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Biochimica et Biophysica Acta, 694 (1982) 1-25 1 Elsevier Biomedical Press

BBA 85225

ANIL INONAPHTHALENE SULFONATE AS A PROBE OF MEMBRANE COMPOSIT ION AND FUNCTION

JAN SLAVIK

Department of Cell Physiology, Institute of Microbiology, Czechoslovak Academy of Sciences, Videhsk6 1083, 142 20 Praha 4 (Czechoslovakia)

(Received September 28th, 1981)

Contents

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

II. Information provided by fluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

III. Theory of ANS fluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

IV. Experimental data on ANS fluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 A. Absorption spectrum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 B. Fluorescence spectrum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 C. Quantum yield . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 D. Fluorescence lifetime . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 E. Fluorescence polarization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 F. Excitation energy transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 G. Time-resolved fluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

V. Interaction of ANS with model systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 A. Interaction with artificial lipid membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 B. Interaction with proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

VI. Interaction of ANS with biological membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

VII. Interpretation of ANS fluorescence in biological membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

VIII. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

References 21

I. Introduction

The fluorescence probe method has assumed a solid position in biomembrane research as one of the few techniques permitting one to study the

Abbreviation: ANS, 1-anilinonaphthalene-8-sulfonate.

0304-4157/82/0000-0000/$02.75 1982 Elsevier Biomedical Press

structure and function of biological membranes in vivo. Its advantages, particularly evident in comparison with EPR spin probes and NMR methods [1], are its high sensitivity, the low degree of membrane perturbation and the primary response of the probe to properties other than environmental mobility.

Application of fluorescent probes to biomembrane studies dates back to the pioneering work of Weber [2,3] and Laurence [4] who, some 30 years ago, used an ANS-like probe to investigate the properties of albumin, and of Newton [5] who, a few years later, used a similar substance to probe a bacterial membrane. However, it was not until 15 years ago that a plethora of papers on fluorescence probes in connections with proteins and biomembranes appeared. At the end of this period a number of reviews were published in which a summary of data and a critique of the applicability of fluorescence probes may be found [6-16].

The fluorescence probe ANS (1-anilinonaphthalene-8-sulfonate) shown in Fig. 1 has remained the most universally used compound in biomembrane research if one disregards fluorescence probes spe- cially designed for studying biomembrane microviscosity (such as e.g. 1,6-diphenyl-l,3,5- hexatriene) or probes used for determining the membrane potential (e.g. cyanine or oxonol dyes). ANS fluorescence is extremely sensitive to changes in the probe environment. The probe binds nonco- valently to membrane proteins as well as lipids. Its negligible interference with membranes and its convenient absorption characteristics make ANS the fluorescence probe of choice for in vivo experiments.

In recent years there has been a revival of interest in fluorescence probes. Among the hundreds of papers dealing with ANS there are a few new ones whose conclusions qualitatively change the possible interpretation of data from biomembranes. As the interpretation of results has always been the weakest point of the method the time may be ripe for writing a critical review focused on understanding the meaning of measurements obtained with ANS. Some information in the review is of a more general character and is

Fig. 1. l-Anilinonaphthalene-8-sulfonate (ANS).

applicable to other fluorescence probes, such as isomers of 1,8 ANS or ANS derivatives (e.g., 2- toluidinonaphthalene-6-sulfonate (TNS)).

II. Information provided by fluorescence

The basic information contained in fluorescence measurements relates to the molecular environment around the chromophore probe. The fluorescence reflects various interactions of the probe with surrounding molecules, especially those present during its excited state, and carries information about the rotations and displacements of the chromophore molecule caused by Brownian thermal motion.

There are a variety of processes that may take place in the excited state of the chromophore. The majority of the chemical and physical properties of the excited molecule are very different from those of the ground state. There may occur various reversible and irreversible chemical reactions caused by the higher reactivity of the molecule and the pK change. The altered physical properties (in the case of ANS namely the increased dipole moment) change the interaction of the chromophore with neighboring molecules thus affecting the en- ergetic state of the chromophore.

The interpretation of experimental data must therefore be based on a detailed knowledge, at the molecular level, of how different properties of the probe-binding site may be reflected in the different measurable spectroscopic properties of the probe such as quantum yield, shape of the emission spectrum (half-bandwidth, wavelength of maximum), lifetime and decay curve, degree of polarization. Much important information may be obtained further from the excitation spectra, from the spectra of sensitized fluorescence and from fluorescence quenching. Time-resolved fluorescence measurement is a great help in the interpretation; it can provide insight into the nature of the processes occurring in the excited state and even permit the experimenter to separate one process from another. One must of course consider the fact that any given fluorescence arises from a great number of fluorescent molecules that may be in different individual microenvironments.

The most fruitful approach to obtaining signifi- cant information about biological membranes is, I

believe, the synthetic, based on understanding the spectroscopic properties of the probe as revealed under a great variety of simple conditions (e.g., different solutions), followed by the use of relatively simple but well defined heterogeneous systems (e.g., liposomes), and finally by extending the complexity of the model to a level where the observed properties in the membrane are interpre- table with some precision [9]. Using the synthetic approach one also has the advantage of fluorescence techniques which consists in the possibility of simultaneous measurements of a number of different fluorescence parameters. A certain change in the immediate environment of the chromophore probe or in its mobility may be reflected in a different way in different measured fluorescence parameters, the mutual comparison of which makes interpretation easier and, at the same time, provides a check on the experimental data. In my opinion this advantage is still insufficiently appre- ciated and exploited.

III. Theory of ANS fluorescence

The best (and most intuitive) explanation of the fluorescence properties of ANS is that based on the solute-solvent interaction model [ 17- 21 ] which can be successfully applied not only to the interpretation of the fluorescence properties of ANS in solutions but also to the interpretation of fluorescence of membrane-bound ANS [10,13,20]. The model can fully explain the various fluorescence properties of ANS, such as the dependence of the position of the ANS emission band on temperature, viscosity, solvent polarity and excitation wavelength, as are observed in solutions and, in corresponding ways, manifest themselves in the fluorescence of membrane-bound ANS [17-19]. Using the solute-solvent interaction model one may readily show that ANS need not necessarily be a 'hydrophobic probe', while ANS fluorescence in a highly polar environment composed of relatively rigid polar molecules (that do not permit a sufficiently fast relaxation of ANS from its excited state) may resemble that in nonpolar environments [10,22-24] in respect of emission maxima, lifetime and quantum yield. ANS may also be bound to polar (charged) binding sites [24,25] and to sites

involved in ion transport across membranes [25- 281.

The solute-solvent interaction model proceeds from assumptions about the relaxation of molecules solvating the ANS molecule. After absorption of light the ANS molecule shifts in about 1 fs from its equilibrium ground state (S O in Fig. 2) to its excited state (nonequilibrium Franck-Condon excited state) designated S' I in Fig. 2. The dipole moment of the ANS molecule in its excited state is greater than in the ground state [17] and hence the interaction of an excited ANS molecule with surrounding molecules is different from that before absorption. Reorientation and translation of nearest-neighbor molecules allow the ANS molecule to relax gradually to its equilibrium state S t (Fig. 2). In solutions of low viscosity where these relaxations are very fast (e.g., in water on a pico- second time-scale [29]) the fluorescence practically always takes place from the equilibrium excited state S t. (In other words, practically all ANS molecules reach this state before the emission of the fluorescence photon.) On emitting the fluorescence quantum the ANS molecule returns to its ground state S~ which finally relaxes to the equilibrium ground state S O (Fig. 2). In more viscous solutions the relaxation of the molecules surrounding ANS may, because of their lower mobility, be so slow that the time required for the relaxation to the equilibrium excited state is comparable to or even greater than the lifetime of the excited state (i.e, a few nanoseconds for ANS). In this case many ANS molecules (possibly all of them) will emit the fluorescence photon before reaching their equilibrium excited state S t . Hence, the emission takes place from one of the energetically higher excited states through which the ANS molecule proceeds to the equilibrium excited state S t and a blue shift of the fluorescence spectrum is observed (e.g., in ice [30]). One of such intermediate nonequilibrium excited states (S'() and the corresponding fluorescence is illustrated by a dashed line in Fig. 2.

