anion) was excited at 700 nm and the fluorescence was measured at 800 nm. The structural formulae of merocyanine dyes are shown in Fig. 1.
determined
and ƒÃm
AND
the
absorbance
experimentally
MATERIALS
chloroplast
399
dye
Fig.
when ƒÃb
the
DISTRIBUTION
as,
membrane
expressed
and
potential
and
expressed
the
defined
parameters,
given
surface
the
be
been
contribution
From
can
of
have
absorbance
is small
phase.
other
Vm
The
the
aqueous
concentration
V and
and ƒÃb of
OF CHARGED-DYE
as follows,
overall
(Vm•áV).
molecules
and
DEPENDENCE
of dyes were
mode with a Absorption spec
with a Union Giken equipped with a spec SM-540.
auramine ƒfrom
merocyanine Kanko-shikiso
were Katayama and
obtained Chem indocyanine Kenkyusho
400
K. MASAMOTO,
K. MATSUURA,
RESULTS
The effect of salt addition on the fluorescence of merocyanine dyes in a chloroplast suspension is shown in Fig. 2. The fluorescence intensity of merocyanine dyes decreased on addition of MgSO4, suggesting that the fluorescence intensity depended mainly on the dye concentration in the aqueous
2.
Emission
Spinach in
2.5ml
MgS04
of
in
each
concentration)
ized
2mM
to
the
figure to
(NK2272),
and level
of
to
Tricine-NaOH 1 ƒÊM
CCCP
was
recorded
the
medium.
510nm
of maximum
the
tary
charge/A2,
some
on
salt
the
after The
(NK2274). emission
23).
Eq. study.
2 by
(data in
not
chloroplast
KCl
slopes
in
shown
7
such
in
KCl
to
most
that
some
(increase
valent
anion) Eq.
7,
in
of
addition were
each
of
(positive
positive
ANS
(mono
those
expected fluorescence
membrane slopes
(34, with
suspension. were
suspended
and dye.
10mM
excited
for
of
and
higher the
dyes
two obtained
(decrease
of
have
are with
concentration)
with
with
NaCl,
of
to were
slopes
3 The
slope
close
salt
Fig.
dyes
the
values
dyes
in
to
MgSO4.
cation)
other
merocyanine
Fluorescence for
these
sus
charged
accord
chloroplast
1mM
slope
fluorescence) in
chlorophyll
8.0),
dyes
for
the
negative
associated
Data
dyes
the
as
3).
were
increasing
of
(Fig.
of
smaller
are
when
3.3 ƒÊM
ratios
(monovalent
slopes
35).
various
The
in fluo
identical
with
for
with
obtained
almost
that
MgSO4
with
calculated
salt-induced
that
twice
The
dyes.
dependence
chromatophore
I. with
auramine ƒ-
yields
was
although
fluorescence
from
in
plots
Table
dyes,
the
shown)
of
subject
agreement
density
of
change
be
be
potential
charge
predicts
should
values
may
good
suspension
Equation
26 ƒÊg
the nature
intensity
pension
absolute
surface
using The
rescence
for
the
in
of
estimated
fluorescence
was
value
change
other
density
the
elemen the
pH
the
charge
uncertainty,
this
than
and
Although
surface
in
10-3
larger
(8)
,
membranes
-4.0 •~
salt-induced
concentration
from
with
is
the
changes
that
was
which
estimated
to
thylakoid
NK2273
suspension (12,
the
and M. NISHIMURA
chloroplast
of
from
values
(pH and
for
use
chloroplast
merocyanine
corresponding
containing
curve
530
spectra
chloroplasts
obtained
with
obtained
phase, since the concentration of anionic dye in the aqueous phase is expected to decrease with the addition of salt (accompanied by an increase in that in the membrane, as shown in Fig. 5). The extents of the fluorescence decrease upon adding the same MgSO4 concentration were different among the three dyes, probably reflecting differ ences in hydrophobicity (hence in partition), as judged from their structural formulae (Fig. 1). The fluorescence intensity change upon salt addition depended on the species and concentra tion of salt added (Fig. 3). The logarithms of fluorescence intensity were almost linearly related to those of salt concentration. As the same fluorescence intensity is expected at the same level of surface potential, the surface charge density of membranes can be estimated from the concen trations of KCl and MgSO4 which give the same salt effect, by an equation derived from Eq. 2 as described in Ref. 7. The surface charge density
Fig.
thus
S. ITOH,
at
intensities
0.01mM The
lower
MgSO4
(final
580
(NK2273), are
normal
dye.
