Anest het ic-Protei n Interact ion : Effects of Volatile Anesthetics on the Secondary Structure of Poly(L4ysine)

AKIRA SHIBATA', KlYO MORITA*, TAKUYA YAMASHITA*, HIROSHI b M A Y A * , AND ISSAKU UEDASX

Received July 12, 1990, from the 'Faculty of Pharmaceutical Sciences, Tokushima Universify, Shomachi, Tokushima 770, Japan, and *Anesthesia Service, VA Medical Center, Salt Lake Cify, UT 84748. Accepted for publication January 14, 1991.

Abstract 0 Effects of volatile anesthetics (chloroform, halothane, and enflurane) on the secondary structure of poly(L4ysine) were analyzed by circular dichroism (CD). The relative proportions among a-helix, p-sheet, and random-coil conformations were calculated by the curve-fitting method on the CD data. Volatile anesthetics partially transformed a-helix to p-sheet but not to random-coil under the present experimental condition. When expressed by the anesthetic partial pressures in the gas phase in equilibrium with the solution, the values that partially trans- formed a to p conformation by 10% were 1.1 . lo-', 4.7. lo-', and 7.9 1 1 O-' atm for chloroform, halothane, and enflurane, respectively. The order of potency is in reasonable agreement with the order of the anesthetic potencies of the agents. The a-to-p transition was completely reversible when anesthetics were purged by nitrogen gas. Volatile anesthetics disrupted the hydrogen bonds of a-helix backbones and rearranged them to form the p-sheet Conformation. The p-sheet confor- mation is stabilized mainly by the hydrophobic interaction among methylene side groups of poly(L4ysine). Volatile anesthetics promoted the transition by enhancing the hydrophobic interaction among side- chains and by rearranging the hydrogen bonds in the peptide backbone.

It has been a matter of controversy whether lipids or proteins are the primary action site of volatile anesthetics. The fluidizing and disordering effect of volatile anesthetics on lipid membranes is well recognized.'" The membrane disor- der leads to the lateral expansion of the membrane and the resulting increased lateral pressure is generally assumed to collapse the electrogenic ion channels and induce anesthesia. Despite the abundance of reports on the anesthetic-lipid interaction, those on the anesthetic-protein interaction are few. The anesthetic effects on proteins include demonstra- tions of the direct inhibitory effects of anesthetics on various water-soluble enzymes. The light-emitting enzymes, lu- ciferases, solubilized from firefly lanterns- and from lumi- nescent bacteriag.10 are inhibited by anesthetics at subclinical concentrations. Lipid proponents conceive anesthesia as non- specific interaction, while protein proponents tend to favor the specific receptor concept.

We5.11 contend that anesthetics affect lipid membranes and proteins indiscriminately, and all macromolecular structures are disordered. The action is nonspecific, in the sense that no lock-and-key relationship exists for the anesthetic binding. The recent clarification of the secondary structure of channel proteins showed that the transmembrane portion of these proteins is in the a-helix conformation.12-14 It has been postulated that the assembly of the a-helix domains forms the ion pore by exposing the hydrophilic side of the helical structure to the channel surface. It follows that the conser- vation of the a-helix conformation is imperative for the channel function.

In water, poly(L-lysine) assumes well-defined a-helix, @sheet, or random-coil conformations according to the exter- nal conditions. The polypeptide has been used successfully as a model for protein structures.15.16 The present study exam- ined the anesthetic effects on the secondary structure of

poly(L-lysine) by circular dichroism (CD). We set the initial conformation of poly(L-lysine) at a-helix and studied how this peptide model responds to volatile anesthetics. The relative proportions among a-helix, @sheet, and random-coil confor- mations were estimated by the curve-fitting method of the CD spectra.

Experimental Section Poly(L-lysine) hydrobromide (MW 32 000) was obtained from

Sigma (St. Louis, MO) and dialyzed against 0.01 M HCl to replace the bromide ion with chloride ion. The preparation was further dialyzed against water to remove excess salt. The Br--Cl- exchange was necessary to avoid high molar absorptivity of Br- in the UV region.