This behavior of ANS in viscous solutions is illustrated by the time-resolved fluorescence spectra in Fig. 3. The solution of high viscosity is represented by propanol at -150C (curves A, B, C), whereas the one of low viscosity by the same solvent (to exclude polarity effects) at room temperature (curve D). The first spectrum (curve A),

,.=,~ =~ ~Z

o.51

SOLVENT "2 RELAXATION

FLUORESCENCE T 3

SOLVENT T4 RELAXATION

H I 0

31 0 RELAXAT'O S~vE"~ N " O ~ - $1

l"11 ABSORPTION FLUORESCENCE ~'1!"$

So 0 SOLVENT RELAXATION

ABSORPTION

Fig. 2. A simplified schematic representation of energy levels (A) attained by an ANS molecule in solution in the course of light absorption and en~ssion (B), according to a solute-solvent interaction model. S O represents the equilibrium ground state of the ANS molecule, S~ the nonequilibrium (Franck-Condon) excited state, S'~' one of the nonequilibrium (partially relaxed) excited states through which the ANS molecule passes during the relaxation process, S] the equilibrium excited state, S~ the nonequilibrium (Franck-Condon) ground state. (The individual energy levels represent in fact sets of different vibrational and rotational levels.) The ~h and ~3 are about 1 fs, ~'2 and ~'4 increase from ps in solution of low viscosity to ns in more viscous solutions. For most of the excited ANS molecules the lag between absorption and emission (the lifetime of the excited state) is of the order of ns (this value also depends on the nature of the solvent.) The changing dipole moment of the ANS molecule is symbolized by the arrow inside the oval.

0

,.,R,

=o o 400 450 500 550 (nm)

EMISSION WAVELENGTH

i _

600

Fig. 3. The gradual decrease of energy of fluorescence photons (i.e., red spectral shift of fluorescence) emitted from ANS molecules in the course of their relaxation as observed in propanol cooled to -150C. The time-resolved fluorescence spectra are recorded at 2 ns, 11 ns and 68 ns after excitation (curves A, B and C, respectively). Curve D represents the fluorescence of completely relaxed ANS molecules (steady-state fluorescence spectrum in propanol at room temperature). (Adapted from Chakrabarti and Ware [17].)

taken at 2 ns (i.e. 'immediately') after the excitation is compared with the spectra of partially relaxed ANS molecules taken 11 and 68 ns after the excitation (curves B and C, respectively). The curve D represents the (steady-state) spectrum of completely relaxed ANS molecules.

In a more detailed explanation vibrational and rotational energies of ANS as well as slight differences in solvation between different ANS molecules (distribution of ground or excited states) must be taken into account (cf. subsection IVB). Further, in the case of membrane-bound ANS the situation becomes somewhat complicated as one observes the fluorescence originating from more than one type of binding site, which may differ in the polarity of the molecules surrounding the ANS molecule and in the rate of the corresponding relaxation processes.

Fleming et al. [30] supplemented the solute- solvent interaction model with an explanation of the dramatic dependence of quantum yield and fluorescence lifetime on solvent polarity. In nonpolar solvents intersystem crossing followed by quenching of excited triplet states is an important nonradiative process which competes with the flu-

orescence [30]. In polar solvents this process is completely absent, the most probable nonradiative path being photo-ionization (in which the photo- product is a solvated electron [30,31]).

In addition to the solute-solvent interaction theory there are theories that try to account for the fluorescence properties of ANS by assuming two different forms of ANS with different types of fluorescence [32,33]. Pentzer [34] claims the existence of a strongly fluorescent coplanar form of ANS with a maximum at 460 nm and a weakly fluorescent noncoplanar form with a maximum at 520 nm. The two forms have the plane of the benzene ring, respectively, parallel and perpendicular to the plane of the naphthalene moiety. Kosower and collaborators [35,36] consider two species of excited ANS, a planar intramolecular charge-transfer state and a nonplanar singlet state. Both Pentzer and Kosower and collaborators believe that the proportion of these two forms depends on solvent polarity.

IV. Experimental data on ANS fluorescence

IVA. Absorption spectrum

The strong absorption band of ANS (Fig. 4) exhibits local maxima at around 270 and 360 nm; the lowest absorption band may be further split into two overlapping bands with maxima at 345- 350 nm and 375-380 nm [29,37-39] (Fig. 4). The absorption spectrum is only very slightly sensitive

B 5C

r " 2~ Z

ToE 2C

1,5 B

8 io

250 300 3.~0 400 450 (nm)

ABSORPTION WAVELENGTH

Fig. 4. Absorption spectrum of ANS dissolved in water (A), in ethanol (B) and bound to protein (corrected for protein absorption) (C). (Adapted from Stryer [37] and Robinson et al. [29].)

to the environment [17,37]. The apparent small red shift of the absorption [29] and excitation [40] spectra in ethanol and after binding to protein [37,40,42] (Fig. 4) compared to that of ANS in water might be explained as an intensification of the absorption band at 375-380 nm. This slight change in absorption spectrum is sometimes used for a more selective excitation of membrane-bound ANS as compared with free ANS; however, the dependence of fluorescence spectra on excitation wavelength may affect the results (see subsection IVB).

IVB. Fluorescence spectrum

The fluorescence spectrum of ANS in solution depends pronouncedly on the nature of the solvent, especially on its polarity (Tables I and II, Figs. 5 and 6) [29,37,43,44]. In low viscosity solvents, where the relaxation of ANS molecules may be complete before the emission of a fluorescence quantum, the position of the fluorescence maximum depends in a simple way on the polarity of the solution only and the difference between absorption and emission maxima (Stokes' shift) may be expressed by macroscopic properties of the solvent (dielectric constant and refraction index) [8,10,45-47] or using the empirical index of polarity-Kosower's Z [483].

In more viscous solvents, where relaxation of ANS cannot be completed during the lifetime of the excited state and the emission often occurs from energetically higher states, a blue shift in the fluorescence spectrum is seen (cf. section III, Fig. 3) [22,23]. At the same time, one may observe a dependence of the fluorescence maximum on the excitation wavelength if the red edge of the absorption spectrum is used for excitation (Fig. 7). This effect may be explained by taking into account that each ANS molecule is solvated in a slightly different way. This so-called ground state distribution of solvated ANS molecules may differ from the distribution of Franck-Condon excited states S' 1, which is, in contradiction to the distribution of relaxed excited states S~, dependent on the excitation wavelength [ 18,19].

.1

Z

>

@

Z

Z

w

m

e

m N

z~ m

, , , ~ N ~ '

r~

[...

Z

z -s_

z

z~

r-i

s

w p.

0~

380 400 420 440 460 480 500 520 540 560

EMISSION WAVELENGTH ( nm )

L

580 600

Fig. 5. Fluorescence spectra of ANS in alcohols. The decrease of the quantum yield is accompanied by a red shift of fluorescence maximum (shown by arrows) as the polarity of the solvent increases. O, n-octanol; B, n-butanol; P, n-propanol; E, ethanol; M, methanol; Eg, ethylene glycol; W, water. (Redrawn from Stryer [37].)

