169

J. Photochem. Photobiol. B: Biol., 13 (1992) 169-185

Fluorescence anisotropy studies of dibucaine . HCl in micelles and bacteriorhodopsin C. T. Lint and C. J. Mertz Department of Chemishy, Northern Illinois University, DeKalb, IL 60115-2862 (USA) I-f. C. Bitting

and M. A. El-Sayed

Department of Chemistry and Biochem~t~, WA)

University of Califonzia, Los Angeles, CA 900241569

(Received July 17, 1991; accepted November 5, 1991)

Abstract Emission and excitation spectra for the local anesthetic drug, dibucaine.HCl in neutral and charged surfactant solutions and in bacteriorhodopsin (bR) have been investigated for A,=266 nm at room temperature. The total fluorescence and fluorescence anisotropy decays of the anesthetic in the same environments were also measured using a picosecond laser/streak camera system (A,, = 266 nm). The total fluorescence decay gave two components for dibucaine micellar and dibucaine bR solutions, one component in the range of 200-500 ps and the other in the range of 1200-3400 ps. Only the nanosecond timescale component was found for the dibucaine monomer surfactant solutions (12UO-3000 ps), indicating that the anesthetic resides in the bulk solution. The fluorescence anisotropy decays of dibucaine in Triton X-100 and in lithium dodecyl sulfate {LDS) micelles are approx~ately 200 ps, which is attributed to dibu~aine solubilized in the micelfar environment. Dibucaine*HCl in anionic monomer solution exhibits an unusually large fluorescence anisotropy, r@),, = 0.22 and a depolarization decay of less than 100 ps. This presumably results from a head-totail exciplex aggregation between the positively charged dibucaine and negatively charged dodecyl sulfate surfactant molecules. The anisotropy decay of dibucaine in bR is 300 ps. This solution was the only one which exhibited a residual fluorescence anisotropy, r( 03) =0.08. This implies that dibucaine is restricted in its rotational motion and suggests protein binding rather than lipid solubility.

Keywords: Bacteriorhodopsin,

dibucaine,

fluorescence

anisotropy,

micelles.

1. Introduction Many local anesthetics are tertiary amines, such as dibucaine, tetracaine, procaine, etc. Dibucaine (2-buto~-~-[Z-diethylamino)ethyl]-4-quinoline carboxamine) is shown in Scheme 1. This represents the neutral free base form of the anesthetic, ~ntaining a quinoline analog and an amide group. Dibucaine can also exist as a monition in +Author to whom correspondence should be addressed.

loll-1344/92/$5.00

0 1992 - Elsevier Sequoia. All rights reserved

170 0

II ,CNHCH2CH2N(C2H5)2

Scheme 1.

which the aliphatic tertiary amine N is protonated, and/or as a dication in which the aromatic N is also protonated. The equilibrium of free base (D), monocation (DH’), and dication (DHz2+) dibucaines depends strongly on the pH of the medium [l] as follows:

(1) Recently, it has been shown [2, 31 that the tertiary amine local anesthetics display rather rich optical properties and, more importantly, that the photophysical properties of uncharged and charged species are quite different and clearly distinguishable from one another. Furthermore, the local anesthetic species exist predominantly as the protonated form, DH+, in hydrophilic environments, and the equilibrium is then shifted to favor the neutral form, D, in hydrophobic environments [2, 31. This indicates that tertiary amine local anesthetics are viable ‘direct’ emission probes for studying the nature of their solubilization microenvironment as well as their interaction in biomembranes. Two main classes of hypotheses which explain the mechanism of molecular action of local anesthetics include: (i) induced lipid structural alterations [4] or (ii) direct interaction with membrane proteins. The observations of an increase in the surface area [5] and fluidity [6], as well as an effect on lipid-phase transition [7] of the membranes upon addition of the anesthetics support the roIe of lipid-matr~ structural alterations in their mode of drug action. As for the hypothesis that proteins have non-specific hydrophobic binding centers for local anesthetics IS], a correlation between affinity for membrane proteins and drug potency has been reported by Garcia-Martin and Gutierrez-Merino [9]. Recently, a spectroscopic study of the interaction of dibucaine *HCl local anesthetics with bacteriorhodopsin in purple membrane suggests [2] that the anesthetic action on bacteriorhodopsin is probably via specific site binding and is not a conformational mechanism. MicelIes have been used as model systems to mimic biological membrane environments for a variety of solutes ranging from hydrocarbons to inorganic ions [10-121. Micelles are self-assembled lipid dispersions of surfactant molecules. As the concentration of surfactant in an aqueous solution increases beyond a critical range (the critical micelle concentration, CMC), self-association results in micelle formation. Steady-state and time-resolved emission spectroscopic techniques at 77 K have previously been employed to characterize the drug species of dibucaine and to identify its location in micellar Triton X-100 (neutral), hexadecyltrimethylammonium bromide (cationic) and lithium dodecyl sulfate (anionic) frozen solutions [13]. The distinct properties observed for the drug species (D and DH”) and their solubilization sites in these micelles are

