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8 Characterization of Potentiometric Membrane Dyes Downloaded by CORNELL UNIV on June 4, 2012 | http://pubs.acs.org Publication Date: May 5, 1994 | doi: 10.1021/ba-1994-0235.ch008

Leslie M. Loew Department of Physiology, University of Connecticut Health Center, Farmington, CT 06030

Dye indicators of membrane potential have been available for the past 20 years and have been employed in numerous studies of cell physiology. "Fast" dyes are able to follow changes in the millisecond range—fast enough to monitor individual electrical events in excitable cells and tissue. Fast dyes include compounds with styryl, oxonol, and merocyanine chromophores that are engineered to stain the plasma membrane. Generally fast dyes have small sensitivities to voltage changes but are very useful for detecting voltage transients or mapping voltage differences along the surface of a cell. "Slow" dyes can measure voltage changes that may accompany hormonal responses in nonexcitable cells or the level of activity in energy-transducing organelles. Slow dyes have delocalized charges, as in the cyanine or rhodamine chromophores, and usually operate via a potential-dependent redistribution between the extracellular medium and the cytosol. Techniques based on these indicators can be used to monitor both spatial and temporal variations in membrane potential with resolutions not possible with the more traditional electrode-based methodologies.

P O T E N T I O M E T R I C M E M B R A N E DYES are e m p l o y e d to study c e l l physiology. T h e p i o n e e r i n g w o r k o f C o h e n a n d his co-workers ( 1 - 3 ) l e d to the availabil ity o f a large n u m b e r o f organic dyes whose spectral properties are sensitive to changes i n m e m b r a n e potential. T h e applications o f these dyes to a variety o f problems i n c e l l biology a n d neuroscience w e r e r e v i e w e d i n a series o f chapters i n a recent book (4).

T h e a i m o f this chapter is to review the

characteristics o f these dyes as d e t e r m i n e d i n m o d e l a n d c e l l membranes. 0065-2393/94/0235-0151$08.54/0 © 1994 American Chemical Society

In Biomembrane Electrochemistry; Blank, M., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1994.

Downloaded by CORNELL UNIV on June 4, 2012 | http://pubs.acs.org Publication Date: May 5, 1994 | doi: 10.1021/ba-1994-0235.ch008

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T h e size o f the voltage-dependent signal, although certainly important, is b y no means the only factor to b e considered i n choosing a dye. T h e intent here is to identify the important parameters o f dye chemistry that enable it to b e a useful a n d practical indicator o f potential. Some b r i e f generalities about the structures a n d physical properties o f the various dye chromophores that f o r m the backbones o f potentiometric dyes are also i n t r o d u c e d . T h i s i n f o r m a t i o n should provide a basis for better initial selection o f suitable dyes f o r a given experimental application a n d h e l p take some o f the mystery out o f the chemistry o f potentiometric dyes. Particular emphasis w i l l b e p l a c e d o n electrochromic a n d N e r n s t i a n redistribution dyes developed i n o u r laboratory. Several o f the methods used to test a n d analyze dye response to changes i n m e m b r a n e potential w i l l b e detailed.

Dye Design Initially, the p r i m a r y m e t h o d f o r finding g o o d potentiometric dyes was trial and error. L a r g e numbers o f dyes were screened o n the s q u i d giant axon b y C o h e n a n d his colleagues (1-3). T h e resultant database revealed several b r o a d rules that c o u l d be u s e d to design n e w generations o f probes. F o r example, the large class o f azo dyes are not particularly suitable because o f their photoinstability a n d their propensity for toxic a n d photodynamic damage to biological preparations. T h e azo dyes require some h y d r o p h o b i c ap pendages to p r o m o t e interaction w i t h the m e m b r a n e , b u t alkyl groups longer than about eight carbons impart too m u c h insolubility for some applications. T h e database also indicated that several different molecular mechanisms are e m p l o y e d b y different dyes to p r o d u c e potential-dependent spectral changes. T h e v a r i e d chemistry u n d e r l y i n g the activity o f these dyes is intrinsically fascinating. A simple yet extremely useful classification o f potentiometric dyes that e m e r g e d f r o m these studies ( 5 ) is based o n the speed o f their response to voltage changes. " F a s t " dyes are able to follow changes i n the m i l l i s e c o n d range—fast enough to m o n i t o r individual electrical events i n excitable cells and tissue. " S l o w " dyes c a n measure voltage changes that may accompany h o r m o n a l responses i n nonexcitable cells o r the level o f activity i n energytransducing organelles. Interestingly, i n addition to identifying the range o f applications accessible to potentiometric indicators, this classification also divides the existing potentiometric indicators into realms o f m e c h a n i s m a n d sensitivity.

