Colloids and Surfaces B: Biointerfaces 13 (1999) 213–218

Requirement of water for anesthetic action: purplemembrane

Akira Shibata a,*, Junko Yoshida a, Hideyuki Ikema a, Kenji Fukuzawa a,Issaku Ueda b,1

a Faculty of Pharmaceutical Sciences, The Uni6ersity of Tokushima, Shomachi, Tokushima 770-8505, Japanb Anesthesia 112A, Department of Veterans Affairs, Medical Center, and Uni6ersity of Utah, School of Medicine, Salt Lake City,

UT 84148, USA

Received 21 December 1998; accepted 27 January 1999

Abstract

Bacteriorhodopsin (bR) in the purple membrane of Halobacterium salinarium is a retinal-containing protein. Thischromophore changes color according to the state of the protein. The dark-adapted membranes, suspended in water,show the absorbance peak (lmax) at 560 nm. Clinical concentrations of volatile anesthetics, halothane, and enflurane,reversibly shifted lmax to 480 nm. When dried membranes were dispersed water-free liquid anesthetics, these spectralchanges were not observed. Addition of water shifted lmax to 480 nm. The [H2O/bR] mole ratio that was necessaryto induce the full spectral change was about 3000 for halothane and 20 000 for enflurane. Addition of water woulddecrease the interfacial energy between the hydrophilic purple membrane and the hydrophobic anesthetics. Thewater-induced interaction between purple membrane and anesthetics is attributable to the positive spreadingcoefficient of water at the anesthetic/membrane interface, Swater(anes/memb), expressed as

Swater(anes/memb)=gmemb/anes−gmemb/water−gwater/anes

where gmemb/anes, gmemb/water, and gwater/anes represent in the interfacial tension between membrane/anesthetic, mem-brane/water, and water/anesthetic phases, respectively. The surface energy of water is 72.8 mJ m−2 (20°C) andconsists of polarity (50.8 mJ m−2), and dispersibility (22.0 mJ m−2). The polarity of water contributes to theinteraction with the polar surface of purple membrane, and the dispersibility contributes to the interaction withhydrophobic anesthetics. The amphiphilicity of water molecules is the principal factor to decrease the interfacialenergy between membrane and anesthetic molecules. The present result shows that anesthetic interaction withbiological phase requires water. © 1999 Elsevier Science B.V. All rights reserved.

Keywords: Anesthetic action; Purple membrane; Water; Interfacial energy

* Corresponding author. Tel.: +1-886-33-7286; fax: +1-886-33-950.E-mail address: ashibata@ph.tokushima-u.ac.jp (A. Shibata)1 This author is also the corresponding author.

0927-7765/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved.

PII: S0927 -7765 (99 )00007 -7

A. Shibata et al. / Colloids and Surfaces B: Biointerfaces 13 (1999) 213–218214

1. Introduction

Effects of anesthetics on ion translocation atmembrane channels have been the target of recentanesthesia research. The transmission of electricsignal generated by visual pigment, rhodopsin, inthe photoreceptor is interesting in connection withthe nerve system. Bacteriorhodopsin (bR) in pur-ple membrane of Halobacterium salinarium func-tions as a right-driven proton-pump, which useslight energy to translocate protons across themembrane and thereby generates a substantialelectrochemical gradient. BR consists of a mem-brane-spanning protein, bacterioopsin and all-trans retinal, a chromophore [1–4] which isattached to lysine 216 through a Schiff base link-age. The secondary structure of the protein ismainly a-helical, with seven transmembrane seg-ments passing through the lipid bilayer of themembrane. The retinal chromophore is central tothe proton pumping activity of the protein. Lightinduced isomerization of the retinal from an all-trans to a 13-cis configuration drives the translo-cation of protons across the membrane. Lightabsorption and storage by bR gives rise to amultistep reaction cycle along which differentspectroscopic intermediates appears: Br�hv�L550 = M412 = N520 = O640�bR [5]. In theL�M reaction, the protonated Schiff base under-goes deprotonation, which is accompanied byprotonation of Asp-85, a residue which communi-cates with the extracellular side of the membrane.Protein structural changes may also occur duringthe photocycle [6]. The spectral behaviors of bRin purple membrane is generally governed by twomain factors: the state of the protonation of thechromophore and the retinal–protein interac-tions. When Schiff base is formed between theall-trans retinal and the protein, max of the chro-mophore shifts from 360 to 560 nm in the dark-adapted state [7–9], and protonation of the Schiffbase can account for part of the shift up to 440nm. Therefore, the retinal–protein interactionsare fine tuners of chromophore absorption andtheir perturbation can shift absorption maximumtowards its blue end in the alkaline pink bR, ortowards the red end of the spectrum, as in thecase of acid blue or deionized blue bRs [10].