171

consistent with a balance between hydrophobic forces, surface polarity and the interfacial electrostatic potential present in the micellar solubilization sites. In order to study local anesthetic-protein interaction, we chose bacteriorhodopsin fbR) which is one of the protein pigments found in the purple membrane of ~alobacfe~um halobium [14, 151. The membrane consists of approximately 25% lipid composition [14]. The protein forms a ho-dimensional hexagonal crystalline lattice in the purple membrane [16, 171, and contains 8 tryptophan and 11 tyrosine residues out of 248 amino acid residues in the polypeptide chain [18-201. It contains an all-trans retinal chromophore attached to lysine-216 via a protonated Schiff base linkage [14, 21, 221. In this study, solubilization sites and acid-base species of dibucaineeHC1 in micelles (lipid dispersions) and bacteriorhodopsin (protein membrane) will be investigated at room temperature and will be compared to those reported previously at 77 K [13]. Emission and excitation spectroscopic techniques are used to monitor the drug species of dibucaine (D or DH+) and to identify its location in micelles (the outer aqueous solution, the water-micelle interface or the micellar hydrophobic region), and in bR (lipid or protein domains). The rotational freedom of dibucaine drug species residing in different microenvironments of micelles and bacteriorhodopsin is determined and confirmed by a 10 ps resolution streak camera detection of fluorescence anisotropic measurements. The results are discussed in light of the molecular basis of pha~acological action, in particular, the mechanism of transporting local anesthetic drugs across membranes.

2. Materials

and methods

Dibucaine free base, dibucaine .HCl, Triton X-100 (TX, neutral surfactant), lithium dodecyl sulfate (LDS, anionic surfactant) and hexade~ltrimethylammonium bromide (HTAB, cationic surfactant) were purchased from Sigma Chemical Co., (St. Louis, MO) and were used without further purification. The aqueous solutions were prepared using phosphate buffers of pH=5.5, 7.0 and 8.0 with an ionic strength of 0.1 M (adjusted with NaCl). (pH=5.5, 0.10 M NaHzP04/0.002 M Na2HP04; pH= 7.0, 0.05 M NaH~PO~/O.O32 M NazHP04; pH = 8.0, 0.005 M NaH2P0,J0.032 M Na2HP0,). The critical micelle concentration of the neutral, anionic and cationic surfactants in aqueous solutions was 0.3, 8.9 and 0.95 mM respectively [23]. The micellar surfactant solutions were prepared in a buffer solution at a concentration of the surfactant 10 times higher than the CMC value. The solution was ultrasonically agitated for 1 h at 45 “C. The monomeric form of surfactant solutions were prepared by using a solution concentration 10 times (and 100 times for the anionic surfactant) lower than the CMC. The concentration of dibucaineaHC1 in the monomeric or micellar surfactant solutions ranged from IX 10W3 to l~lO_’ M. The procedure for the growth and purification of bR was a combination of those previously outlined by Oesterhelt and Stoeckenius [24] and Becher and Cassim [25]. Steady-state excitation and emission spectra were recorded at room temperature on a SPEX Fluorolog model 1902. Consistency between the steady-state and timeresolved experiments was maintained by using 266 nm as the excitation wavelength for the emission studies. The excitation spectra were monitored at the A,, determined from the A,_= 266 nm emission spectra, unless otherwise noted in the text. For excitation at 266 nm, no fluorescence impurities were detected for the solvents or detergent