IVC. Quantum yieM

Besides the polarity of the environment (Tables I and II, Figs. 5 and 6) the quantum yield of ANS fluorescence may reflect other properties of the molecules surrounding ANS. Generally this depen-

;= 48(

47(

46E

z Q 450

>=

440

19~ K

IS3 K

,s~, ~ ~ 7;'r-- ,~e K

77 K

I I I I i I 360 580 400 420 440 460

EXCITATION WAVELENGTH ( nm )

Fig. 7. Dependen~ of the fluorescence maximum on the excitation wavelength in propanol at different temperatures. (Re- drawn from Azumi et al. [19].)

dence of the quantum yield 3' is expressed as

kF k F+kQ[Q] +kxc+kts c+kP+ kEx

(l)

where k F is the rate constant of fluorescence, kQ that of dynamic quenching, k~c that of internal conversion, kmc that of intersystem crossing, k e that of photochemical reaction and complex for-

0.4

0.3

.d LIJ

T- 0.2

I- z < 0.1 E l

A

OI 0

VOLUME

56O

c 540

=E

520 ~- X

500 z _o 03

480 u') =E LU

460

emission maximum

~ ~ q u a n l u m yield

- - ;~ I I I 20 40 60 80 IO0 (%)

CONCENTRATION OF ETHANOL

IN WATER

I0

6 ~: Lid

hi h

2 -J

13 . J h i >-

p. z

o

0.6

0.4

02

B

" "7 / \ _ ~ ' / L : aMum I y ie ld

0 20 40 60 80 I00 (%

VOLUME CONCENTRATION OF DIOXANE

IN WATER

12

56O

IO

540

520

=E 6

500 ~ ~: 4 tu

480 ~ ~_

m 460 _ J

Fig. 6. Dependence of the quantum yield, the lifetime and the position of fluorescence maximum on the relative concentration of ethanol (A) and dioxan (B) in water. (Compiled from Robinson et al. [29], Stryer [37], Kosower et al. [35} and Turner and Brand [44].)

mation in the excited state, kE, I that of energy transfer, [Q] denotes the concentration of the quencher; all these quenching processes are discussed below in respective order. The quantum yield may be further decreased by static quenching when part of the fluorescence molecules are degraded to nonfluorescent complexes in the ground state. This phenomenon affects the quan- tuna yield only, other fluorescence parameters being unaltered.

The fluorescence of ANS, similarly to that of other fluoresence compounds, may be quenched by various substances present in the medium or in the membrane. Also, diverse impurities may play a surprisingly large role in spite of their minute concentrations. Generally, the longer the fluoresence lifetime, the higher the sensitivity to dynamic fluorescence quenching. (Therefore, it is recommended that the direct influence of biologi- cally active compounds on ANS fluorescence be tested for instance in ethanol, where the lifetime of ANS is longer and comparable to that of membrane-bound ANS, rather than in water where the quenching can be overlooked because of the short ANS lifetime. In other words, in polar solvents k p becomes large [30] and therefore kQ[Q] may be negligible.)

The quenching of ANS by water may be employed for estimating the accessibility of membrane-bound ANS to water molecules. The iso- topic effect of water is used here, the exchange of H20 for 2H20 increasing the quantum yield 2-3- times (similarly it increases 4-times when CH3OH is exchanged for CH302H), while the position of the fluorescence maximum remains unaffected [9,29,44,49]. The mechanism of this quenching is not clear [29].

The quenching of ANS fluorescence due to water-dissolved oxygen or nitrogen can usually be neglected (in normal conditions) [50].

An increase of temperature leads to higher rate constants k w and k~s c and hence to a decrease of quantum yield up to about 1% per 1 K depending on the nature of the solvent [34,50]. The decrease is more pronounced in nonpolar solvents such as dioxane [21,51,81 ].

The strong dependence of kls c and kp (photo- ionization) on the solvent polarity has already been mentioned in section lII and is thoroughly

discussed by Fleming et al. [30]. The quantum yield may be further decreased

due to increased excitation energy transfer (higher kj~v) as discussed in detail in subsection IVF.

In the range from 2 to 11 pH has no direct influence on ANS fluorescence [52-54]. Only below pH 2 a drop of the fluorescence intensity can be observed [34,55], caused by the pK of the sulfonate group of ANS lying between 0 and 1 [55,561.

The value of k o (may be determined graphically from the linear dependence of the reciprocal value of the quantum yield on the concentration of the quencher, see Eqn. 1) can in principle be used for the determination of viscosity [57] as follows. If the quenching of ANS fluorescence is a diffusion- controlled process (dynamic quenching) then from the equation for kQ (ideal case, Ref. 57)

8RT kQ -- 3000~7 T (2)

viscosity ~/ may be calculated (R is a gas constant, T temperature, ~'o lifetime in absence of the quencher). However, this technique is rarely used because there exists a far more precise and convenient way to determine viscosity based on polarized fluorescence measurements (subsection IVE).

In special cases the measurement of the quantum yield may be slightly affected, depending on the geometrical arrangement, by changes in the degree of polarization [58].

IVD. Fluorescence lifetime

The fluorescence lifetime of ANS depends strongly on the properties of the molecules surrounding the ANS molecule, the dependence being similar to that of quantum yield and reflecting a competition of radiative fluorescence transition with other, nonradiative, pathways for the deactivation of the excited state of ANS. The lifetime can be defined in a way similar to the definition of the quantum yield by means of rate constants

1 = (3)

k F + kQ[Q] + klc + ktsc + kEx

There is a close connection between quantum

yield and lifetime, expressed by the equation

1" = ~,'r o (4)

(T O denotes the intrinsic (natural) fluorescence lifetime of the chromophore, or, in other words, % = 1 /k v can be interpreted as the fluorescence lifetime in the absence of nonradiative transitions). For ANS in solution, % is somewhat dependent on the properties of the solvent, ranging between 30 and 80 ns [17,29,59].

As the quantum yield cannot be determined directly but only by measuring fluorescence intensity, Eqn. 4 may prove to be useful in distinguish- ing some cases of ANS interaction with biological membranes. For instance, fluorescence enhancement accompanied by a commensurate increase of the fluorescence lifetime may be interpreted as an increase in quantum yield. If the lifetime remains unchanged after fluorescence enhancement we may be dealing with an increase in the number of bound ANS molecules, all of them with the same quantum yield. A decrease of fluorescence intensity followed by a decrease of the lifetime suggests an increased excitation energy transfer, a photochemical reaction or other quenching processes. A decrease of the quantum yield together with an unchanged lifetime may be, besides the already mentioned decrease of the number of bound ANS molecules, explained by static quenching, reabsorption of fluorescence, inner filter effect or by another trivial process.

The decay of fluorescence of ANS in biological membranes is not exponential, with the consequence that the two commonly used techniques (phase-shift and pulse) may yield different results. The phase-shift technique provides approximately the average lifetime of all decay components. The pulse techniques which are becoming more popu- lar resolve the decay into as many components as are put into the computer program. Special care must be taken in the application of deconvolution programs used for increasing the time resolution of the apparatus. These programs are usually designed to decompose the fluorescence decay curve into two or three exponentially decaying components giving, at the same time, the relative intensities (weights) of these components (cf. section VII).

I VE. Fluorescence polarization

The degree of polarization may be defined as the difference between fluorescence intensities observed through a polarizer (analyzer) oriented parallel (lip) and perpendicular (I1), respectively, to the plane of polarization of the exciting beam, divided by the sum of these two intensities

Ill -- I

P -- Ill + I

The polarization of fluorescence reflects the mobility of the fluorescent molecules and the transfer of excitation energy between them. In the case of rigidly held, independent, randomly oriented molecules the theory gives the value of the degree of polarization (for linearly polarized excitation light [60])

3 COS2 fl - 1 po - (5 )

3cos2/3+ 3

depending only on the angle fl between the absorption and emission oscillators (dipole transition moments). This angle is constant for a given compound. The situation is approximated in practice by examining highly viscous (mostly frozen) solutions with low concentrations of ANS. After excitation with linearly polarized light the degree of polarization in glycerol equals 0.43 [38], in propanol 0.46 [39]. Corresponding values of/3 calculated from Eqn. 5 range from 20 to 25 [38,39,61].