Among blue-shifted bRs, with respect to the na-tive one, there exists a series of perturbed bRsabsorbing maximally near 480 nm, which can beobtained by addition of volatile anesthetics [11–15]. The effect of anesthetics upon the structure orconformational stability of the fully hydrated pur-ple membrane is the sum of several differing andcompeting interactions, such as the membrane–water, the membrane–anesthetic, and anesthetic–water interactions. The molecular process of thephoto-conversions in bR closely relates to hydra-tion of the purple membrane [16–19]. Water inthe binding site plays a major role in controllingpKa of the protonated Schiff base and its counte-rions [17]. This study deals with the controllingmechanisms of anesthetic interaction with purplemembranes. Our results show that the interfacialwater is absolutely required for anesthetic–bio-logical membrane interaction.

2. Experimental

The purple membrane fragments with diameterof :550 nm, isolated from Halobacterium sali-narium strain S9, were suspended in distilled wa-ter, and then freeze-dried. The dried membranefragments were kept frozen at −20°C. The de-frosted membranes were dispersed in 50 mMTris–HCl buffer pH 7.4 at 10.5 mM. The bRconcentration was determined by using molar ab-soptivity coefficient of 62 700 M−1 cm−1 at 560nm for the dark-adapted form. For dispersion inliquid anesthetics, halothane (2-bromo, 2-chloro-1,1,1-trifluoro ethane, Ayerst, Philadelphia, PA)or enflurane (2-chloro-1,1,2-trifluoroethyl difl-uoromethyl ether, Anaquest, Madison, WI), thefrozen membranes were defrosted under nitrogengas at relative humidity of 50–70%, which wasmonitored by a hygrometer because of the impor-tance of the presence of a trace of water for themembrane functional states [16–19]. The ab-sorbance spectra were obtained by a JASCO V-550 spectrophotometer (Tokyo, Japan) in a 1.0cm light-path cuvette with a tight-fitting Tefloncap. The measurements were carried out undercontinuous stirring of the sample solution. Thezeta potential of purple membrane fragments were

A. Shibata et al. / Colloids and Surfaces B: Biointerfaces 13 (1999) 213–218 215

Fig. 1. Absorption spectra of purple membrane dispersed in water. The anesthetic concentrations are: (A) Halothane, 1: control; 2:3.0 mM; 3: 7.5 mM; 4: 10.0 mM; and 5: 15.0 mM. (B) Enflurane, 1: control; 2: 7.0 mM; 3: 11.0 mM; and 4: 17.0 mM. Thedark-adapted peak at 560 nm decreased by the addition of anesthetics dose dependently, with appearance of 480 nm peak.Halothane 15.0 mM induced irreversible change with appearance of 380 nm peak.

measured by a Laser-Zee Model 500 zeta poten-tialmeter (Pen Kem, New York).

3. Results and discussion

The responses of the purple membrane in thebuffer solution to halothane (A) and enflurane (B)are shown in Fig. 1. At the dark-adapted state,addition of anesthetics decreased the intensity ofthe 560 nm band with appearance of the 480 nmband in agreement with previous reports [11–14].The shift from 560 to 480 nm was reversible byeliminating the anesthetics by evaporation. Theblue-shift by the saturating concentration ofenflurane was 480 nm (Fig. 1B). Halothane fur-ther shifted the 480 nm band to 380 nm when itsconcentration exceeded 10 mM (Fig. 1A). Thelmax at 380 nm signifies unbound retinal. Thischange was irreversible. The spectral behaviors ofthe bR in the purple membranes are dominatedby the degree of protonation of the Schiff baseand the retinal–protein interactions. The specialorganization of charged residues around the reti-nal is very sensitive to protein structural modifica-tion [13]. Therefore, the spectral change of bRinduced by anesthetics is considered to be due tothe modification of retinal–protein interactions.There are no data whether anesthetics act directlyat the chromophore vicinity.