172

solutions. The reported spectra are the difference spectra between the sample solution with and without local anesthetic. The spectra were recorded using a quartz tube of 2 mm inner diameter with front surface excitation geometry, so that the inner filter effect (light attenuation) was minimal. Measurements of fluorescence anisotropy can reveal the average angular displacement of the fluorophore during the lifetime of the excited state. Two models [26], namely the hindered fluorophore model and the anisotropic rotator model, might be used to describe the decay of the emission anisotropy of dibucaine=HCl in micelles and bacteriorhodopsin. The hindered fluorophore model treats the emission anisotropy as r(t) = (rO-r,)

exp( - f/~a) +r,

where r, and r, are the limiting values of the emission anisotropy in a free and restricted rotational motion, and TV is the rotational correlation time. The anisotropic rotator model treats the emission anisotropy as r(t) =r,E

i

gi exp( -t/r~)

where cgi= 1. Multiexponential decay of the emission anisotropy could be interpreted [27] in terms of different local anesthetic species solvated in different microenvironment sites. Time-resolved fluorescence and anisotropy decay measurements were performed with the fourth harmonic (266 nm) of a passive/active mode locked Ndf3:YAG laser (Quantel 471, Santa Clara, CA). The samples were excited with a spot size of approximately 2 mm, vertically polarized laser pulses of approximately 35 ps and approximately 70 mJ. A half-wave plate and a clean up polarizer were placed in front of the sample. UV transmitting filters (340-420 nm) were placed in front of the detector to block bR fluorescence at h < 325 nm and a CuS04 solution filter was used to remove scattered laser light. The fluorescence was collected at a right angle from the excitation source. The fluorescence components polarized parallel, I,,(f) and perpendicular, 1,(t) to the polarization of the excitation source were collected independently. The detector consisted of a 10 ps resolution streak camera (Hamamatsu C979, Hamamasu City, Japan) coupled to a reticon (Princeton Applied Research intensified 1420, Princeton, NJ). The detection system was interfaced to a computer (Digital LSI 11/233 Maynard, MA) and the data analysis used a VAX computer. Nonlinear time and intensity responses of the streak camera/reticon system were calibrated. The temporal response function has been measured to be 40 ps (FWHM). All measurements were performed at room temperature. Several scans were taken to check the reproducibility of the intensities and decays. Data analysis was performed using a non-linear least squares computer program [28]. The total fluorescence polarization anisotropy r(t) is defined in terms of the fluorescence intensities I,,(t) and II(t), as follows:

I,, and

I, are the components of the fluorescence intensities, parallel and perpendicular to the excitation polarization respectively. The factor G corrects for the efficiency of the detector towards vertically and horizontally polarized light. G is the ratio of the vertical to horizontal emission when the excitation is horizontally polarized. The total fluorescence intensity decays from this experiment were in agreement with those found by Vanderkooi [l] for dibucaine in buffered solution.

173

3. Results

and discussion

3.1. Spectral properties of dibucaine species in hydrophobic

and hydrophilic environments In this study, the steady-state spectral properties of free base dibucaine (D) in methylcyclohexane (hydrophobic) and dibucaine.HCl (DH+) in deionized water (hydrophilic environment) were shown to be unique for 266 nm excitation at room temperature (spectral data in Table 1). Neutral dibucaine (1X 10e4 M) in methylcyclohexane displays some structural features with A,,=355 nm in the broad band emission which extends from 300 to 440 nm (refer to Table 1). The excitation spectrum for this sample monitored at 350 nm exhibits a broad band emission with A,,=296 nm. On the other hand, the emission spectrum of 1 X10e4 M DH+ in water (a hydrophilic environment) has a single band centered at A,,=419 nm (a 64 nm red shift from that of D in a hydrophobic environment). The excitation spectrum of DH+ in water monitored at 400 nm exhibits a band maximum at 328 nm. This same spectral maximum was observed in the absorption spectra [3]. The spectral band at 328 nm has been previously reported as the effective origin of the rr,r* absorption of DH+ in a hydrophilic environment [3]. In a hydrophobic environment the neutral dibucaine (D) emission at 355 nm presumably originates from a n,rr* charge-transfer state (the n orbital from the oxygen of the carbonyl group). In a hydrogen-bonding solvent, the n,rr* charge-transfer state shifts further to the blue depending upon the bond strength. These distinct spectroscopic properties are essential for this investigation and will be used to distinguish the action species and action sites of dibucaine in micelles and bR, as described below. 3.2. Solubilization sites and dibucaine species in Triton X-100 Dibucaine.HCl (1 X 10W4 M) in an aqueous solution of monomeric Triton X-100 (0.03 mM) displays an emission band at 419 nm and its excitation maximum is at 328 nm (Table 1). This indicates that the monoprotonated species, (DH+) is the predominant form of dibucaine while dissolved in Triton X-100 monomer solution. The total fluorescence decay shows a single exponential component of T= 3OOOk 100 ns. This is in agreement with the fluorescence lifetime r= 3.33 rt 0.07 ps reported by Vanderkooi [l] for 2.5 x low5 M dibucaine.HCl in phosphate buffer, 0.05 M, pH 6.7. For 1 x lop4 M dibucaine.HCl in Triton X-100 micellar solutions (3 mM, neutral micelles), two new fluorescence peaks are observed at 307 and 341 nm, in addition to the peak at 419 nm (Table 1). When the fluorescence emission is monitored at 310 nm, the excitation spectrum shows a band maximum at 291 nm. These spectral characteristics suggest the partition between DH+ and D in neutral micellar solution. DH+ exists in the extramicellar solution, as detected by the emission peak at 419 nm. Deprotonation of DH+ is supported by the fluorescence peaks at 307 and 341 nm, which suggest solubilization of D in a more hydrophobic environment in the Triton X-100 micelles. It is believed that the blue shift in the fluorescence peak at 307 and 341 nm from the maximum at 355 nm for D in a non-polar solvent can be attributed to solubilization of D in two different sites. (i) The emission at 341 nm does not deviate far from the non-polar solvent results, thus suggesting that D is located in the micellar hydrocarbon region. (ii) The peak at 307 nm is significantly blue-shifted due to a proposed hydrogenbonded D at the micellar interfacial region. The total fluorescence decay shows two computer fitted exponential components of r1 = 3400&300 ps and T,=500f30 ps with an intensity fraction of I1 = 0.8 and I2 = 0.2, suggesting that two forms of dibucaine are in two different microenvironments. The long component has been identified as