The actually measured degree of polarization is lower than P0 because of the already mentioned depolarization processes; rotational depolarization and depolarization due to excitation energy transfer. The former is caused by the rotational components of thermal (Brownian) motion as a result of partial rotation of the ANS molecule during the time between light absorption and emission, the latter is discussed in subsection IVF. As the extent of this depolarization depends on the extent of the rotation, which in turn reflects the mobility of the molecules surrounding ANS, polarized fluorescence may be employed for determining the viscosity of biological membranes (whatever the meaning of viscosity of membranes may be). Perrin's

10

equation [60]

1 1 1

relating the fluorescence lifetime ~-, measured degree of polarization p and the parameter describ- ing the rotation of the fluorescent molecules (rotational relaxation time p) may be employed. Know- ing ~- and p we may calculate p, which in solutions is directly related to solution viscosity

RTp (7) ~= 3V

where V denotes the molar volume of the fluorescent molecules. The last two equations hold for free, independent, spherical molecules with exponentially decaying fluorescence after excitation with linearly polarized light, which is certainly not the case with membrane-bound ANS. Thus, both the theory of microviscosity determination in biomembranes and the interpretation of experimental data are far more complicated than might appear at first sight [62].

The experimental results of fluorescence polarization should be corrected for depolarization of the excitation light and of the fluorescence light, caused by the passage through the suspension of membranes or cells [13].

donor. (The excitation spectrum of a dye generally coincides with the long-wavelength part of its absorption spectrum.)

There are basically two different energy transfer processes. The so-called trivial one is the energy transfer by reabsorption (the fluorescence quantum emitted by one molecule is absorbed by another molecule) and it is usually insignificant. The second one is the nonradiative singlet-singlet excitation energy transfer (FOrster transfer [63]) which occurs with no intermediate light emission engaged. The intensity of this energy transfer depends on the fluorescence properties (lifetime and spectrum) of donor molecules, absorption spectrum of acceptor molecules, their mutual orientation, their distance apart and properties of the medium separating the donor and acceptor (expressed e.g., in the form of the refraction index) (e.g., Refs. 8, 13, 63, 64). A quantum mechanical calculation shows that the probability of energy transfer depends on the sixth power of the distance between the donor and the acceptor. The probability of excitation energy transfer (and accordingly the value of rate constant k ET) abruptly decreases after reaching a certain char- acteristic limiting distance called the F0rster radius R 0 (Fig. 8). (For the couples ANS-tryptophan the R 0 values are about 2.3 nm [40], for ANS-ANS 1.4 nm [38] to 2.4 nm [65].) This steep decrease with

I VF. Excitation energy transfer

The transfer of excitation energy from one molecule (donor) to another (acceptor), which emits the fluorescence quantum, leads to a depolarization of fluorescence, changes in the quantum yield, the lifetime and excitation spectra. The depolarization of fluorescence (because of differences in orientation of donor and acceptor molecules) is indistinguishable from that caused by rotation. However, in solutions the depolarization due to energy transfer is concentration dependent, decreasing as the dye concentration is re- duced (distance between molecules increases). As the observed fluorescence appears after excitation of both acceptor and donor molecules, the excitation spectrum is formed by a superposition of excitation spectra of both the acceptor and the

IOC

8c

6o

z uJ

4O

2O

o

o_

0

k

O,SR o R o 15 R o 2Ro

DISTANCE ( rel. un i ts )

Fig. 8. Illustration of the pronounced dependence of the probability of excitation energy transfer on the distance between the two chromophores.

II

separation of the interaction between donor and acceptor makes the F~Srster radiationless singlet- singlet excitation energy transfer meaningful only in the nanometer range.

For membrane-bound ANS the major role in the excitation energy transfer is played by transfer between tyrosine and especially tryptophan protein residues and the fluorescent probe and between the probe molecules themselves [65,66]. En- ergy transfers between ANS and quinones, flavins, porphyrins are less important. Investigation of protein-bound ANS is somewhat complicated because of the double absorption band of ANS, whose short-wavelength part coincides with the absorption band of proteins [37,67,68].

IVG. Time-resolved fluorescence

In the nanosecond range the time-resolved fluorescence spectra of ANS provide invaluable information about the course of relaxation processes, while time-resolved polarization measurements yield information on the movement proper of the fluorescent probe itself [64,69]. Information de- rived from time-resolved spectra assists in estimating what kinds of nonradiative processes compete with the ANS fluorescence [29,30] (see also section II). Time-resolved fluorescence spectra permit one to separate out the contributions of scattered light and Raman scatter (which 'decays' on the fs scale). It also permits one to separate fluorescence components with different lifetimes (e.g., the fluorescence spectrum of free ANS (~ < 1 ns) can be distinguished from the fluorescence of lipid-bound (~- approx. 5-7 ns) or protein-bound (I" approx. 14-20 ns) ANS; see section VII). Time-resolved spectra may be useful for the investigation of sensitized ANS fluorescence, mainly caused by excitation energy transfer from tyrosine and tryptophan residues to ANS, since the sensitized fluorescence appears only after a certain time (in the pico- and nanosecond range) needed for the transfer of excitation energy.

V. Interaction of ANS with model systems

VA.Interaction with artificial lipid membranes

Fluorescence is drastically increased on adding ANS to a suspension of lipopsomes or planar lipid

membranes. This enhancement is caused by the movement of a great number of ANS molecules from water into the lipid bilayer, the lower polarity of the environment of these molecules resulting in a pronounced increase in their quantum yield. Detailed studies using X-ray diffraction methods [70,71], NMR [72-75] and excitation energy transfer [68,76] show that ANS binds to membranes in the region of the phospholipid polar heads. The nonpolar part of the ANS molecule is submerged in the direction of the fatty acid alkyl chains so that the center of the aromatic ring of ANS lies approximately at the level of the lecithin carbonyl group [74] while the sulfonate group projects out- ward and lies at the level of polar choline heads [70,71,73], both absorption and emission oscillators being almost parallel to the plane of the bilayer [74,77].

As the number of bound ANS molecules only increases with increasing ANS concentration to a certain saturation limit, special specific binding sites for ANS are assumed to exist in the membrane. A model of this binding site, as proposed by Haynes and Staerk [78], supposes that one ANS molecule binds to a pre-existing binding site; a pocket formed by four polar heads of lecithin (Figs. 9 and 10). Sphingomyelin, with its choline polar head, binds ANS in a similar way to lecithin [54,79] and it is believed that the positive charge of the choline residue plays an important role in the binding [80]. Nevertheless, electrostatic interaction is not absolutely decisive here since phosphatidylethanolamine with a positively charged head practically bind no ANS [78,81]. Other lipids (phos- phatidylserine, phosphatidylinositol, cardiolipin and gangliosides) bind ANS much less avidly than sphingomyelin or lecithin [81,79,248]; phos- phatidic acid liposomes bind no ANS [80]. On increasing the cholesterol:lecithin ratio another type of ANS-binding site appears. In these binding sites ANS projects further out and it is more accessible to water; (therefore, it has a lower quantum yield - higher quenching [78,82]). Cholesterol alone or mixtures of cholesterol with phosphatidylethanolamine do not bind ANS at all [78].