Fast Dyes. Generally, the mechanisms underlying the fast dye re sponses involve potential-dependent intramolecular rearrangements o r small movements o f the dye f r o m one c h e m i c a l environment to another. These


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reactions have the requisite speed but usually do not p r o d u c e very large changes i n the spectra o f the dye. T h e largest reported fluorescence response to an action potential was greater than 2 0 % relative to the resting fluores cence level (6). Typically, however, good fast dyes respond w i t h fluorescence changes o f only 2 - 1 0 % per 100 m V and transmittance changes of 0 . 0 2 - 0 . 1 % p e r 100 m V . A sampling of some o f the best fast dyes is i n c l u d e d i n C h a r t I, w h i c h w i l l be referenced i n the following discussion. A great deal o f effort has b e e n invested to develop fast dyes that e m p l o y an electrochromic ( 7 - 9 ) mechanism. Briefly, electrochromism is possible i f there is a large shift i n electronic charge w h e n a chromophore is excited f r o m the g r o u n d to the first excited state; i f the direction of charge movement fies parallel to an electric field, the energy o f the transition w i l l be sensitive to the field amplitude. T h u s , i f the c h r o m o p h o r e is oriented so that the charge redistribution is perpendicular to the m e m b r a n e surface, an electrochromic dye s h o u l d be an indicator o f membrane potential. These ideas are s u m m a rized i n F i g u r e 1. T h e reason this mechanism is attractive is that it lends itself to a theoretical molecular orbital treatment ( 9 ) that aids i n the design o f appropriate targets for organic synthesis. T h e aminostyrylpyridinium chro m o p h o r e best fits the criterion o f a large charge shift u p o n excitation a n d can easily be m o d i f i e d w i t h sidechains that assure an appropriate orientation i n the membrane. Variations o n this c h r o m o p h o r e have b e e n the targets o f synthetic efforts i n o u r lab (10-12) a n d i n Grinvald's lab (6, 13). C h a r t I includes two examples: d i 4 - A N E P P S (JO) a n d R H 4 2 1 (6), w h i c h have b e e n particularly successful i n a variety o f applications. N o t e that the potentiomet ric responses o f these dyes i n different biological preparations may be complex superpositions o f several mechanisms w i t h only a m i n o r contribution f r o m electrochromism (14). Nevertheless, the styryl dyes have e m e r g e d as the most popular fast fluorescent potentiometric indicators. A m o n g their special attributes are relatively g o o d photostability, a h i g h fluorescence q u a n t u m y i e l d for the m e m b r a n e - b o u n d dyes, a n d almost no fluorescence f r o m aqueous dye. Absorbance measurements are often desirable, especially for complex preparations w i t h nonexcitable satellite cells, because the large b a c k g r o u n d fluorescence f r o m the membranes o f these u n i n v o l v e d cells attenuates the relative fluorescence response w i t h a corresponding degradation o f the signalto-noise ratio; a transmitted light signal is used i n absorbance and is not significantly affected b y b a c k g r o u n d staining. T h e oxonol class of dyes n o w has e m e r g e d as the most sensitive for detecting fast potential-dependent absorbance changes, although some merocyanine dyes, the earliest class o f successful fast indicators, are also still i n use. T h e oxonol c h r o m o p h o r e is defined b y its delocalized negative charge. T h e mechanism for the potentialdependent response has been d e t e r m i n e d for several o f these probes; it involves a movement between a b i n d i n g site o n the m e m b r a n e surface a n d an aqueous region adjacent to the m e m b r a n e (15, 16). T h e merocyanine chro-


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ν DI-4-ANEPPS JPW-211

JPW-1120-2 WW-401

JPW-1034 RGA509

Chart I. A representative sampling of good fast dyes. The name of the general chromophore is given in the box above each structure. The common names of the dyes are listed under each structure.