333 418,(sh)365-400d 418

5.5 8.0 7.0 7.0 7.0 7.0 7.0 7.0

1x10-s 1x10-’

1 x 1o-4 1 x 1o-4

27 /.LM 270 /.LM 30 PM

HTAB(9.5)(mM) HTAB(9.5)(mM)

LDS(O.O89)(mM) LDS(89)(mM)

bR(27 bR(27 bR(27 bR(30

“Emission monitored at A,, from emission maxima [a.=266 bTotal fluorescence decay; one or two components. Wuorescence anisotropy decay. d(sh) =shoulder peak.

PM) /.LM) PM) /.LM)

418 371

291 330,(sh)300” 328

295 329

329 329,(sh)300f

328 328

301

343 328 291=

328

296

Excitation &,(nmY

2300 + 100, 300&30

1200 + 100 1900 * 150, 200 f 30

23OOk200 2200 + 200, 500 * 50

3000& 100 3400 f 300, 500*30

&)

b

nm], except, “310 nm and f425 nm.

358 418

7.0 7.0

1 x 10-4 1 x 10-4

HTAB(O.O95)(mM) HTAB(gS)(mM)

419 419

7.0

TX(3)(mM)

1x1o-4 1x10-4

1 x 1o-s

418 419 419,(sh)307d (sh)341d 353

355 419

Emission &,(nm)

microenvironments

7.0 7.0 7.0

pH

in different

1x1o-3

1 x 1o-4 1 x 10-4

cone(M)

Dibucaine . HCl

results for dibucaine.HCl

Surfactants TX(3)(mM) TX(O.O3)(mM) TX(3)(mM)

