Langmuir 1994,10,784-789
784
Synthesis and Physicochemical Study of Hepatitis A Viral-VP1 Protein Peptides I. Marth,f I. Haro,? F. Reig,'p+ and M. A. Alsinat Peptide Department, CSIC-CID, Jordi Girona Salgado 18-26,08034 Barcelona, Spain, and Physicochemical Unit, Faculty Pharmacy, Placa Pius XII sln, 08028 Barcelona, Spain Received May 25,1993. In Final Form: October 2 8 , 1 9 9 P The synthesis, on a solid support of the Hepatitis A viral protein peptide (HAV-VPl(ll-25)), is described. The interaction of the peptide with (HAV-St-YPl(11-25)) and without a stearoyl residue attached to the amino group terminal with phospholipids has been studied, using liposomes and monomolecular layers as biomembrane models. Chan es in the fluidity of the bilayers induced by these peptides are determined by means of the polarizable p r o k s such as 8-anilino-1-naphthalenesdfonicacid (ANS) and 1,g-diphenyl1,3,5-hexatriene (DPH). The integrity of membranes has also been ascertained with carboxyfluorescein (CF). The hydrophobic derivative HAV-St-VPl(11-25) inserts into the bilayers without disruption of the vesicle structure. In this way liposomes can be used as carriers for small immunogenic peptides.
Introduction Synthetic peptides containing one or more epitopes of different *al proteins have been extensively studied as potential immunogens to prepare vaccines. The immunological response is highly dependent on the way the peptide fragment is presented to the T cell by antigenpresenting cells. It has been widely accepted that the conformations and orientations imposed on the peptides by the membrane surroundings meet functionalatructural requirements between than the radnom organization in solution. This assumption appears justified by correlations between membrane binding and biological potency.' Moreover, as in general receptors occupy only a small fraction of the totalcell surface, it is logical to assume that when a peptide approaches a cell the first contact is much more likely to be with the lipid phase than with the receptor. For this reason, the interaction found with a lipid bilayer can be taken as the initial steps in the peptide/ receptor contact. The first step in this interaction is the electrostatic accumulation of the peptide a t the surface followed by membrane binding. In both cases these processes are bound to conformational changes in the peptide and the lipid. The accumulation depends mainly on the peptide concentration, the surface potential of the membrane, the peptide charge, and the temperature. This membrane interaction induces preferred conformations and orientations in the peptide~.~93 With some exceptions most synthetic peptide vaccines described so far need the presence of some type of adjuvant to induce an immune response. One of these approaches can be used of liposome/peptide combinations. As one of the most common criteria applied to select epitopes in protein sequences is hydrophilicity: the affinity of these peptides for lipids is low. In order to increase the binding affinity between peptides and lipids, some hydrophobic peptide derivatives include the tripalmitoyl+ CSIC-CID. t Faculty Pharmacy. e Abstract published in Advance ACS Abetracts, January 1,1994.
(1) Sargent,D. F.; Schwyzer, R. M. Roc. Natl. Acad. Sci. U.S.A. 1986,
83.5174-6178. -. .- -.
cysteinyl r e s i d ~ e .Nevertheless, ~ the easiest and cheapest way is to link directly an alkyl chain to the amino terminal group of the peptide. To this end of VP1 protein fragment was selected according to the hydrophilicity profile and the literature references.6 The sequence corresponding to the 11-25 fragment was synthesized, and in addition an stearoyl residue was attached to a sample of this peptide. Here we report the study of physicochemical interactions between these two peptides and dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidyglycerol (DPPG), determined using liposome fluorescent probes (ANS, DPH, CF) and lipid monomolecular layers of phospholipids.