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I

Styryl

I

RH-421

Chart I.—Continued.


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Figure 1. An electrochromic dye undergoes an internal charge migration upon excitation (tap). In the presence of the electric field produced by a potential drop across the membrane, the energy required for the excitation is altered (bottom) resulting in a spectral shift.

m o p h o r e is characterized b y neutral a n d zwitterionic resonance structures. Merocyanines are often highly solvatochromic and c a n theoretically respond to membrane potential v i a electrochromism. H o w e v e r , the only thorough experimental investigations o f merocyanine mechanisms p e r f o r m e d to date have b e e n w i t h just one dye, M - 5 4 0 ( C h a r t I). These studies indicated that dye reorientation possibly c o u p l e d w i t h aggregation at the m e m b r a n e surface underlies the potentiometric response o f this early m e m b e r o f the merocya nine family o f potentiometric dyes ( 2 , 17, 18). Probes c a n b e designed w i t h variable overall charge a n d h y d r o p h o b i c i t y as appropriate f o r different applications. T h e rationale f o r these ideas w i l l b e illustrated f o r the styryl dyes, o f w h i c h Joe W u s k e l l i n this laboratory has


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made many variants. Structures o f some o f these probes are shown i n C h a r t II. D i - 4 - A N E P P S is a good general purpose fast probe that has several beneficial attributes (based o n both p u b l i s h e d (JO, 1 9 - 2 6 ) a n d u n p u b l i s h e d results i n this laboratory as w e l l as those o f L . B . C o h e n , B . M . Salzberg, G . Salama, J. P i n e , J. D i x , P . Schrager, W . W e b b , D . Gross, D . Zecevic, J . Jalife, J . M . D a v i d e n k o , W . M i i l l e r , a n d H . W i n d i s c h ) . D i - 4 - A N E P P S is reasonably consistent i n giving a relative fluorescence change o f « 1 0 % p e r 100 m V i n a variety o f different c e l l types a n d experimental protocols, a n d it can be used i n a dual wavelength ratiometric excitation m o d e to normalize out artifacts due to uneven staining or photobleaching ( 2 7 ) . A s w i t h other styryls, only the m e m b r a n e - b o u n d dye displays appreciable fluorescence. I n several experi mental protocols, it has b e e n persistent for hours u n d e r continuous perfusion w i t h dye-free m e d i u m . D e c r e a s i n g the size o f the c h r o m o p h o r e as i n di-4A S P P S results i n a b l u e shift o f the spectral characteristics o f the response o f about 30 n m ; unfortunately a 4 0 % r e d u c t i o n i n sensitivity also occurs. A decrease i n the chain length o f the hydrocarbon tails as i n d i - 2 - A N E P P S increases the water solubility o f the dye. T h i s is necessary for thick tissue preparations where the dye must penetrate through many c e l l layers; the more water-soluble dyes, o f course, w i l l give less persistent staining o f the preparation. T h e opposite situation w i l l pertain to a dye like d i - 8 - A N E P P S , where l o w solubility requires a high molecular weight surfactant like P l u r o n i c F 1 2 7 ( B A S F Corporation) to promote staining (28, 29). I n addition to persistence, we discovered that another attribute o f d i - 8 - A N E P P S is a very slow rate o f internalization. I n several c e l l types, d i - 4 - A N E P P S was incorpo rated into intracellular organelles over times as short as 10 m i n ; d i - 8 - A N E P P S is retained exclusively o n the plasma m e m b r a n e over periods o f hours. D i - 4 - A N E P E P a n d d i - 4 - A N E P E Q represent dyes w i t h varying head-group charges. These charges can subtly change the location o f b o u n d dye i n the membrane w i t h the effect o f sometimes significant improvements i n sensitiv ity. G e n e r a l l y it is true that positively charged dyes are especially w e l l suited for experiments that require microinjection to localize dye to just one c e l l i n a complex preparation (30, 31).