H20

MCH

Solvents

Fluorescence

TABLE 1

0.5 0.5

0.3 0.7

0.5 0.5

0.8 0.2

300

200

70

200

0.16

0.06

0.22

0.06

0.08

175

DH+ solubilized in the aqueous solution phase. The much shorter fluorescence decay has been attributed to solubilization of the drug in a hydrophobic environment. It has been observed [3, 131 for D in a hydrophobic environment, that there is a significant contribution to the non-radiative intersystem crossing process at 77 K, as compared to DH’ in a hydrophilic environment. The partition of neutral dibucaine (D) in the hydrophobic region of Triton X100 micelles and charged dibucaine (DH+) in the hydrophilic region of extramicellar phase is shown to be concentration dependent for neutral micellar solutions, Figure 1 shows the emission (top) and excitation (bottom) spectra of 1 x low3 M (left) and 1 X lo-’ M (right) dibucaine *HCl in the micellar Triton X-100 solutions. At 1 x 10m3 M, the emission (Fig. l(a)) and excitation (Fig. l(b)) maxima are located at 418 nm and 343 nm respectively. The emission suggests that only DH+ is present in the the extramicellar aqueous phase; however, the excitation maxima may represent combination of DH+ and D present in neutral extramicellar and intramicellar sites respectively. At 1X lo-’ M dibucaineeHC1, however, the emission (Fig. l(c)) and excitation (Fig. l(d)) maxima are located at 353 nm and 301 nm respectively, indicating that the D species is located in the intramicellar hydrophobic phase. At low anesthetic concentrations, D is the dominant species residing inside the micellar environment. When the hydrophobic micellar sites are saturated at high dibucaine concentration, DH+ is then seen in the aqueous extramicellar phase. 3.3. Solubiltiation sites and dibucaine species in HTAB micelles For 1 x lop4 M dibucaine.HCl in both monomeric (9.5X lo-’ M) and micellar (9.5 x 10e3 M) HTAB solutions at pH = 7.0, the room temperature fluorescence band has a maximum at 419 nm and its excitation spectral peak is located at 328 nm (refer to Table 1). This confirms the solubilization of DH+ in the extramicellar aqueous phase. One would expect positively charged dibucaine, DH+, to have little association with positively charged detergent monomers and micelles. The spectral characteristics do not suggest the existence of different local anesthetic species solvated in different microenvironment sites of HTAB micelles. However, the total fluorescence decay of 1 x lop4 M dibucaine.HCl in micellar HTAB solution displays two computer fitted exponential components of r1 = 22OOk 200 ps and Q= SOOrt 50 ps with an intensity fraction of Z1=0.5 and Z2= 0.5, whereas that in monomeric HTAB solution shows a single exponential component of 71= 2300 + 200 ps. The dynamic lifetime measurements indicate that besides DH+ species solvated in the extramicellar aqueous solution of HTAB micelles, there are D species which penetrate in the hydrophobic micellar region. To verify the existence of anesthetic drug species solvated in different micellar microenvironments of HTAB micelles, we studied the pH effect on the drug partition in HTAB micellar solution. Figure 2 shows the emission (top) and excitation (bottom) spectra of 1 x lo-’ M dibucaine .HCl in the micellar HTAB solutions at pH = 5.5 (left) and pH=S.O (right). At high pH, neutral dibucaine (D) is the highly favored form and is expected to reside in the hydrophobic region of HTAB micelles. This is shown in the location of the emission maximum at approximately 371 nm (Fig. 2(c)). The red shift in the emission maxima from that observed for D in a non-polar solvent (i.e. from 353 to 371 nm) is most likely to be due to the cationic intramicellar environment. When the fluorescence emission is monitored at 425 nm (Fig. 2(d)), the partition of DH + in the extramicellar aqueous phase is evident from an excitation peak at 329 nm. In addition, a shoulder excitation spectral band associated with D is observed at approximately 300 nm. On the other hand, the location of DH+ in the

04

0.02E

06

Wavelength

O.OOE 00

2.46E

(nm)

280

425

570

Fig. 1. Emission spectra: (a) 1 X lo-’ M; (c) 1 x lo-’ M dibucaine-HCI in Triton X-100 micellar solutions at pH 7.0 with 266 nm excitation. Excitation spectra: (b) and (d) were obtained by monitoring 42.5 nm of emission spectra in (a) and (c) respectively.

04

1.72E

05

200

280

I

300

I

425’

I

I

I

06

(nm)

280

05X

O.OOE 00

l.lOE

0.0&x

Wavelength

400’

1

570

425

Fig. 2. Emission spectra: (a) pH 5.5; (c) pH 8.0 of 1X10-’ M dibucaine.HCl in hexadecyltrimethylammonium 266 nm excitation. Excitation spectra: (b) and (d) were recorded by monitoring 425 nm of emission spectra

-E

za

C

x

O.OOE 00

4.50E

bromide micellar solutions in (a) and (c) respectively.