Chloroform (containing ethanol 0.6%) was obtained from Nakarai Chemical (Osaka, Japan). Halothane (2-bromo-2-chloro-l,l,l- trifluoroethane), containing 0.01% (vlv) thymol, and enflurane (2- chloro-1,1,2-trifluoro difluoromethylether) were gifts from Takeda (Osaka, Japan) and Dainabot (Tokyo, Japan), respectively. Anesthet- ics were treated by an activated aluminum oxide column to remove water. The stabilizer thymol, contained in halothane, was removed by this procedure. Anesthetics were added to the poly(L-lysine) solution in a 5-mL glass container by a microsyringe. To ensure the a-helix conformation, the solution pH was adjusted to 11.4 by NaOH. The poly(L-lysine) concentration was 2.75 * M when expressed by the L-lysine residue. The concentration of poly(L-lysine) was confirmed by colloid titration17 and by micro-Kjeldahl nitrogen analysis. The container was tightly capped and vigorously mixed for 2 h.

Circular dichroism was measured by a JASCO 5-600 spectropola- rimeter (Tokyo) at 22.0 2 1.O"C under continuous purge with nitrogen. A Teflon-stoppered air-tight cuvette with a light-path length of 1.0 mm was used for all determinations.

Observed ellipticity (Oobs) was converted to mean residue ellipticity ([el) by the following equation:

where C is the molarity of the poly(L-lysine) residue and L is the cuvette light path length in centimeters. When the structure of poly(L-lysine) in aqueous solution is a mixture of a-helix, psheet, and random-coil, the mean residue ellipticity is described as a sum of the contributions from each structure as follows:17-26

where [el is the experimentally obtained mean residue ellipticity and [el,, [elp, and [el, are the mean residue ellipticities of the reference spectra of a-helix, p-sheet, and random-coil conformations, respec- tively, f is the relative proportion, and Xf i = 1.

Results In the wavelength region 190-250 nm, the CD spectrum of

the polypeptide in the a-helix structure is resolved into three Gaussian bands a t -190, 206, and 222 nm. These bands are assigned to r-72CL, n--72Cll and n-72C transitions, respectively. From the frequency, shapes, and intensities of the CD spectra in this wavelength region, the proportion of a-helix, p-sheet,

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and random-coil conformations in the poly(L-lysine) confor- mation in aqueous solution can be estimated.26

Figure 1 shows the CD spectra of a-helix, psheet, and random-coil conformations of poly(L-lysine). The spectrum of a-helix was determined at a concentration of 2.75 * lov4 M poly(L-lysine) residue at pH 11.4. This sample was heated to 52 "C for 15 min, then cooled to 22 "C to obtain the spectrum for the psheet.18@ The spectrum of the random-coil was obtained at pH 5.7.

Both Psheet and random-coil showed low ellipticity at 206 nm, with an equal value of --4000 deg - cm2 - dmol-' (Fig- ure 1). In contrast, the a-helix structure showed a minimum (maximum negative ellipticity band) at this wavelength,ls --32 000 deg - cm2 dmol-'. The three spectra in this figure were used to resolve the CD spectra of poly(L4ysine) into each conformation in the presence of anesthetics.

Figures 2-4 show the effects of chloroform, halothane, and enflurane on the CD spectra of poly(L-lysine) in the a-helix conformation. For comparison, the control CD spectra of a-helix and Psheet in the absence of anesthetics are included in these figures. These figures show that the addition of anesthetics decreased the absolute value of the negative ellipticity bands of the T-# and n-?r* transitions. The de- crease in the intensities of the negative ellipticity bands at 206 and 222 nm was pronounced at high concentrations of chloroform. The decrease was accompanied by the appearance of a new broad minimum at 218 nm. These data suggest that the anesthetics decreased the proportion of the a-helix struc- ture.

Next, we calculated the relative proportion of the secondary structure of poly(L-lysine) according to the curve-fitting meth- od.2&26 Figure 5 shows the comparison between the experi- mentally obtained and computer-generated spectra in the presence of 1.7 * M halothane and 8.2 * lOP3M enflurane according to the curve-fitting procedure. The experimental spectra are almost identical with the computer-generated data points obtained between 195 and 250 nm, with a 1-nm interval. These figures show 73% a-helix and 27% &sheet for halothane and 87% a-helix and 13% psheet for enflurane. The relative proportion of the random-coil was null with all anesthetics in the present experimental conditions.

6 n

I - .-

E o 4 U

CUE u 2

lo 0

0 a, U

P

r X v

L I I I I I I

190 210 230 250

WAVELENGTH (nm) Flgure 1-Typical CD spectra of poly(L4ysine) in aqueous solution (2.75 . M L-lysine residue): (1) random-coil; (2) p-sheet; and (3) a-helix.

n

I - l-

; U

E cv 0 0 a, -0

P

0 I

X F

v

7 s

190 210 230 250

WAVELENGTH (nm) Flgure 2-The CD spectra of poly(L4ysine) with 3.7. lo-' M (trace 2) and 5.9. M (trace 3) chloroform. The spectra of the p-sheet (1) and a-helix (4) are shown as references.