J. Biochem.
SURFACE
POTENTIAL
DEPENDENCE
OF CHARGED-DYE
DISTRIBUTION
401
Fig. 3. Dependence of the fluorescence intensity of NK2273 on salt con centration in chloroplast suspension. The logarithm of the ratio of fluores cence intensity after salt addition (f) to that before addition (fr) is plotted. The same basal medium as in Fig. 2 was used. Open circles, KCl; closed circles, MgSO4.
TABLE ‡T.
Slope
experiments
similar
of the to
fluorescence those
in
intensity Fig.
3
with
dependence spinach
on
salt
chloroplasts
concentration. and
The
chromatophores
slopes
were of
obtained
from
Rhodopseudomonas
sphaeroides.
cationic dyes and negative slopes with anionic dyes) indicated higher fluorescence yields from the dye in the bulk aqueous phase. In some cases, the values of slope ratios were smaller than two, which suggested the possibility of mixed fluores cence from dye molecules in membranes and in
Vol.
89,
No.
2, 1981
the bulk aqueous phase (see also " DISCUSSION"). The concentrations of dyes in the bulk phase can be directly measured in the supernatant after centrifugation of chromatophore suspensions incu bated with dyes. The logarithm of the distribution ratio of dyes (D) was linearly related to that of
402
K. MASAMOTO
salt concentration (Fig. 4), as predicted by Eq. 8. However, the slopes were smaller than the pre dicted values. At the same KCl concentration D became larger with increase of the expected hy drophobicity of the dye species (Fig. 1). These data, like the fluorescence intensity measurements, indicate the surface potential dependence of the distribution of dye molecules. The salt-induced changes of absorption spectra of merocyanine dyes in chloroplast suspension were also measured (Fig. 5). The MgSO4-induced absorbance increases had peaks at 607nm for NK2273, at 573nm for NK2272, and at 551nm for NK2274. Those in chromatophore suspension were at 605nm (NK2273), 570nm (NK2272), and 549nm (NK2274) (data not shown). The ab sorption of NK2272 at 570nm was mainly due to the monomeric dye in the membrane phase since the dimer had an absorption peak at 520nm and only a small absorbance at 570nm (32). In the concentration range of dyes used under the ex perimental conditions in the present study the spectra show little contribution of absorption due to dimers. Therefore the absorbance change of dyes with salt addition can be treated as simple
Fig.
5.
Absorption
plasts
(36 ƒÊg
1mM
NaCl,
plasts
were
plasts
and
spectra chlorophyll) 0.01mM
subtracted
from
membrane
dyes in
the
suspended 3.3
spectrum
The in
concentrations
3, 0.81mM
in 4,
of and
in
chloroplast
3ml
indicated
by
of
the
added in
suspension.
of 2mM
,ƒÊM dye.
MgSO4
1.48mM
is also
the
red
shift
of
Fig. 4. Dependence of distribution ratios of mero cyanine dyes on KCl concentration. Distribution ratios of the dyes between chromatophore membranes and the aqueous phase were determined by the centrifugation method described in " MATERIALS AND METH ODS." The volume factor is involved in the values obtained.
were
with
and M. NISHIMURA
spectral bands, which is a result of the dependence of the absorption peak of merocyanines on the
of merocyanine
MgSO4
S. ITOH,
partition of monomeric dye between the mem brane and aqueous phase. The partition of merocyanine dyes into the
(Adye+chloroplasts-Adye-Achloroplasts
indicated). 0.17mM
of
, K. MATSUURA,
The mixed
Chloro
Tricine-NaOH
spectra
of dye
suspension
of
(pH and
8.0),
of chloro
dye plus
recorded
over
the
spectral
were
0.01mM
in
1, 0.034mM
chloro region in
2,
5.
J.
Biochem.
SURFACE
POTENTIAL
DEPENDENCE
OF CHARGED-DYE
DISTRIBUTION
403
Fig. 6. Dependence of the absorbance difference of NK2273 on the salt concen tration in chloroplast suspension. The conditions were the same as those in Fig. 2. The changes of absorbance difference (606-582nm) on salt additions are plotted.
the absorption spectra of merocyanine solved in various straight-chain primary
dyes dis alcohols.
Small values of the estimated dielectric constants indicate that the dyes are located in the non aqueous
phase.
The ance
salt-concentration
of
NK2272
582nm)
is
increased in
value the Fig. and
7.