570

with

2

178

extramicellar aqueous phase of HTAB at pH 5.5 is seen in an emission band at 418 nm (Fig. 2(a)) and an excitation spectral peak at 329 nm (Fig. 2(b)). These results are consistent with steady-state and time-resolved emission spectroscopic techniques at 77 K. It was illustrated [13] that in cationic micellar solution, (HTAB), dibucaine exists as (i) the monocation species (DH+) where the anesthetic is solubilized at the counterion layer of the extramicellar aqueous solution; and (ii) D is solubilized in the hydrophobic region with close proximity to the micellar interface. 3.4. Solubilization sites and dibucaine species in LDS micelles Figure 3 illustrates the room temperature emission (top) and excitation spectra (bottom spectra) of 1 X lop4 M dibucaine .HCl in monomeric (Fig. 3(a) and 3(b)) and micellar (Fig. 3(c) and 3(d)) LDS solutions at pH=7.0. The emission and excitation maxima for DHC in LDS micellar solution are observed at 418 nm (Fig. 3(c)) and 329 nm (Fig. 3(d)) respectively. Unlike DH+ in the neutral and cationic monomeric surfactant solution, an unexpected emission band is observed at 358 nm (Fig. 3(a)) and an excitation maximum at 295 nm (Fig. 3(b)) for DH+ in anionic (LDS) monomeric surfactant solution. The unexpected emission band at 358 nm is proposed to result from a head-to-tail exciplex aggregation between the positively charged dibucaine and negatively charged surfactant molecules. The total fluorescence lifetimes of DH+ in LDS monomeric surfactant solutions shows a single exponential decay of T= 1200 + 100 ps. This is two to three times shorter than the total fluorescence lifetime of DH+ in the neutral and cationic monomeric surfactant solution. The shorter lifetime indicates a stronger molecular coupling to LDS as suggested by the formation of heat-to-tail exciplex species. The total fluorescence decay of dibucaine .HCl in LDS micellar solution at room temperature gives two components of pi = 19OOk 150 ps and Q= 200+30 ps with an intensity fraction of I1 =0.3 and 12= 0.7. The long lifetime component, ~-i is only slightly shorter, but the short component, TVis at least 2.5 times shorter than that of dibucaine.HCl in Triton X-100 and HTAB micellar solution. In LDS solution the monoprotonated dibucaine (DH+) can act as organic counterions which may result in a strong drug-surfactant coupling. Steady-state and time-resolved emission spectroscopic studies of dibucaine .HCl in LDS micellar solutions at 77 K have provided evidence for the existence of DH+ acting as counterions in the vicinity of the micellar surface [13]. The short lifetime component represents DHC anchored at the micellar interface via the tertiary amine group and the quinoline analog of this species located in more hydrophobic regions of LDS micelles. The lifetime shortening may be due to a strong coupling between the anchored dibucaine and the micellar interface. The fluorescence anisotropy decay will serve as further evidence for this strong coupling (see section 3.6). 3.5. Dibucaine species in the lipid or protein domain of bacteriorhodopsin (bR) The fluorescence of bR and its retinal-free form bacterioopsin (b0) is characteristic of tryptophan (Trp) in the polypeptide chain [29]. An excitation spectral maximum at 291 nm and emission maximum at 333 nm for Trp in bR is observed in Fig. 4(e) and 4(a) respectively. The dibucaine emission maximum (Ann+ = 419 nm; hn = 355 nm) is at a considerably longer wavelength than that of tryptophan in bR. In fact, the emission band of Trp overlaps appreciably with the absorption band of dibucaine (327 nm), and energy transfer from Trp to protein-bound dibucaine in bR may be observable. Figure 4 shows the emission (left) and excitation (right) spectra of 27 PM bR aqueous solution with the following amounts of dibucaine.HCl added to the solution: 0 PM dibucaine.HCl (Fig. 4(a) and 4(e)); 6.75 PM dibucaine.HCl (Fig. 4(b) and 4(f));

h

ii -c

‘ZJ

05

05

(B)

280

-

(A)

4

267.50

I

425

I

I

t

I

I

335

_

05 m

(nm)

280

O.OOE 00 .

Wavelength

I

570

i -Tz

x C

7.60E

m

’

425

570

Fig. 3. Emission spectra of 1 X 10e4 M dibucaine- HCl in lithium dodecyl sulfate: (a) monomeric ([LDS] = 8.9 X 10P5 M); (c) micellar ([LDS] = 8.9 x 1W2 M) solutions at pH 7.0 with 266 nm excitation. The CMC value of LDS is 8.9~10~~ M. Excitation spectra: (b) and (d) were obtained by monitoring 350 nm of emission (a) and by monitoring 425 nm of emission (c) separately.

O.OOE 00 200

ii s -c

x c

9.90E

OBOE 00

6.10E

2

180 a.ooE 04

(El x .ZZ 2 a, 5

‘-“I

O.OOE 00 1

35:s

200’

390

290

04

x s 6 +

O.OOE 00

iao

300

280

390

I

0.01E 05

500

I 500

Wavelength

(nm)

Fig. 4. Emission spectra: (a) 0.0 PM, (b) 6.75 PM, (c) 27 PM, (d) 270 PM dibucaine-HCl in 27 /IN bR aqueous solutions at pH 7.0 with 266 nm excitation. Excitation spectra: (e), (f), (g) and (h) were recorded by monitoring at 400 nm of emission spectra (a), (b), (c) and (d) separately.