F

'O X Om I

U I \

190 210 230 250

WAVELENGTH (nm) Figure &-Same as Figure 2, with 2.7 - lo-' M halothane (trace 2). The spectra of the p-sheet (1) and a-helix (3) forms are shown as references.

In Figure 6, the dose-dependent a-to-p relative proportions of the secondary structure of polyblysine) for chloroform, halothane, and enflurane are shown. The anesthetics trans- formed the a-helix into the psheet dose dependently. The anesthetic concentrations that transformed a-helix to 10% psheet were 3.63, 2.63, and 5.62 mM for chloroform, halothane, and enflurane, respectively.

Discussion Before discussing the anesthetic effect on the a-to-p tran-

sition of poly(L-lysine), a short discussion on the anesthetic concentration is in order. Because inhalation anesthetics are given as a vapor, the aqueous concentration for clinical anesthesia is not well established. The clinical dose is ex- pressed as the minimum alveolar concentrations (MAC) of

1038 I Journal of Pharmaceutical Sciences Vol. 80, No. 11, November 1991

I I I I I 1 J

190 210 230 250

WAVELENGTH (nm) Flgure &Same as Figure 2, with 8.2 * M enflurane (trace 2). The spectra of the p-sheet (1) and a-helix (3) forms are shown as references.

100-

90

n 8 v

80- c1

70

n

I - F

E" -0

E hl

0

-

-

I I

E O,,

- 5 ' ' I I I

200 220 240 WAVELENGTH (nm)

Flgure Calculated (dots) and experimentally obtained (continuous line) CD spectra of poly(L4ysine) with (A) 1.7 - lo-' M halothane and (B) 8.2 M enflurane (halothane: [el = 0.73[01, + 0.27[4, + 0[0],; enflurane: [el = 0.87[13J, + 0.13[4, + O[OJ,).

anesthetics that induce surgical stage anesthesia in 50% of the population. The MAC values27 for do s are reported to be

and 2.2.10-' atm for enflurane when expressed by the partial pressure. By using the water-gas partition coeffi- cients,28 the present data for the 10% a-to+ change translate approximately into MAC values of 1.1 lop2, 4.7 * lop2, and 7.9 * atm for chloroform, halothane, and enflurane, respectively. The order is in reasonable agreement with the order of the clinical anesthetic potency of these agents.

The well-established correlation between the anesthetic potency and oil-water partition coefficients gives an impres- sion that the action site of anesthetics is apolar, such as the lipid membrane interior. Nevertheless, the original demon- strations by Overton29 and Meyer30 were performed not with

0.77 * lo-' atm for chloroform, 0.87 * 10- 5 atm for halothane,

1 10

CONCENTRATION (mM) Flgure &Relative proportions of a-helix and p-sheet forms for poly(~- lysine) as a function of the concentration of anesthetics. Key: (0) enfiurane; (A) chloroform; (0) halothane.

apolar hydrocarbons, but with polar olive oil. Hansch and co-workers31 showed in their work on QSAR (quantitative structure-activity relationship) that apolar oils are not suit- able for representing the action sites. They have shown that octanol, which is a weakly polar organic solvent, is much superior to apolar hydrocarbons in representing the property of the site of action of anesthetics. This suggests that the action site is not the apolar core, but is inclined to be the hydrophilic surface of macromolecules.

All inhalation anesthetics presently in clinical use are weakly polar molecules. These anesthetics are designed by replacing hydrogen atoms in alkanes and ethers by halogens, to make the molecules to be nonexplosive, but deliberately retaining part of the hydrogen atoms to make the molecule weakly polar.32 It has been known that weakly polar mole- cules show stronger anesthetic potency compared with their apolar counterparts.32 Because of their polarity, these mole- cules are amphipathic and tend to localize at the water- macromolecule interface.

The localization of inhalation anesthetics a t the macromol- ecule-water interface was demonstrated by Kaneshina et a1.33 by 'H NMR and by Yoshida et a1.34 by 'H and 19F NMR. From the analyses of the chemical shift in the frequency domain, they showed that the hydrophobic ends of the inha- lation anesthetics are solubilized into the lipid core of the surfactant micelles, but their hydrophilic ends did not lose contact with water. By two-dimensional nuclear Overhauser effect 'H NMR, Yokono et a1.35 demonstrated that only the hydrophobic methoxy end of an inhalation anesthetic, meth- oxyflurane, formed a cross-peak with the hydrophilic choline head of dipalmitoylphosphatidylcholine vesicle membranes. No other cross-peaks were observed. The hydrophilic end of this anesthetic stayed in the aqueous phase. This does not mean that apolar molecules are not anesthetic. Apolar mol- ecules penetrate into the hydrophobic core of lipid mem- branes, expand the membrane laterally, decrease the surface charge density, and weaken the water-membrane interac- tion.