Therefore, with membranes composed of a mixture of different lipids, most ANS molecules would be bound in pockets formed by four lecithin

12

A B

Fig. 9. A tentative two-dimensional space-filling model of the packing of ANS molecule in the pocket formed by four lecithin molecules (A) or in the shallow binding site formed by lecithin and cholesterol (B). 1, area of sulfonate group of ANS; 2, aniline part of ANS; 3, naphthalene moiety of ANS. (Inspired by Haynes and Staerk [78].)

or sphingomyelin molecules, and a smaller part of the ANS would be bound in the shallow binding sites associated with the modification by cholesterol [78]. Since the fluorescence quantum yield of ANS bound to four lecithin or sphingomyelin heads is considerably larger than that found on binding to other types of binding sites [78], this fluorescence will predominate. This accounts for the fact that ANS fluorescence does

OeO 0_0 0_0 0 o o o o_:o 5"5 o o-o o 0-0"0-010~0 0 0 ~@ e~) 0 0

0o-o oo oo'oo ooo oo %ooc o oo.

oc o ooo

2.1 nm 3.1 nm

(0) (b) Fig. 10. Two different arrangements of bound ANS molecules (closed circles) in the layer of lecithin molecules (open circles denote phospholipid heads), according to the model of Kumbar and Maddaiah [32].

not significantly depend on the lipid composition of the liposome [78,83] (Table lIl). The relatively homogeneous fluorescence can be characterized by a fluorescence lifetime of about 7 ns, a degree of polarization of 0.2 and a fluorescence maximum at 490 nm. The corresponding rotational relaxation time of ANS calculated from Eqn. 6 is around 10-15 ns. Trfiuble [76,84] estimated that 1 molecule of ANS is bound to every 15-40 molecules of a phospholipid mixture (depending on the temperature) and Radda [85] calculates 1 ANS to every 6 phospholipid molecules.

The presence of cations increases the fluorescence intensity [26,55,76~81,86-88], trivalent cations being more active than bivalent cations and these again more than univalent ones [76,81]. The increase has a sigmoid dependence on cation concentration [81,55]. A similar fluorescence enhancement is induced by a pH decrease [12,54,79]. The increase of fluorescence intensity is usually not explained by a direct interaction of ANS with cations [88], but by reaction of cations with the phosphate and carboxyl lipid groups [55,79,87]. Such a reaction decreases the electrostatic surface potential and thus facilitates the binding of ANS molecules [55,88,90,91]. Hence, the increase of fluorescence intensity may be caused by an increase in the number of bound ANS molecules [12,79,86,87]. The character of the fluorescence (spectrum, quantum yield, lifetime and polarization) remains unchanged [92,78].

Depending on its polarity, an artificially im- posed 100 mV transmembrane potential can cause small shifts up or down of fluorescence intensity, not exceeding 10 4 of the initial value [77,93]. This change is directly proportional to the voltage applied (in the 150 mV region) [93,94] and takes place within 1 ms of the application of the external voltage [77]. The effect is explained either as a direct interaction of the electrical field with the dipole of the chromophore [95,96] or as a consequence of pushing or pulling ANS inside or out- side the membrane (no electrophoretic movement is assumed here [97]).

The binding of ANS to the liposome itself probably takes place in two steps (Fig. 11). The very fast adsorption of ANS (in about 2 ms according to Ref. 98, less than 0.1 ms according to Ref. 99) to the surface of the liposome in the

13

TABLE I l i

FLUORESCENCE OF ANS BOUND TO L IPOSOMES

L iposome Tern - Spect ra l Quantum L i fe t ime Degree o f Rotat iona l

p repared pera - max imum y ie ld (ns ) po la r i za t ion re laxat ion

f rom ture (nm) t ime (ns )

(C)

Ref .

Lec i th in

Lec i th in +

cho les tero l

Lec i th in +

card io l ip in

Card io l ip in

Gang l ios ide

Sph ingomye l in

L ip id mix ture

P ro teo l ip id

- 490 7 .8 0 .206 18 a

- - - 6 . 7 - -

- 492 0 .2 - - -

15 - 0 .28 b, 0 .37 c 6 .8 b, 9 c 0 .07 --

30 -- 0 .24 b 5 .8 b 0 .07 --

0 .29- -0 .31 c 7 .0 - -7 .5 c 0 .07

50 -- 0 .24 5 .7 0 .07 --

. . . . 0 .19(0 .21 d)

- - - - - - 5 . 7 - - - -

- - - - - - 7.4 0 .16 16

25 -- -- -- 0 .102 --

37 -- -- -- 0 .087(0 .067 d) _

30 490 0 .4 5 .6 0 .15 8 a

25 476 -- 9 --

-- -- -- 4 .22 , 7 .14 f - -

1 0 - - - - - - 0 . 2 1 ~

20 -- -- -- 0 .18 ~, 0 .21 b

30 -- -- -- 0 .14 e, 0 .171

40 -- -- -- 0 .12 e, 0 .14 b

50 -- -- -- 0 .10 ~, 0 .12 b

60 -- -- -- 0 .08 e, 0 .10 b _

20 - - - 0 .15 ~ -

30 - - - 0 .11 c _

- - - 7 . 0 -

- - 0 .2 2 and 7.5 s _ _

20 480 - 7 .2 0 .11 6 a

- 470 0 .2 - - -

- - 6 . 5 - -

- - 1 2 . 5 - -

2 5 0.015 - - -

45 0 .025 - - -

- 470 - - 0 .2 -

- 485-489 - 3 -4 and 6 -7 s _ _

- 475 - 11 .2 0 .25-0 .3 40-70 a

- - 0 .67 14.5

61 ,82

64

246

78

78

78

233

247

9

140

140

28

248

234

249 ,

249 ,

249 ,

249 ,

249 ,

249 ,

251

251

61

78

83

68

247

247

241

241

252

237 ,

253

106

250

250

250

250

250

250

238

a Ca lcu la ted us ing Per r in ' s equat ion (Eqn . 6) , P0 taken as 0 .43 ; b d ipa lmi toy l lec i th in ; c d imyr i s toy l lec i th in ; d lyso lec i th in ; e egg lec i th in ;

f l aser and f lash lamp exc i ta t ion , respect ive ly ; g two exponent ia l decay components .

lecithin head region at sites marked as type B in Fig. 11 is (after 50 ms [98] or less than 500 ms according to Ref. 100) followed by a further submersion of ANS molecules into deeper binding sites (marked as type A in Fig. 11), which are probably identical with the above mentioned

pockets formed by four lecithin heads (Fig. 9). This relatively fast process is sometimes fol-

lowed by a slow fluorescence enhancement (tens of seconds up to tens of minutes). The extent and the rate of this enhancement depends on the ionic composition of tire solution, on the temperature

14

and on the lipid composition [97-102]. This slower enhancement of fluorescence is explained by an increased accessibility of ANS to the inner mono- layer binding sites which increases abruptly in the temperature region around the lipid phase transition [98,99,101]. However, it is difficult to ascribe a definite role to this mechanism in the interaction of ANS with biological membranes, where it is believed that only carrier transport is responsible for ANS translocation and for accessibility of these inner binding sites [25,85,100,103-105].

The model presented in Fig. 11 may also be used to account for the fluorescence of ANS bound to liposomes at relatively high ANS concentrations. Here it may happen, particularly at a low pH, that most of the binding sites marked A in Fig. 11 are already occupied and some ANS molecules have to remain bound to binding sites marked B [73]. As the ANS fluorescence from these B sites has a low quantum yield and a shorter lifetime (because of quenching by water molecules) and a lower degree of polarization (due to the relatively high mobility of ANS molecules at these binding sites) one observes a decrease in the average lifetime and in the degree of polarization, together with an increase in fluorescence intensity.

The fluorescence of ANS bound to proteolipids resembles more the fluorescence of protein-bound ANS than that of the lipid-bound ANS, in particu- lar with respect to the strong excitation energy transfer from the protein part of the molecule to ANS [106]. On the other hand, the fluorescence of ANS bound to lipoproteins is similar to that of lipid-bound ANS [107,108].