Slow Dyes. Slow dyes generally operate b y a potential-dependent partitioning between the extracellular m e d i u m a n d either the m e m b r a n e o r the cytoplasm. T h i s redistribution o f dye molecules is effected v i a the interaction o f the voltage w i t h the ionic charge o n the dye. U n l i k e fast potentiometric indicators, slow redistribution dyes must b e charged. T h r e e chromophore types have y i e l d e d useful slow dyes: cyanines, oxonols, a n d rhodamines. E a c h o f these chromophores has special features that suit different kinds o f experimental requirements. A set of important slow dyes is depicted i n C h a r t III. T h e cyanine class o f symmetrical dyes w i t h delocalized positive charges was originally i n t r o d u c e d b y A l a n W a g g o n e r a n d has p r o v i d e d extraordinarily




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DI-4-ANEPPS JPW-211

JPW-1063 Di-4-ANEPEQ

JPW-1064 Di-4-ANEPEP

Chart IL A collection of styryl dyes engineered to meet specific experimental demands.



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JPW-1113 Di-2-ANEPPS

JPW-1153 DI-8-ANEPPS

DI-4-ASPPS

Chart

II.—Continued.



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TMRE JPW-179

Rhodamine-123

^ ^ ^ ^ ^ ^ ^ Di-0-C6(3) Chart III. A representative sampling of good slow dyes.


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8.

Oxonol-Vl

Bis-oxonol DiS-BaC2(3) Chart

III.—Continued.


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sensitive probes for potential changes i n populations o f nonexcitable cells ( 5 , 32-34). A large n u m b e r o f these dyes w i t h varying hydrocarbon chain lengths, numbers o f methine groups i n the b r i d g i n g polyene, a n d heterocyclic n u c l e i are available. D e p e n d i n g o n the nature o f the dye a n d its concentra tion, potential-dependent uptake can effect either an increase o r a decrease i n fluorescence intensity. I n general, t h e fluorescence o f these dyes is enhanced u p o n m e m b r a n e b i n d i n g ; thus accumulation o f dye leads to fluo rescence enhancement. H o w e v e r , at h i g h d y e : l i p i d ratios many o f the dyes have a tendency t o aggregate, w h i c h results i n fluorescence self-quenching. Self-quenching occurs i n the case o f di-S-C2(5) (Chart III), w h i c h can lose 9 8 % o f its fluorescence w h e n a c e l l or vesicle preparation is p o l a r i z e d to 100 m V (32, 3 5 , 36). D i - 0 - C 6 ( 3 ) has less tendency to aggregate a n d displays an increased fluorescence q u a n t u m y i e l d as i t binds to the plasma a n d organelle membranes. T h e lipophilicity o f d i - 0 - C 6 ( 3 ) is responsible for its use as a stain for m i t o c h o n d r i a a n d endoplasmic reticula ( 3 7 ) . H o w e v e r , dye association w i t h intracellular organelles can lead to cytotoxicity o r misinterpreted fluo rescence changes (38). A n i o n i c oxonols also show enhanced fluorescence u p o n b i n d i n g to m e m branes, b u t , because o f their negative charge, b i n d i n g is p r o m o t e d b y depolarization. M o r e importantly, the negative charge lessens intracellular uptake o f oxonol dyes, w h i c h solves some o f the difficulties encountered w i t h the cyanines. Oxonols are, however, less sensitive than the cyanines. O x o n o l V I , depicted i n C h a r t I I I , is a m o n g several similar oxonols developed b y C h a n c e et al. ( 3 9 , 40) for d u a l wavelength absorbance measurements o n energy-transducing organelle suspensions. Bis-oxonol a n d its relatives w i t h barbituric acid n u c l e i have b e e n used i n fluorescence experiments o n cell suspensions (41-43). T h e m e m b r a n e potential o f i n d i v i d u a l cells can b e m o n i t o r e d w i t h a fluorescence microscope. F o r this purpose, however, i t is preferable t o use a permeable redistribution dye w i t h spectral characteristics that have m i n i m a l environmental sensitivity. T h u s , the fluorescence intensity w i l l reflect the degree o f N e r n s t i a n accumulation o f dye only a n d can, therefore, b e readily interpreted. T h e plasma m e m b r a n e potential can b e distinguished f r o m the organelle m e m b r a n e b y s i m p l y using the microscope t o identify appropriate regions o f the c e l l (44). R h o d a m i n e - 1 2 3 ( C h a r t I I I ) was i n t r o d u c e d as a mitochondrial stain b y C h e n a n d co-workers (45-47); i t has b e e n used largely i n qualitative studies o f m i t o c h o n d r i a l membrane potential and has b e e n especially effective i n flow cytometry applications. O u r laboratory (44) has synthesized two rhodamine dyes, T M R E ( C h a r t III) a n d T M R M , that are very similar to rhodamine 123 except t h e free amino groups are substituted w i t h m e t h y l substituents. T h i s substitution makes the dyes m o r e permeable than rhodamine 123 a n d also blocks any poorly reversible h y d r o g e n - b o n d i n g interactions w i t h anionic sites i n t h e mitochondrial i n n e r m e m b r a n e a n d matrix. These qualities c o m b i n e d w i t h