Fig. 2 shows the changes in the absorbance ofpurple membrane at 560, 480, and 380 nm bandsas a function of halothane concentration. Theabsorbance and the spectral shift of purple mem-brane in the presence of anesthetics are consideredas a reliable indication of the interaction betweenanesthetic and biological membranes.

At 480 nm, the Schiff base linkage is still in theprotonated form with strained all-trans retinal[4,7]. Because the Schiff base linkage is intact, theblue shift from 560–480 nm by anesthetic is at-tributable to a change in the higher order struc-

Fig. 2. Effects of halothane eoneentrations on the absorbanceof purple membrane dispersed in water. Symbols: open circles,560 nm; closed circles, 480 nm; and half-closed circles, 380 nm.

A. Shibata et al. / Colloids and Surfaces B: Biointerfaces 13 (1999) 213–218216

Fig. 3. Absorption spectra of purple membrane dispersed in liquid anesthetics. The dried membrane fragments were suspended inliquid anestheties. The added water concentrations are: (A) halothane, 1: control; 2: 0.029%; 3: 0.043%; and 4: 0.051% (v/v). (B)Enflurane, water concentrations are: 1: control; 2: 0.10%; 3: 0.25%; and 4: 0.43% (v/v).

ture of the protein with weakening of the retinal–protein interaction. Deprotonation of the Schiffbase shifts lmax to 380 nm.

When the dried purple membrane was dispersedin liquid anesthetics, halothane, and enflurane, inthe absence of water, these spectral changes didnot occur. It has been reported that dehydrationof the purple membrane fragments did not affectthe hydrogen-bond network among the helix rods[16]. These hydrogen bondings stabilize the a-heli-cal conformation.

Fig. 3 shows the dark-adapted spectra of driedpurple membrane dispersed in halothane (A) andenflurane (B). In these liquid anesthetics, lmax was556 nm, only slightly blue shifted from the controlpurple membrane dispersed in water. The slightshift is attributable to the dielectric effect of hy-drophobic anesthetic molecules. When anestheticswere removed by vacuum evaporation, and thendispersed in water, the dark-adapted lmax resumedto the control value of 560 nm.

Addition of small amount of water to the pur-ple membrane dispersion in liquid anesthetics in-duced large spectral changes. In liquid halothane,addition of B0.05%(v/v) water decreased the 556nm band intensity and shifted the band to 480nm. This change was reversible. Further additionof water in halothane was accompanied with ap-pearance of the 380 nm band. The mole ratiobetween the added water and bR, [H2O]/[bR], was2.7×103, or the added amount of water is about

3000-fold in excess of bR. In liquid enflurane, theamount of added water was B0.43%(v/v). The[H2O]/[bR] mole ratio was 2.3×104 and theadded amount of water was about 20 000-fold inexcess of bR. In enflurane, 380 nm band did notemerge.

Fig. 4 shows the change in absorbance of thepurple membrane dispersed in liquid halothane at560, 480, and 380 nm bands as a function ofwater concentration. The spectral shift from thenative 560–480 nm was similar to the aqueoussolution of purple membrane, and the anesthetics

Fig. 4. Effects of water concentrations on the absorbance ofpurple membrane dispersed in liquid halothane. Symbols:open circles, 560 nm; closed circles, 480 nm; and half-closedcircles, 380 nm.

A. Shibata et al. / Colloids and Surfaces B: Biointerfaces 13 (1999) 213–218 217

were added later (Fig. 2). The great excess ofwater over bR to induce spectral changes suggeststhat the added water molecules form interfaciallayers between the purple membrane and liquidanesthetics.