181

27 PM dibucaine.HCl (Fig. 4(c) and 4(g)); and 270 /.LM dibucaine.HCl (Fig. 4(d) and 4(h)). It is clearly seen that the excitation energy of Trp in bR shows significant quenching by the presence of the dibucaine species. As the concentration of dibucaine increases (from top to bottom of Fig. 4), the emission maximum at 333 nm and excitation maximum at 291 nm of Trp in bR reduce in intensity, while the intensity of the emission maximum at 418 nm and excitation maximum at 328 nm of the dibucaine species increases. The strong energy coupling between Trp in bR and the added dibucaine suggest that the solubilization sites of the dibucaine species in bR is likely to be in hydrophobic protein regions. It is important to note that 27 PM dibucaine.HCV27 /.LM bR aqueous solution exhibits an excitation shoulder at approximately 300 nm and maximum at approximately 330 nm (Fig. 4(g)) and a broad emission of approximately 365-440 nm (Fig. 4(c)). This suggests that dibucaine exists in both the protonated (DH+) and neutral (D) forms in bR solution. Protonated dibucaine (DH+) may exist in the aqueous phase and in the hydrophilic regions of bR while D is solubilized in hydrophobic regions (probably as a protein-bound dibucaine). The observation of multiexponential decays in the total fluorescence decay of 30 PM dibucaine.HCl in 30 PM bR (aqueous solution at pH=7.0) strongly supports the existence of several dibucaine species in various solubilization sites of bR. The total fluorescence decay resulted in two computer fitted exponential components of 71= 2300 rf: 100 ps and Q = 300 f 30 ps with an intensity fraction of II =0.5 and I,=OS. The possible existence of protein-bound dibucaine will be illustrated by the following fluorescence anisotropy study. 3.4. Fluorescence anisotropy decays of dibucaine . HCI in micelles and bacteriorhodopsin Fluorescence anisotropy measurements have been used to reveal the rotational diffusion of emitting dipoles in a variety of environments (aqueous solution [30], microemulsions [31], and biomembranes [32]). Jahnig (331 has evaluated fluorescence anisotropy measurements in order to characterize the structural order of lipids and proteins as follows. In an isotropic environment, the final distribution of the emitting dipoles is isotropic and r(t) decreases to zero. Time-resolved fluorescence anisotropy measurements in membranes has shown r(t) to reach a finite value rm, where the final distribution is anisotropic. While the final r(t) value provides structural information, the relaxation time supplies kinetic information on the microviscosity (77) of the probe environment (large +j results in a long relaxation time). The dynamics of molecular interactions have been used to infer molecular orientation restraints from neighboring molecules, and the degree of order in the hydrocarbon core of micelles [34]. The present study employed picosecond fluorescence anisotropy measurements to characterize the solubilization site of dibucaine in micelles and bR. Picosecond fluorescence depolarization lifetimes were reported by Fleming et al. [30] for rose bengal in MeOH and i-PrOH (180 and 890 ps respectively). Short rotational relaxation times of 104 ps at 20 “C and 79 ps at 30 “C were found for oxypyrene trisulfonate (OPS) in water [35]. Both of these studies suggested an increase in the molecular volume of the charged dyes due to solvent attachment. It is expected that the local anesthetic, dibucaine would exhibit a much shorter ( < 100 ps) fluorescence anisotropy decay in an aqueous solution or in the bulk extramicellar solution. The experimental results did not indicate anisotropic decays for solutions of 1 X 1O-4 M dibucaine.HCI in neutral (Triton X-100) and cationic (HTAB) monomer solutions or in cationic micelles. The anisotropic decay for dibucaine was probably shorter than the 10 ps time resolution of the experimental set up. Recently, steady-state emission measurements at 77 K have identified the solubilization site of dibucaine in the aqueous surfactant