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the general environmental insensitivity o f rhodamine fluorescence a n d the l o w tendency o f T M R E and T M R M to aggregate, makes t h e m good " N e r n s t i a n " indicators of m e m b r a n e potential. That is to say, the ratio o f fluores cence intensities measured i n two compartments separated b y a m e m b r a n e (viz. F / F ) , w h e n p r o p e r l y corrected for b a c k g r o u n d dye b i n d i n g , can be inserted into the N e r n s t equation to provide a direct measure of the potential difference between the compartments ( A V ) : 1

2

m

A V

=

RT

F, In— F F


2

(1) W

R is the ideal gas constant, Τ is the absolute temperature, a n d F is Faraday s constant. This approach was successfully a p p l i e d to the plasma membrane potential o f several different c e l l types (44) a n d was used to follow changes i n mitochondrial m e m b r a n e potential via digital i m a g i n g microscopy (48). A l though ordinary w i d e - f i e l d microscopy is successful i n m o n i t o r i n g these kinetic events, the actual magnitude of m i t o c h o n d r i a l membrane potential cannot be quantitated because it is not possible to obtain an accurate measure o f fluorescence intensity that emanates f r o m such a small compartment w i t h the b r o a d depth o f field set b y the microscope optics. T h i s limitation can be overcome i f confocal microscopy is e m p l o y e d w i t h its very narrow d e p t h o f field. A l t h o u g h this approach has b e e n successfully demonstrated (48-50), the h i g h excitation light intensities necessitated b y the technique (at least i n the current generation of confocal microscopes) limits its usefulness for measurements o n living cells. T h u s , single measurements can be p e r f o r m e d , but the accumulation of large image sets for either kinetics or t h r e e - d i m e n sional reconstructions leads to fading o f the dye or phototoxic effects o n the cell.

Dye Characterization T o determine i f a dye w i l l be a useful potentiometric probe i n biological applications, i n f o r m a t i o n o n its chemical, physical, spectral, a n d toxicological properties must be gathered i n addition to data o n the sensitivity to m e m brane potential. A n u m b e r o f m o d e l m e m b r a n e systems are e m p l o y e d to characterize dye properties as w e l l as some simple biological preparations.