ANS type B type A e

0.1 - 2 ms 50 - 500 ms

Fig. l l. The interaction of an ANS molecule with a lecithin liposome. The adsorption of ANS molecules to the phospholipid heads (binding sites marked B) is quickly followed by submersion to deeper binding sites (marked A). (Redrawn from Tsong [981.)

VB. Interaction with proteins

The fluorescence enhancement after addition of ANS to certain protein molecules can be explained, as in the case of liposomes, by a stoichiometric binding of ANS to specific binding sites on the protein molecule.

The majority of protein molecules possess one to two specific binding sites for ANS [37,41,54,106,109-124] but albumin has three [125] or five sites [126-128] and some other proteins have even more [129-131]. On the other hand, some proteins bind practically no ANS [37,79,121 ]. The binding sites are often in the active centers of proteins [37,109,112-114,120,124,132] and binding of ANS may inhibit the function of the protein [41,42,109,114,120,124,133-135]. The ANS fluorescence then reflects changes occurring in these centers and therefore it can be employed for de- tecting structural and functional alterations of proteins.

However, at present there is no general model for ANS binding to protein molecules. ANS is believed to be tightly bound (large rotational re- laxational times of ANS [24,28,37,39,54,136,137]), to binding sites localized not too deep in the protein interior (accessibility of binding sites to water molecules is about the same as in the case of lipid-bound ANS [9,49]). The intense excitation energy transfer from tyrosine and tryptophan residues to the ANS molecule [37,67,68,110,113, 116,117,119,126,138] ind icates average tryptophan-ANS distances from about 1.6 to about 2.7 nm [38,40,49,106,119,139].

It is difficult to define the nature of the protein-binding site for ANS and to determine whether it is 'hydrophobic' or 'hydrophilic'.

The concept of hydrophobic binding sites widely accepted in the past was usually based on a simple comparison of the maximum of the fluorescence spectrum of protein-bound ANS with that of ANS in alcoholic solvents. However, as we know from the solute-solvent interaction model the polar but relatively rigid binding sites which do not allow a sufficiently fast relaxation of excited ANS molecules may display the same fluorescence lifetime, quantum yield and position of the fluorescence maximum of ANS as the nonpolar ones. Only a detailed study of these relaxation processes (e.g.,

15

TABLE IV

FLUORESCENCE OF ANS BOUND TO PROTEINS

Prote in Spect ra l Quantum L i fe t ime Degree o f Rotat iona l

max imum yie ld (ns) po la r i za t ion re laxat ion

(nm) t ime

(ns)

Ref.

A lbumin 465 - 14.8 - -

- 0 .7 - - -

- 0.68 - - -

- - 16 0 .31-0 .44 a > 100 b

- - 15.6, 16.7 c _ _

- - - 0 .26, 0 .28 -

- - 16 - 1 0 5 - 1 1 0

- - - 0 .20-0 .25 a _

- - - 0 .210, 0 .255 -

- - 15 .9 - -

4 6 5 0.75 - 0 .22-0 .31 a _

470 0 .519 - - -

480 0.9 16 0 .30 100

- - - 0 . 1 3 -

A p o h e m o g l o b i n 457 0.92 - 0 .396 43.5 c

455 0.99 16.7 - -

Apomyog lob in - - 16.4 - -

454 0.98 - 0 .396 28.7 e

455 0 .99 16.4 - -

478 0 .90 18.7 - -

Apophosphory lase 470 0 .34 - - -

Cho l ines terase 470 - - 0 .22 -

Chymotryps in - - 11.9 - 45

Dehydrogenase 480 0, 8 17 .9 -18 . I 0 . 2 0 40 b

- 0 ,83-0 .96 - - 188

480 0.75 - -

465 0 .056 - -

a -Fetoprote in - - - 0 .26-0 .32 a _

G lobu l in 480 0,62 - -

G lycopbor in 468 - 0 .215 -

Immunog lobu l ins 475 0 .03-0 .15 - -

Luc i fe rase 480 0.39 - -

Mye l in bas ic

p ro te in - 0.4 - -

Myos in 468 0.48 10.1 - -

Perox idase - 0.85 5.9 and 17.9 f -

Phosphory lase 490 0 .12 -

- - 8.7 and 19.2 g 153-165

Tropomyos in 494 - - 0 .22 -

Tubu l in 460 0.48 - - _

M ix ture o f

membrane prote ins - - - 0 .20 -

470-475 - 14 - -

138

132

106

38

234

254

39

156

255

247

65

124

28

140

37

132

158

37, 43

132

132

147

133

24 ,54

163

136

120

124

156

51

117

41

41

106

143

113,

147

137

118

116

52

237

158

a Depend ing on the number o f ANS molecu les bound (probab ly because o f depo lar i za t ion due to exc i ta t ion energy t rans fer ) ;

b ca lcu la ted us ing Per r in ' s equat ion (Eqn . 6), Po taken as 0.43; laser and f lash lamp exc i ta t ion , respect ive ly ; d at 20 and 30C,

respect ive ly ; * es t imated; f two components w i th we ights 1.24 and 4.45, respect ive ly ; s two components , average va lue 15.4 ns.

16

by means of time-resolved spectroscopy) could shed more light on this controversy. So far the hypothesis of hydrophobic ANS-binding sites has been favored, based on Stryer's experiments with apomyoglobin [43]. In these experiments ANS was displaced from its complex with apomyoglobin after addition of hemin which is known to bind to a highly nonpolar binding site [36,43]. Exposure of new binding sites observed after protein denaturation [140,141] may also be used in support of this hypothesis.

The hypothesis of rigid hydrophilic ANS-binding sites [17,40] derives from the relatively large degree of polarization and rotational relaxation time of ANS. It could easily explain the high accessibility of binding sites to water molecules [9,49]. Chymotrypsin is the only protein for which a direct X-ray determination of ANS-binding site has been performed [24]. It was found that ANS is bound to the protein surface, in a polar region near an array of charged amino acid residues. ANS interacts strongly with a disulfide group and it is accessible to polar solvent (water) molecules which seem to be ordered and relatively immobi- lized [24].

The complexity of the composition and structure of protein molecules explains why some proteins, besides their 'main' binding site with high affinity for ANS, may contain other ANS-binding sites with lower affinity and specificity [137,142]. The fluorescence of ANS from these binding sites has a lower quantum yield, a lower degree of polarization and a shorter fluorescence lifetime (i.e., higher mobility of bound ANS, higher accessibility of water to these binding sites) [137].

The fluorescence enhancement after ANS addition is biphasic with some proteins [t43- 149], the equilibrium being reached only after tens of minutes [143,146]. There is no plausible explanation for this phenomenon.

In comparison with lipid-bound ANS the fluorescence of ANS-protein complexes is relatively pH-independent [52,106,124] (e.g., albumin pH 5.5-11.5 [127,150]), and independent of the ionic composition of the medium [51,106]. The range of this independence is probably limited by denaturation changes of the protein molecule [51,123,129,136]. A number of binding sites not accessible to ANS in the native state of the protein

only become accessible after denaturation [140,141]. The enzymic degradation of proteins (e.g., by trypsin or pepsin) gradually decreases the number of ANS-binding sites [106,151].

ANS has been used for the investigation of the properties of albumin [38,39,65,125,126,128, 132,138,149,150,152-157], alphafetoprotein [156], apohemoglobin [37,132], apomyoglobin [36,37, 43,132,158], aminotransferase [ 115], collagen [ 159], different dehydrogenases [109,114,124,129, 134,136,148,160-163], chymotrypsin [24,40], esterase [42,133], elastin [159,164], glycophorin [117], glycosidase [121], some globulins [51,112,123], hormones [165,166], histones and nucleoproteins [167], hydrogenase [168], kinase [ 122,130,169,170], lactalbumin [ 171], lipoproteins [I07,108,172,173], luciferase [41], lysozyme [68], myosin [143,144,174], peroxidase [113,158], phos- phorylases [137,142,147,175], phosphatase [176], polylysine and polyarginine [177], nucleic acid synthetases [110,146], anthranilate synthetase [ 178,179], thyroglobulin [ 131], transaldolase [ 111], tropomyosin [118], tubulin [116,119] and other proteins [44].