182

(Triton X-100, LDS and HTAB) solutions [13]. The results did not indicate any intermolecular complexation or interaction for dibucaine in the Triton X-100 and HTAB monomer solutions at 77 K [13]. It was also concluded that the partition of the local anesthetic in HTAB micelles at 77 K [13] was controlled predominantly by the surface potential of the positively charged micelles, therefore DH+ was found to be solubilized in the bulk solution. The room temperature fluorescence anisotropy decay of dibucaine in anionic (LDS) monomer solution was found to be 70 ps and for the r&lax = 0.22 (Fig. 5(a)). This anisotropy decay suggests a strong interaction aqueous solution which may result from an intermolecular head-to-tail exciplex between positively charged dibucaine (DH+) and negatively charged surfactant monomers. It was further noted that the fluorescence anisotropy has a rise time of 160 ps, which suggests that the head-to-tail exciplex of DH+ and LDS may be a photo-induced species (generated by the laser pulse). DibucaineoHCl (1 X 10v4 M) in neutral Triton X-100 (Fig. 5(b)) and anionic LDS (Fig. S(c)) micellar solutions displayed anisotropy decays of 200 ps and r(t)=0.06. We propose that the anisotropic decay indicates that D is solubilized within the micelle. Fluorescence anisotropy decay of 30 PM DH+ in 30 PM bR aqueous solution shows r= 300 ps and r(f),.,,%= 0.16 (Fig. 5(d)). This solution was the only one in this study which exhibited a residual anisotropy, r, = 0.08. All fluorescence anisotropy decay curves for the surfactant solutions have r, =0 (Figs. 5(a)-5(c)). The presence of residual anisotropy is evidence that the dibucaine species is unable to fully rotate into random positions during the lifetime of the emitting dipoles when incorporated in bR. These results suggest that dibucaine is bound to the protein domain of bR and is not located in bR’s isotropic lipid bilayer.

4. Conclusion

Room temperature steady-state emission and excitation measurements and timeresolved fluorescence dynamic properties of the local anesthetic drug, dibucaine*HCl in neutral and charged micelles (lipid analog) and in bR purple membrane (protein domain) lead to the following observations. (i) Under physiological conditions, dibucaine is shown to exist in the free base form (D) while solubilized in the hydrocarbon region of neutral, Triton X-100 micelles. (ii) In cationic, HTAB micellar solution, dibucaine exists as the monocation species (DH+) where the anesthetic is solubilized in the extramicellar aqueous solution and D is solubilized in the hydrophobic region with close proximity to the micellar interface. (iii) In the anionic, LDS micelles, interfacial solubilization is most consistent with a site in which the tertiary amine group of the monocation dibucaine (DH+) is anchored at the micellar interface with its quinoline analog penetrating the hydrophobic region. (iv) In bR, dibucaine in the aqueous phase (DH+) probably deprotonates and enters bR’s hydrophobic environments (lipids or proteins) as the neutral dibucaine (D), in much the same manner as it enters the hydrophobic region of micelles [13]. The results of this study are in excellent agreement with those obtained using low temperature (77 K) emission spectroscopic techniques [13], suggesting that the micellar structural integrity is maintained at 77 K Unlike the dibucaine-lipid (micelles) anisotropy measurements, dibucaine in bacteriorhodopsin is the only sample in this study which exhibited a residual fluorescence This suggests that in bR, dibucaine is restricted in its rotational anisotropy, rol =0.08. movement and implies protein binding rather than lipid solubility. This interpretation is also consistent with the previous conclusion [2] that the action site of the local

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Fig. 5. Fluorescence anisotropy (solid line) and computer-generated (for curves (c) and (d) only) decay for (a) 1X10m4 M dibucaine-HCI in 8.9X lo-’ M LDS monomeric solution; (b) 1 X 10e4 M dibucaine.HCI in 3 x 10m3 M Triton X-100 micellar solution; (c) 1 X 10e4 M dibucaine-HCl in 8.9X10s2 M LDS micellar solution; (d) 30 PM dibucaine-HCl in 30 PM bR solution. The wavelength of excitation is 266 nm and of detection is 340-420 nm.

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anesthetic, dibucaine-HCl, is residing near or at the chromophore due to fluorescence quenching of the retinal Schiff base of bR. The partition of neutral dibucaine (D) in the hydrophobic region of micellar solutions and charged dibucaine (DH+) in the hydrophilic region of extramicellar phase is shown to be concentration dependent. At low concentrations, D is the dominant species situated inside the micellar environment. When the hydrophobic micellar sites are saturated at a high dibucaine concentration, DH+ is then seen in the aqueous phase. The saturation concentration of dibucaine in micellar solutions is approximately 1 x 10v5 M, which corresponds well with the con~ntration needed to exert narcotic action 1361. Moreover, the observed deprotonation of dibucaine’HC1 in a hydrophobic media (lipid or protein domain) should offer some insight into the mechanism of transporting the protonated form of dibucaine across membranes.

Acknowledgments

Financial support from the Illinois Department of Commerce and Community Affairs, Northern Illinois University Graduate School and College of Liberal Arts and Sciences is acknowledged. M. A. El-Sayed and H. C. Bitting wish to thank the Department of Energy (Office of Basic Energy Sciences; Grant No. DE-FG0388ER13828) for financial support.

References

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