Relationship
calculated
between surface
absorbarice
potential.
The
difference data
(•›,
2
for
The
values
MgSO4)
solid
of
are
curves
of parameters
surface
replotted
potential
from
were (see
the
calculated
calculated
Fig.
with
salt
constant
Eq.
medium.
values
The peak
positions of membrane-associated dyes reflect the microenvironment of dye molecules, especially the polarity, if the contribution of the refractive index term is small. The probable polarity of the microenvironment, estimated from the positions the absorption peaks, was in the following increas ing order: NK2273 (dielectric constant=4 for chloroplasts, 7 for chromatophores), NK2272 (4 and 9), and NK2274 (10 and 11), as expected from their structures. The local dielectric constants were calibrated in terms of the peak positions of
Vol.
89, No.
2, 1981
the
were
(de
By
using
the
fluorescence
data,
calculated The
from data
of
Eq. Fig.
replotted
in
Fig.
7 against
the
calculated
6
values
exp
(-z1
FƒÓ0/RT
).
The
solid
line
represents
2.
text).
of the
concentration
concentration.
the
theoretical
curve
calculated
was
4 ƒÊM
and ƒ¢ƒÃb(606-582)
-50mM-1•cm-1.
dielectric
at
absorbance
6 against
from
appropriate
from
ansoro
reference
The
potential).
potentials
each
6. salt
of the
for of
the
Fig.
surface
obtained
surface at
are KCl; •œ,
v
in
(with
increasing
the
of
606nm
shown
with
crease
dependence
at
chosen
to
eters
used
were
Po
(Vm/V).
shown) chloroplasts of
physical
fit
the
of ƒ¢ƒÃm
in
the
change in
the
6 and
parameters
7)
except
Param 150
analysis, its
as for
for the
surface (data
same
be
(Vm/V)
and
and
CT
to Po
chromatophores
essentially
11.
curve.
fluorescence
absorbance
(Figs.
and
for ƒ¢ƒÃm(606-582)
dependence were
Eq.
measured
experimental
50
As
salt-induced potential
was
Values
were
from
not
those the
in
values
involved.
DISCUSSION
The distribution
of charged
amphipathic
ions into
404
K. MASAMOTO,
photosynthetic
membranes
potential. as to in
the
exact
the
of
the
distribution
in
this
study.
the
case
the
Gouy-Chapman
the
theory
surface.
in 8.0)
(5),
in
10mM (8).
with the
other
methods
20
However,
the to
25mM
values
in
by by
the Ā-poten
calculated
surface
parallel
at
various
(8). The
distribution
determined Eq.
by
8
according
According Fig.
this
gests
that
(Ja)
of
to
the
not
at
Fig.
using
charge/A2
of
explain
some
an
salt
in
However,
the
predicted
values
surface
30%
increase) lower
KCl
of
-1.9 •~
effect
of o opposite
the
partition
be
than obtained
concentrations.
and
to
that
of
the
due
for
to ƒ¢ƒÐ.
The
other
4).
by
Smaller
dyes
calculations
as
in
The
dye
bulk
in
the
(13,
deviation.
29).
was
in
the
It
was
due
which
to
result
that
the
aqueous
observed
deviation
and
when
the
In
tional
to
sites
and
the the
the
in
calculated
At
the from
binding
far
binding,
NK2273
of
2
mechanisms mitochondrial
saturation is
propor binding
zero
surface the
the
12 ƒÊM.
same
should
as
if
membranes
we
true
treat
the
Examples and
adequately
include
dye the
hold
binding.
membrane were
dis
at
Therefore,
conditions
between which
mem
at
was
interaction
molecules
equiv
the
concentrations,
and
experimental
is
to
of total
constant salt
This
Po
number
of
between
below
conditions
interactions
dye
as the
of
changes
partition
binding
is
of
given
far-from-saturation our
dye
binding
binding
ratio
to phase.
of
treatment
the
distribution due
aqueous
product
concentrations
of
the being
treatment
(29).
and
mentioned
the
been
than
treated
the
dye-membrane
above
with
to
under
the
small.
theory.
membrane
that
accounted
the
the
may
(Fig.