Some data on the fluorescence of ANS bound to these proteins are presented in Table IV.

The dimer of ANS (bis-ANS) [180-183] may have a much higher affinity for some proteins than has ANS [181]. As bis-ANS may be present in commercially available ANS (e.g., 0.3% of the Mg salt of ANS [183]) and as it can be readily formed by chemical reaction catalyzed by some proteins (e.g., by albumin [181]) special care must be taken in the interpretation of experimental data.

VI. Interaction of ANS with biological membranes

Whole cells stained with ANS resemble in most cases a luminous ring under the fluorescence microscope (leukocytes [ 184,185], thymocytes [ 186], mouse neuroblastoma cells [187], yeast cells [28]). A luminous disk was observed with hepatocytes [188]. There is a rapid fluorescence enhancement observed within several hundredths of a second up to seconds on addition of ANS to a suspension of membranes or whole cells. This, usually more than a 100-fold, increase is followed by a slower enhancement lasting from tens of seconds to tens of minutes. Such a biphasic increase in fluorescence

intensity has been observed in erythrocytes [9,26,52,85,104], mitochondria [40,49,189,190], Escherichia coli [191,192], sperm cells [193], ascites cells [25], neuroblastoma cells [194], gastric vesicles [195], hepatocytes [188,196], rat hepatic microsomes [197], skeletal sarcoplasmic reticulum [198], chromatophores of Rhodospirillum rubrum [199], mouse cells [200] and pulmonary macrophages [201]. On the other hand, a monophasic increase was observed with leukocytes [184] and yeast cells [28].

The biphasic fluorescence enhancement may be explained either as a slow equilibration of ANS binding (one set of binding sites reaches equilibrium with ANS rapidly, the second one only slowly) or as a transport of ANS to the inner binding sites of the membrane [56,85,189,198] or inside the cell [25,202,203]. A carrier mechanism for ANS transport is assumed by many [25,85,100,103-105], the possible candidate in erythrocytes being the band 3 protein [27]. Never- theless, such transport has been demonstrated con- vincingly only in hepatocytes [188] although many cells display the biphasic fluorescence enhancement.

The inhibition of the transport of some anions in the presence of ANS (C1- [104,204], SO42 [25,104,204], SCN- [26,104]) has been explained either directly as a competitive inhibition of transport or as a consequence of ANS binding to the positively charged anion-binding sites in the membranes (or in their proximity) [25-27,205] followed by a change of the surface charge in the vicinity of these binding sites, making the binding of anions more difficult [25,27,104].

The number of ANS molecules bound to the membrane is strongly influenced by the surface charge of the membrane [13,26,14]; it is inversely proportional to the value of the negative membrane surface potential [12,206]. The fluorescence intensity strongly increases in a sigmoid manner after addition of 1-10 mM cations [9,52,81,86,87, 140,206-210]. The higher their valency the greater the fluorescence enhancement [81,206,209]. A similar enhancement is observed on lowering the pH [52,67,79,81,193,208,211-214]. These responses are similar to those of ANS in liposomes and are explained as a facilitation of ANS binding (reversible [87] increase of the number of bound ANS

17

molecules [86,87,196,206], no new types of binding sites being exposed [196]) brought about by neu- tralization of phosphate and carboxyl groups in lipids [79,81,87,196,206], or possibly of carboxyl groups of sialic acid or of neuraminic acid in the gangliosides [79,193]. The quantum yield and the lifetime remain unchanged [206], only the binding constant changes [196]. In special cases a protein conformational change may play an important role in these pH and cation induced fluorescence enhancements [ 123,215].

The response of ANS fluorescence intensity to transmembrane potential is similar to that observed with model lipid membranes. The changes are very small, generally not exceeding 0.1% of the fluorescence intensity [94,96,216-222] and they take place within milliseconds [94,216,217,221,222] of the potential change.

There are some interesting but at present largely controversial data about relations between pronounced changes of ANS fluorescence and the energization of the membranes of mitochondria, submitochondrial particles, bacteria and chloro- plasts. These experiments are discussed in many reviews [8,9,12,14,223]. The observed energy-lin- ked fluorescence changes are interpreted either as a transmembrane electrophoretic movement [224] or as a redistribution of electrical charges in the vicinity of ANS-binding sites [225] or by a model of four different membrane states which is now the most widely accepted idea [9,202,226-228]. In this model the aplectic and symplectic states correspond, respectively, to energized and nonenergized membranes. The postulated four membrane states differ in the quantum yield and the lifetime of ANS fluorescence, in the binding constant of ANS, in the extent of the excitation energy transfer from tryptophan residues to ANS and also in the accessibility of water molecules to bound ANS [9]. But the existence of such states has not yet been con- firmed [ 189].

Estimates calculated on the basis of literature data give about 105-106 [229,230] ANS molecules bound to an Escherichia coli cell, 10 7 to an erythrocyte [86,104,231] or leukocyte [184], 108- 109 [193,232] to a sperm cell, 109-10 I [194] to a mouse neuroblastoma cell, 10 l to a macrophage [201]. Expressed per g of membrane protein there are 150-200 /~mol ANS for mitochondrial frag-

18

ments [53], 100 /~mol for skeletal muscle microsomes [140], 110 gmol for hepatoma cells [208], 50 /~mol [283] or 100 #mol [231] for erythrocytes, 16-20 #mol for Escherichia coli [191]. Expressed per membrane surface area one obtains 1 ANS molecule bound for every 4 nm 2 of mitochondrial surface [53] or for 5 nm 2 of erythrocyte membrane

[231]. These values correspond to a surprisingly large value of about one-fourth of the membrane surface being covered with ANS molecules for erythrocytes [231], mitochondria [53] and microsomes of sarcoplasmic reticulum [140].

Some additional data about the fluorescence of membrane-bound ANS are shown in Table V.

TABLE V

FLUORESCENCE OF ANS BOUND TO B IOLOGICAL MEMBRANE

Membrane Temperature Spect ra l Quantum L i fe t ime

(C) max imum yie ld (ns)

(nm)

Degree o f

po la r i za t ion

Ref .

Asc i tes cel ls - - - 7.8

Ee l e lec t rop lax 20 - - -

20 - - 8.2 a

Ery throcyte

- - 7 and 19

_ 9 a

- 9

480

Escher ich ia co l i 21 - - 1.1 b, 3.5 c

25 - - 2.5

37 490 - 5.4

(ves ic les) 21 - 11.6

F ib rob las t - 7.9

L iver cel l - - - 7.9

Lymphocytes . . . .

M ic rosomes

(ske le ta l musc le ) 25 - -

37 - - -

( l iver) 25 475 - 9.4

( Tet rahymena pyr i fo rmis ) - - - 6-7 d

( l iver) - - 7.9

M i tochondr ia

(hear t ) - 470 - -

( l iver ) - - - 8.7

(hear t , f ragments ) - - 0.1 -

Mye l in - 490 0.4 10 .4 -12 .9

Neurob las toma cel ls . . . .