Lipid Vesicles. L i p i d vesicles are c o m p o s e d o f l i p i d bilayers that separate the b u l k aqueous m e d i u m f r o m an entrapped aqueous compartment. T h e y are easily p r e p a r e d b y mixing p h o s p h o l i p i d w i t h the appropriate aque ous buffer. T h e vigor o f the mixing operation determines the n u m b e r and size distribution o f bilayers that comprise each vesicle. F o r example, extensive sonication o f egg phosphatidylcholine produces an optically clear suspension



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o f unilamellar vesicles w i t h an average diameter o f about 250 Â (51). T h i s simple m o d e l system can b e used to assess the spectral properties o f m e m b r a n e - b o u n d dyes a n d their b i n d i n g affinities. I n addition, it is possible to generate w e l l c o n t r o l l e d m e m b r a n e potentials across the vesicle m e m branes a n d thereby calibrate the spectral response o f a potentiometric probe. A fixed dye concentration can be titrated w i t h aliquots o f l i p i d vesicles and the fluorescence or absorption spectra can be m o n i t o r e d . A l m o s t a l l dyes that are sensitive to m e m b r a n e potential w i l l display a significant difference i n the spectra o f m e m b r a n e - b o u n d versus free aqueous forms. (Notable exceptions are the N e r n s t i a n rhodamine dyes.) A l o n g w i t h the spectral characteristics o f the m e m b r a n e - b o u n d dyes, these experiments provide information o n the b i n d i n g equilibria. A thorough analysis o f such data ( 3 9 ) can be used to generate b o t h b i n d i n g constants a n d b i n d i n g numbers (i.e., the n u m b e r o f b i n d i n g sites p e r vesicle). W e use the fluorescence o f a 1-μ M dye solution titrated w i t h l i p i d vesicles to determine the ratio o f the dissocia tion constant to the b i n d i n g n u m b e r , K /n, to serve as a convenient quantitative measure o f the relative affinities o f potentiometric dyes: d

C

+

K /n A

F is the fluorescence o f the dye i n a l i p i d vesicle suspension w i t h a l i p i d concentration o f C ; F a n d F are the l i m i t i n g dye fluorescence readings i n the absence o f l i p i d a n d at very h i g h l i p i d concentrations, respectively. T h e fluorescence as a f u n c t i o n o f l i p i d concentration is fitted to this equation v i a nonlinear regression. T h e results o f such measurements for a series o f styryl dyes (10), as w e l l as the spectral characteristics o f the dyes i n several solvents, are given i n T a b l e I. A s expected, the m e m b r a n e affinity o f the dyes increases w i t h the length o f the c h r o m o p h o r e or the n u m b e r of carbons i n the a p p e n d e d hydrocarbon chains. A l s o noteworthy is the striking increase i n fluorescence q u a n t u m y i e l d for the m e m b r a n e - b o u n d dyes; this is probably due to the w e l l - o r d e r e d i m m o b i l e environment o f the membrane, w h i c h inhibits nonradiative decay processes that require molecular m o t i o n i n the excited state. c

0

œ

T h e kinetics o f dye b i n d i n g to the m e m b r a n e is o f practical importance. C o n t r a r y to expectation, the rate o f association w i t h the bilayer is not directly related to the b i n d i n g affinity. Indeed, dyes w i t h the longest hydrocarbon chains have the slowest b i n d i n g kinetics (29), w h i c h is presumably due to the presence o f stable oligomeric aggregates o f the dye i n aqueous solution. T h e rate o f dissociation o f a m o n o m e r f r o m these aggregates is rate l i m i t i n g w h e n the hydrocarbon chains are longer than three or four carbons. A n o n i o n i c macromolecular detergent, P l u r o n i c F 1 2 7 , can be used as a catalyst for staining cells w i t h these sluggish dyes. Interestingly, dyes w i t h l o n g hydrocar-