The structural integrity of lipid membranes and proteins in water is maintained by their interaction with water. Lipid bilayer membranes cannot be formed without water. Among

Journal of Pharmaceutical Sciences I 1039 Vol. SO, No. 7 1, November 1991

a milliard of possible conformations of proteins, only a single structure comes up when dissolved in water. We5.11 proposed that anesthetics break the water structure in the hydration shell of macromolecules. For this reason, anesthetics are expected to ehange the conformation of proteins, as well as lipid membranes.

From the large increases in the entropy of the firefly luciferase by inhalation anesthetiq7 Eyring et a1.s proposed that these anesthetics broke the hydrogen bonds among water molecules clystered at the protein surfaces. They estimated that -50 water .molecules are released from the luciferase surface by clinical concentrations of inhalation anesthetics.

The anesthetic action on hindering formation of hydrogen bonds has been demonstrated by Sandorfy and co-work- ers37-40 with IR spectroscopy. They3'3-40 have shown that anesthetics disrupt >N-H-.O=C<, N-H--N<, O-H--N<, and 0-H.-0-H hydrogen bonds by forming competitive proton donor-acceptor complex. For instance, H,O-X hydrogen bonds may be replaced by anesthetic-X hydrogen bonds. The importance of hydrogen bonds in the anesthesia mechanisms has also been proposed by Sax and Pletchefil for local anesthesia and by Klemm42 for alcohol intoxication.

The present study showed that anesthetics partially trans- formed the a-helix structure of polyblysine) into the psheet conformation (Figures 2-51. The intramolecular hydrogen bond in a-helical poly(L-lysine) is the >C=O*-H-N< type, where the >C=O moiety is a strong proton acceptor and the H-N< moiety is a relatively weak proton donor. At a first glance, the present data appear to suggest that the anesthet- ics broke the intramolecular hydrogen bonds of the peptide backbone by forming competitive hydrogen bonds between the anesthetics and the peptide bonds. These anesthetics are able to form hydrogen bonds in competition with the peptide bonds. The following two schemes are possible for disruption of the hydrogen bonds.

>C=O-H-N< (a-helix) + A

(3) f>C=O.-A (random coil) + H-N< \A-.H-N< (random coil) + >C=O

where A designates anesthetics. If these exchanges occur, the intra- and intermolecular hydrogen bonds in the peptide backbone must disappear aRer anesthetic interaction, and poly(L-lysine) should be transformed into the random-coil form. In the CD spectra, these interactions should accompany the changes in the peak intensities of 195 nm (random-coil), together with 206 and 222 nm (a-helix). Yet, the data showed a transition to 218 nm (psheet). The change was a-helix to psheet, and not to random-coil (Figure 5) . The reaction sequence should be:

>C=O--H-N< (a-helix) + A

Both a-helix and psheet are supported by the hydrogen bonds among the peptide backbone, and the a-helix to /%sheet transition occurs by changing the hydrogen bond partners within the peptide backbone. The competitive formation of hydrogen bonds between anesthetics and the peptide bonds may not be the cause of this a-to-p transition.

In general, the thermodynamic stability of the psheet is supported by the hydrophobic interaction (bonding) among the hydrocarbon side-chains of the component amino acids.16 With polyb-lysine), the four methylene groups associate

together to form the psheet. These hydrophobic contributions of side-chains for conformational stability are almost negli- gible in the a-helix structure.15

The a-helix structure of poly(L-lysine) transforms into the psheet form by heating. In this context, anesthetic action may be viewed as equivalent to adding heat to the system. The heating process may supply enough energy to disrupt hydro- gen bonds in the helical backbone to rearrange the hydrogen bonds into psheet, and enough enthalpy to liberate the methylene side groups of poly(L-lysine) from water to link together (hydrophobic bonding). The equivalence between anesthetica and addition of heat has been demonstrated in the membrane fluidization phenomenon of anesthetics, where both heat and addition of anesthetics increased the fluidity. With protein, we7 have shown that anesthetic interaction with solubilized firefly luciferase was accompanied by large increase in entropy change (AS'), which is equivalent to the addition of heat.