- - - 6 .7 a

P lasmodium - 460 - -

Sarco lemma 10 475 0.47 9.8 a

Sarcop lasmic re t i cu lum 20 478 - -

40 - -

Submi tochondr ia l

par t i c les - 480 0.08 - - - - 5 and 9 e

Yeast 30 500 - 5.2 a

0 .235

0.31

0.28

0.31

0.3

0.21

0.12

0 .104

0.25

0.20

0.18

0 .14

0 .125

247, 256

254

257

233

64

258

9

52

259

230

28

259

247

247

260

140

140

92

234

247

0.08 252

247

0 .194 53, 190

106, 206

0.21 194

0 .219 261

0.22 187

0.4 239

- 237 ,238

.0 .30 139, 245

0.28 139

- 262

0 .18-0 .22 40

0.15 28

a Average va lue, cf. Tab le V I I ; b phase method; c modu la t ion method; d average va lue depend ing on ANS concent ra t ion , cf. Tab le

V I I ; two components .

Vll. Interpretation of ANS fluorescence in biological membranes

Fluorescence microscopy of whole cells confirms that in most cases ANS is chiefly bound to the cell surface; and since the membrane lipids and membrane proteins are practically the only components of cell surface structures that are known to possess high affinity for ANS, these are the most important candidates for ANS binding. Transport of ANS to the inside of the cell may be inhibited by transport inhibitors or by a substan- tial decrease in temperature. Based on these assumptions, ANS fluorescence may be considered as a superposition of the fluorescence of molecules of ANS bound to membrane lipids and of membrane proteins, of free ANS, and also in some cases of ANS transported to the inside of the cells. For excitation wave-lengths of 365 nm and longer the intrinsic fluorescence of membranes or cells may often be neglected compared to the ANS fluorescence.

One might conclude from the preceding sections that if there were in membrane-bound ANS fluorescence a fraction with a fluorescence lifetime of about 7 ns, degree of polarization 0.2, spectral maximum 490 nm and quantum yield about 0.3, it could be ascribed to the fluorescence of ANS bound to membrane lipids. Similarly, a fraction with mean fluorescence lifetime somewhere between 14 and 20 ns, degree of polarization about 0.3, spectral maximum at 480 and quantum yield higher than 0.4, with a strong dependence on the excitation wavelength (due to the excitation energy transfer from tryptophan residues) should originate from protein-bound ANS. There might also be present fluorescence of free ANS with a lifetime of about 0.5 ns, practically zero polarization, with a spectral maximum at 515 nm and a very low quantum yield of about 0.004.

Surprisingly, such kinds of fluorescence are re- ally observed. Indeed, there are only two main types of ANS binding sites observed in biological membranes corresponding to membrane proteins and membrane lipids [8,13,16,15,56,81,82,106,197, 206,215,234-239] and the fluorescence may be easily decomposed into the above-mentioned components.

As the number of protein binding sites for ANS

19

on biological membranes is lower than the number of lipid binding sites but their affinity for ANS is greater [237,240], starting with a low ANS concentration the probe would bind first to the proteins and only subsequently to the lipids [237,240,241]. At commonly used ANS concentrations most of the protein binding sites are already occupied by ANS and the fluorescence of lipid- bound ANS gradually predominates in the total fluorescence because of the higher number of lipid ANS-binding sites.

This is the reason why the fluorescence of membrane-bound ANS often so strongly resembles that of liposome-bound ANS [16,28,235,242], especially at higher ANS concentrations [12,16]. The responses to pH changes and changes in the ionic composition of the medium are in both cases very similar [140]. Nevertheless, the strong transfer of excitation energy from tyrosine and tryptophan protein residues confirms the proximity of ANS- binding sites to proteins. This energy transfer has been shown for erythrocyte membranes [40,67], fragmented mitochondria [49], submitochondrial particles [40], sarcoplasmic reticulum and microsomes [139,140], E. coil [191], human spermatozoa [193], neuroblastoma cells [194], myelin membranes [106,206] and brain plasma membranes [210].

Let us make a simple calculation based on data available from E. coli membranes. If each protein molecule is surrounded by roughly 600 lipid molecules in the membrane [241] and if 1-3 binding sites for ANS exist in the average protein molecule and about 5-20 sites are present per 100 average lipid molecules [84,85,244] we arrive at a ratio of kNSprotein:ANSlipi d equal to 1 : 10 to 1:100. Be- cause of the differences in quantum yield, the ratio of fluorescence intensities is somewhat higher.

Quantitative experimental observations based

TABLE VI

THE POLARIZATION DEGREE OF ANS BOUND TO MICROSOMAL MEMBRANES

Skeletal muscle microsomes, data taken from Ref. 140.

Microsomes 0.14 Microsomal lipids 0.094-0.106 Microsomal proteins 0.162-0.169

20

>

.<

Z

l.u

0 D

Z .,<

0

<

eq

I

~:~ i-~ ~1:

I

o I

e, i

~o

c, I

J

e~

I r i :~ I

I

Z .,<

c~ e~

~ ~ o

on enzymic degradation of membrane proteins or lipids or on detailed study of ANS fluorescence confirm these calculations. Enzymic digestion of erythrocyte membranes [86], myelin membranes [139], membranes of the sarcoplasmic reticulum [139,140,245], skeletal muscle microsomes [81] or rat hepatic microsomes [80,235] with proteolytic enzymes after staining with ANS caused only sub- tle changes in fluorescence [81,86,206,235]; while the removal of phospholipids by applying a phos- pholipase caused a pronounced decline in fluorescence intensity [81,81,86,140,206,235] to less than one-half of its original value [80,81], accompanied by a blue spectral shift of the emission maximum [80,81], changes in the excitation spectrum [140] and an increase in the polarization of fluorescence [139,245]. In detailed study of fluorescence the small differences in the position of emission maximum (e.g., 495 nm for membrane lipids and 475 nm for membrane proteins of E. coli [241]) or in the degree of polarization (Table VI) can be used for quantitative purposes only in special cases. The best way seems to be an analysis of the fluorescence decay curve. This curve is usually broken down into two or three exponentially decaying components ascribed to the exponentially decaying fluorescence of lipid-bound ANS and protein- bound ANS. The weights of these components yield, after a correction for the differences in quantum yield, a quantitative representation of ANS bound to lipids and protein of the membrane (Tables VII and VIII). The most detailed work in

TABLE VIII

THE RELATIVE INTENSITIES (WEIGHTS) OF DIFFER- ENT EXPONENTIALLY DECAYING COMPONENTS OF FLUORESCENCE OF SARCOLEMMA BOUND ANS (AS IN REF. 237)

Component: I II III

Lifetime Lifetime Lifetime 4 ns 7 ns 14-16 ns

Sarcolemma membrane 0.07 0.60 0.33

Protein extract (solution) 0.15 0 0.85

Lipid extract (liposomes) 0.15 0.65 0.20

21

this area is that of Zierler and Rogus [237], whose main results are summarized in Table VIII. All these data confirm this expected dominance of fluorescence originating from ANS bound to pockets formed by four lecithin or sphingomyelin molecules in the total fluorescence of biological membrane-bound ANS.

VIII. Conclusion

In the preceding sections it was demonstrated that the fluorescence of ANS bound to biological membranes may be explained using simple models such as liposomes and water-soluble proteins, em- ploying the molecular model of ANS bound to pockets formed b3)four lecithin or sphingomyelin heads. The fluorescence may be taken as a sum of fluorescence originating from lipid-bound ANS and from protein-bound ANS. The similarities between the fluorescence of ANS bound to the lipids of a biological membrane and that of ANS bound to liposomes, represents a further confirma- tion of the membrane fluid mosaic model of Singer and Nicolson.

I personally see a great future in applications of time-resolved fluorescence spectra and time-resolved polarization measurements which will permit the direct study of the relaxation processes, i.e., the movement of molecules, on the nanosecond scale. Taking advantage of differences in excitation spectra one may choose for further studies only that component of the total (steady-state) fluorescence in which one is interested. This should make the interpretation of results much easier and would allow to treat more complex problems, such as membrane energization.

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