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b o n tails flip across the l i p i d bilayer at slower rates than slower dyes a n d are r e q u i r e d for many c e l l types to prevent r a p i d internalization. T h e vesicle system can support a valinomycin-mediated K diffusion potential, w h i c h affords a q u i c k a n d simple means for calibrating a potentio metric dye response. T h e potential can be generated b y adding valinomycin, a highly K - s e l e c t i v e ionophore, to a vesicle suspension i n w h i c h the i n n e r a n d outer potassium concentrations are unequal. T h i s u n e q u a l concentration can be achieved most s i m p l y b y p r e p a r i n g the vesicles i n a solution at one [ K ] and diluting the vesicles into a solution p r e p a r e d w i t h a different [ K ] . T h e m e m b r a n e potential can be calculated directly f r o m the N e r n s t equation (eq 1). T h i s approach was originally a p p l i e d to the cyanine dyes by W a g g o n e r a n d H o f f m a n a n d their colleagues for b o t h l i p i d vesicles a n d erythrocytes (32). I n general, the effectiveness o f a dye i n a c e l l suspension can be assessed w i t h a l i p i d vesicle experiment; however, possible complications (such as the accu mulation of dye b y m i t o c h o n d r i a and other organelles) require that such comparisons be made w i t h care. +

+

+


+

Spherical Lipid Bilayer. Spherical or hemispherical 2 - 4 - m m diameter l i p i d bilayers can be p r e p a r e d b y applying hydrostatic pressure to a hydrocarbon solution o f l i p i d that covers the tip o f an electrolyte-filled tube suspended i n the electrolyte (52). This m o d e l m e m b r a n e is particularly convenient for determination o f the response of a m e m b r a n e - b o u n d dye to a directly a p p l i e d voltage (16, 17). A diagram o f the i m p l e m e n t a t i o n o f this m e t h o d for screening potentiometric dyes is shown i n F i g u r e 2 (21). I n this latest version o f an apparatus that has continuously evolved over the last 13 years, we induce a m e m b r a n e potential b y applying voltage to a pair o f external parallel plate electrodes. E a r l i e r versions a p p l i e d the voltage directly to the m e m b r a n e v i a a voltage-clamp circuit connected to an electrode inside the tube (53). T h i s apparatus can be used to determine the sensitivity o f a dye as w e l l as to gain insight into the dye mechanism. Dragsten a n d W e b b ( 1 7 ) used polarized light a n d signal averaging o f the fluorescence response to a voltage pulse to show that the mechanism o f M - 5 4 0 ( C h a r t I) involved a voltage-driven reorientation o f the molecule along the m e m b r a n e surface c o u p l e d to an orientation-dependent dye aggregation. A series o f fast oxonol dyes were examined and showed absorbance changes w i t h magnitudes that were depen dent o n the offset a p p l i e d to a train o f voltage pulses (15, 54, 55). T h i s observation was interpreted as evidence for the voltage-dependent occupation o f two sites near the m e m b r a n e surface ( " o n - o f f " mechanism). W e used the spherical bilayer to investigate the mechanism o f the styryl dyes (10, 12, 53, 56). A square wave voltage a p p l i e d to the m e m b r a n e p r o v i d e d the reference for a l o c k - i n amplifier that was able to extract a small voltage-dependent optical signal f r o m a large b a c k g r o u n d . T h i s signal p e r m i t t e d us to determine the complete wavelength dependence o f the absorbance a n d fluorescence


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response. A n example o f a fluorescence excitation response spectrum f o r d i - 4 - A N E P P S is given i n F i g u r e 3. A detailed investigation o f the wavelength, polarized light, voltage, a n d dye concentration dependences o f the potentio metric response l e d us to conclude ( 3 5 , 5 6 ) that many o f the styryl dyes operated v i a an electrochromic mechanism (although this is not the dominant

Table I. Spectroscopic and Binding Properties of Charge-Shift Probes Structure and Abbreviation


No.

so-

(di-4-ASPPS) ΛΛ

w ^3

(fddii--44 -- A ACS PPnB

Biomembrane Electrochemistry - ACS Publications - American

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