calculating
the
liposomes
potential
have
situation
from
in
effects
molecules
especially
slopes
fully
decrease
(rather were
the
dye
tribution
7
rather
involved
the
-47
be
in
both
have
that
was of
consideration
errors be
that
to
calcu
The surface charge density obtained from Fig. 3 was about four times as large as those reported elsewhere (8, 12, 23). Divalent cations are known to interact with phospholipid membranes more strongly than monovalent cations (36, 37). This may result in a difference in the discreteness-of charge effect between KCl and MgSO4 due to the non-ideal behavior of ions in the aqueous layer adjacent to the membrane surface, such as charge neutralizing binding. This situation may be re sponsible for the large a values obtained (Ref. 7).
potential.
elementary
experiment of
cannot
from
Eqs.
deviation,
deviations
of
in
This
the
presence
10-3
(13)
branes
increase
calculated
ANS
into
the
be
the
potential
from
may
the Fig.
concentrations ƒ¢ƒÐ
and
slopes
surface
alent
molecules an
the
be
increase
of
potential
of ƒ¢ƒÐ
to
concentration. part
encountered
due
the sug
density
dye
of
We
KCl
of
charge
7)
of
in
when ƒ¢ƒÐ
taken
salt
and
Eq.
between
should
Gouv-Chapman
Table
This
example,
can
value
The
gives
increase
values
30%. KCl
the
(7).
always
the
by
by
for
values
charged
For
100mM
the
in
1/2
smaller.
surface
of
neglected.
density
NK2273
of
slopes
1 and
but
partition
be
charge
4 by
to
were
changes
given theory.
the
close
obtained
the
due
should
relationship, be
ions
is
Gouy-Chapman
respectively,
actually
amphipathic
potential
the
3 should
MgSO4,
slopes
the
surface to
to
and
and
of
the
not
4)
phase
smaller the
buffer
the Ā-potentials of
were
7.0
theory
pH-profile
was lower
discreteness-of-charge
obtained
than
the
suggested
potentials
density
of
were
9mV
example (ƒ¢ƒÐ/ƒÐ=0.3,
potential
emissions
(see
for
KCl
surface
potentials
Deviations binding
of
(Fig.
surface
from
pH
salt
surface
smaller
phase
mem
at
it At
2,
mat
surface
when
membrane
Tris-HCl
KCl
of
and M. NISHIMURA
Eq.
100
(calculated
and
the
by at
difference
fluorescence
potential
chloroplasts
The
that
mV)
of
the
monovalent
larger
and
from
the
and
mat
charge
were
these
necessarily
Gouy-Chapman
of
similar
potential;
at
the
values
12).
pH's
in
values
using
was
and
pH
calculated
tial
of
NaCl
-25mV
change
not
obtained
mobility mat
considered
comes
the
to
phase
a
(-38mV).
calculated
is equal bulk
-29mV
neutral
(8,
88
(2),
and
at
is
The Ā-potentials
electrophoretic
-17mV
detect
potentials
values
and
S. ITOH,
NK2273
gave
lated
demonstrated
potential
the
4),
determined
it
surface
between
brane
were
However,
that
difference
to
of
absorbance
dyes
useful
surface
case
molecules,
experimentally
potential.
the
remain
potential
intensity,
The
surface
changes
dye
surface
charged
are
surface
8
the
of
the
quantitative
charged
fluorescence
relationships
(pH
and of
to
uncertainties
mechanism
between
changes
I
some
distribution
parallelisms
the
responds
Although
K. MATSUURA,
charged
explained
liposomes
(14,
(with
by 38)
NK2272)
(15). and of
Merocyanine dyes, as well as cyanines and oxonols, have been reported to respond to mem
J.
Biochem.
1
SURFACE
POTENTIAL
DEPENDENCE
OF CHARGED-DYE
brane potential changes in the microsecond range in bilayer lipid membranes and axons (30-32), In those studies, the spectrum of absorption change of NK2272 agreed with the spectral change taking place upon dimer-to-monomer dissociation on the membrane (a trough at 520nm and a peak at 570nm in the difference spectrum) (31, 32). The spectral change of NK2272 under our experimental conditions (Fig. 5) was not in accordance with the dimer-to-monomer change. It was satisfactorily explained by the increased amount of the monomer dye in the membrane phase. The response in absorbance of merocyanine dyes to the surface potential change is probably due to the partition change, Absorbance depends on the dyes in both the membrane and the aqueous phase (Fig. 7, cf. Eq. 11), so when using dyes as membrane potential probes, careful consideration is necessary of experimental conditions such as salt concentration and dye concentration, as well as the amount of membrane preparation. With careful choice of the experimental conditions, however, merocyanine dyes should be useful as surface potential probes in view of the sensitive changes in partition of the dye molecules.
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