The question remains whether the anesthetic-induced @sheet was maintained by the intra- or intermolecular hydrogen bonds. Experimentally, this question can be re- solved by the dependency of the CD spectra on poly(L-lysine) concentrations. The psheet, formed by intermolecular hydro- gen bonds, is characterized by a decrease (upward shift) in the absolute value of the negative ellipticity bands in 200- 225-nm region when the polyb-lysine) concentration is di- luted. In the present study, its dependency on peptide con- centration was not observed (not shown). In addition, the ratio of the a-to-p transformation, induced by anesthetics, was moderate and -70% remained as a-helix even above the clinical concentrations (Figure 6). These results suggest that the anesthetic-induced transformation was caused by the exchange in the intramolecular hydrogen-bonding partners and by the increased hydrophobicity around the poly(L-lysine) side-chains.

The anesthetic dehydration of macromolecular surfaces has been shown by a series of IR studies on the hydrogen bonds by Sandorfy and CO-workers.3740 We434 have also shown by Fourier transform IR spectroscopy of phospholipid mem- branes that anesthetics increased the intensity of the free water 0-H stretching and bending bands and shifted the P=O stretching band to lower frequencies. Anesthetics released the hydrogen-bonded water molecules from the phosphate moiety by a competitive mechanism. Due to the anesthetic binding, the vibration of the P=O stretching became slower. Differential scanning calorimetry in partially hydrated phospholipid membranes showed that anesthetics decreased the number of surface-bound water molecules that freeze at subzero temperature and increased the number of free water molecules that freeze at 0 "C.46 High-precision solution densimetry of aqueous solutions of crystalline bovine serum albumin47 and poly(~-lysine)4* showed that anesthetics increased partial molal volume of the protein and polypeptide, which is attributable mainly to destruction of the hydrogen bonds among the water molecules electrostricted at the surface charges. Because the structure of the electrostricted water molecules is maximally compressed by the electrostatic field, the destruction of the structure results in the expansion of the system volume. The cloud point of nonionic surfactant micelles was decreased by the addition of volatile anesthetics, which indicates loss of hydrogen-bonded water molecules from the micellar hydrophilic moiety.

These interfacial dehydrating effects of anesthetics must have assisted the interaction among the hydrophobic side- chains to form the /%sheet conformation. The dehydration constitutes the molecular basis of anesthesia mechanisms.

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Trudell, J. R. In Molecular Mechanisms of Anesthesia. Pro ress in Anesthesiology, Vol. 2; Fink, B. R., Ed.; Raven: New fork , 1980; pp 261-270. Miller, K. W.; Roth, S. H. In Molecular and Cellular Mechanisms of Anesthetics; Roth, S. H.; Miller, K. W., Eds.; Plenum: New York, 1986; pp 261-266. Simon, S. A.; McIntosh, T. J.; HInes, M. L. In Molecular and Cellular Mechanisms ofdnesthetics; Roth, S . H.; Miller, K. W., Eds.; Plenum: New York, 1986; pp 297-308. Ueda, I.; Kamaya, H. Anesth. Anulg. 1984, 63, 929-945. Ueda, 1. Anesthesiology 1965,26,603-606. Ueda, I.; Kamaya, H. Anesthesiology 1973,41, 425436. Franks, N. P.; Lieb, W. R. Nature 1985,316,349451. Adey, G.; Wardley-Smith, B.; White, D. Life Sci. 1976, 17, 1849-1854. Curry, S.; Lieb, W. R.; Franks, N. P. Biochemistry 1990, 29, 46414652. Kama a H ; Ueda, I.; E ing, H. In Molecular Mechanisms of Anest iesk Progress in zesthesiology, Vol. 2; Fink, B. R., Ed.; Raven: New York, 1980; pp 429-433. Catterall, W. A. Science 1984,223, 653-661. Guy, H. R.; Seetharamulu, P. Proc. Natl. Acad. Sci. U S A . 1986, 83,508-512. Fasman, G. D. Prediction of Protein Structure: Principles of Protein Conformation; Plenum: New York, 1989. Be chok, S. In Poly-a-amino Acids; Fasman, G. D., Ed.; Marcel Deiker: New York, 1967; DD 293336.

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Walton, A.; Blackwell, j.-Biopolymers; Academic: New York, 1973; DD 376406. Terayama, H. J. Polym. Sci. 1952,8,243-253. Greenfield, N.; Fasman, G. D. Biochemistry 1969,8,4108-4116. Saxena, V. P.; Wetlaufer, D. B. Proc. Natl. Acad. Sci. U S A .

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Acknowledgments Supported in part by the VA Medical Research Service and NIH

grants GM25716 and GM27670.

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