The lack of interaction between the purplemembrane and liquid anesthetics in the absence ofwater may be related to the surface properties ofthe purple membrane fragments with large specificsurface area (:12 000 m2). We found the zetapotential of the purple membrane, dispersed in 50mM Tris–HCl buffer pH 7.4 at 22°C was −43mV and hydrophilic. Taneva et al. [13] also men-tioned strong hydrophilicity of the purple mem-branes. Therefore, the lack of interaction indicatesthat the interfacial energy is large between thehydrophilic surface of the purple membrane andhydrophobic anesthetics. In other words, anes-thetic molecules can not spread on the membranesurface. The spreading coefficient of anestheticsover the surface of membrane, Sanes/memb, is writ-ten [20],

Sanes/memb=gmemb−gmemb/anes−ganes (1)

where gmemb and ganes are surface tension of themembrane and anesthetic phases, and gmemb/anes isthe interfacial tension between the two phases.The spreading coefficient is the difference betweenthe work of adhesion between the membrane andanesthetics, Wa(memb,anes) and the work of co-hesion of anesthetics, Wc(anes).

Sanes/memb=Wa(memb,anes)−Wc(anes) (2)

where

Wa(memb,anes)

=gmemb+ganes−gmemb/anes and Wc(anes)=2ganes

(3)

The sign of the spreading coefficient deter-mines the interaction. The sign has to be positive(Sanes/memb\0) for liquid anesthetics to spread onthe purple membrane surface. The inability ofanesthetics to induce spectral change indicatesthat the spreading coefficient is negative, andgmemb−ganesBgmemb/anes. Anesthetics do notspread on the purple membrane surface.

Addition of water to this system blue-shiftedlmax depending on the water concentration (Fig.3). The added water molecules are distributed tothe hydrophilic/hydrophobic interface betweenthe purple membrane and the liquid anesthetic.This would decrease the interfacial energy andincrease the wettability of the purple mem-brane by anesthetics. The spreading coefficientof water at the anesthetic/membrane interface,Swater(anes/memb), is written

Swater(anes/memb)=gmemb/anes−gmemb/water−gwater/anes

(4)

where gmemb/anes, gmemb/water, and gwater/anes repre-sent the interfacial tension between membrane/anesthetic, membrane/water, and water/anestheticphases, respectively.

The spreading coefficient, Swater(anes/memb) is thedifference between the work of adhesion of waterto the purple membrane, Wa(memb,water)anes,and the work of cohesion of water in the liquidanesthetic phase, Wc(water)anes. It is also the dif-ference between the work of adhesion of theanesthetic to water, Wa(water,anes)memb, and thework of cohesion of water in the membranephase, Wc(water)memb.

Swater(anes/memb)=Wa(memb,water)anes

−Wc(water)anes

=Wa(water,anes)memb

−Wc(water)memb (5)

The present result (Fig. 3) shows the spreadingcoefficient, Swater(anes/memb), is positive only in thepresence of excess water. The works of adhesionof both membrane-water and anesthetic-water arelarger than the work of cohesion of water.

Therefore, we will establish that Swater(anes/

memb)\0. This proposition will be tested by thefollowing equation. From Eq. 4

gmemb/anes\gmemb/water+gwater/anes (6)

The following experiment was carried out in thethree component system of anesthetic/purplemembrane/water to examine the proposition.

The wettability of a membrane by a liquid isgoverned by the relative magnitudes of the inter-

A. Shibata et al. / Colloids and Surfaces B: Biointerfaces 13 (1999) 213–218218

molecular forces between the membrane and liq-uid. The purple membrane fragments transferredcompletely into the aqueous phase (data notshown). The purple membrane fragments totallytransferred into the water phase. Water has amuch stronger affinity to the purple membranecompared to the liquid anesthetic. The resultshows that water molecules wet the purple mem-brane at the membrane/anesthetic interface. Thecause of the blue-shift of the absorbance of thepurple membrane by the addition of smallamount of water (Fig. 3) is the decrease of theinterfacial energy between membrane and anes-thetic molecules.

The surface energy of water is 72.8 mJ m−2

(20°C), and consists of polarity (50.8 mJ m−2)and dispersibility (22.0 mJ m−2) [21]. The polarityof water contributes to the interaction with thepolar surface of purple membrane, and the dis-persibility contributes to the interaction with hy-drophobic anestehtic. The amphiphilicity of watermolecules is the principal factor to decrease theinterfacial energy between membrane and anes-thetic molecules and promotes the interaction be-tween anesthetics and the purple membrane. Thepresent result shows that anesthetic binding tobiological phase requires presence of water.

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