Experimental Section Chemicals. N-[(9-Fluorenylmethoxy)carbonyl] (N-Fmoc) amino acids and Kieselguhr-supported poly CNJV-dimethylacrylamide)resin with [4-(hydroxymethyl)phenoxy]aceticacid as the handle were purchased from Novabiochem (Cambridge, England). NJV-Dimethylformamide (DMF) and piperidine/DMF (20%) were from Milligen. Washing solventa such ae isopropyl alcohol, acetic acid, and diethyl ether were obtained from Merck (pa). Trifluoroacetic acid (TFA)was supplied by LKB (Ultrosyn Chemicals). Stearic acid was purchased from Merck (biochemistry grade). Coupling reagents (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (Bop) and (benzotriazol-1-y1oxy)tripyrrolidinophosphoniumhexafluorophosphate (pyBop)were obtainedfrom Fluka and Novabiochem, respectively. Amino acid analyses were carried out in a Pico-Tag system (Waters). HPLC was performed in a Perkin-Elmer Series 250 LC pump connected to a LC-235 diode array detector and a LCI-100 integrator. Analytical HPLC was performed on a 250 X 4 mm Spherisorb ODs-2 (10pm) column eluted with mixtures of acetonitrile (0.1% TFA)/H20 (0.1% TFA), at a flow rate of 1 mL/min. ANS, DPH, DPPC, and DPPG were from Sigma, and used without further purification. CF (Sigma)was purified by column chromatography on Sephadex LH-20. Acetate buffer (0.25 M) was prepared with sodium acetate (Sigma) adjusted at pH 7.4 with acetic acid (0.1 M) (Sigma). Phosphate buffer saline (PBS) solution at pH 7.4, conductivity 15.4 mS-cm-l, was prepared.
--I
(2) Schwyzer, R. M. Biochemistry 1986,25,6335-6342. (3) Park, N. G.; Lee, S.; Oishi, 0.; Aoyagi, H.; Wnuaga, S.; Yamashita, 5.;Ohno, M. Biochemietry 1992,31,12241-12247. (4) Hoop, T. P.; Woods,K. R. Roc. Natl. Acad. Sei. U.S.A. 1981, 78, 3824-3828.
(5) WiesmWer, K.-H.;Beseler, W. G.; J u g , G.Int. J . Pept. Protein Res. 1992,40, 255-280. (6) Emini, E. A,; Huges, J. V.; Perlow, D. S.; Boger, J. Induction of HepatitisAviruaneutralizii antibodybyaviruaspecificsynteticpaptide. J. Virol. 1988,55,83&839.
0743-7463/94/2410-0784$04.50/0Q 1994 American Chemical Society
Hepatitis A Viral-VP1 Protein Peptides
Langmuir, Vol. 10,No. 3, 1994 785
Peptide Synthesis. Peptide HAV-VPl(l1-25) was synthesized by solid-phase methodology both on a poly(N,N-dimethylacrylamide) resin (0.1 mequiv/g) in a continuous flow synthesized (Biolynx 4170 LKB) and on an alkoxybenzyl resin (0.8 mequiv/g) in the manual way. Esterification of the f i s t amino acid to the resin-bound linkage agent was carried out using the pentafluorophenyl active ester, the preformed symmetrical anhydride, or the New Castro’s coupling reagent pyBop. Esterification of the preformed anhydride was catalyzed by the presence of 4-(dimethylamino)pyridine (DMAP),the reaction conditions being similar to those found by Atherton et al.7 to minimize racemization. Fmoc amino acidswere used either as pentafluorophenyl esters or as carboxyl-free derivatives mixed with DIPCD-HOBt. Aliquots of peptide/resin were removed during the course of the synthesis, the yield of each coupling being at least 95% according to Kaiser’s test.8 Qualitative analysiswas also provided by monitoring the dibenzofulvene/piperidineadduct at 340 nm. Typical acylation and deprotection traces were obtained which also indicated satisfactory progress in the assembly. Each of the steps involved in the synthetic cycle was controlled by an OlivettiM-240 personal computer. Dry synthetic protected peptide/resin was treated with TFA (95%)in water, in order to cleave the desired peptide. “Acylation with Stearic Acid. Dry peptidehesin (500 mg, 0.05 mequiv) of HAV-VPl(l1-25) was well swollen in DMF for 1h, and the solvent was decanted off. Stearic acid (0.111111101, 28.5 mg) was dissolved in a minimum amount of solvent (1mL of DMF/dichloromethane, 1:l) followed by the addition of Castro’s coupling reagent ( p y B o ~ (0.1 ) ~ mmol, 52 mg). The solutionwas then poured intothe soaked peptide/resin, and DIEA (0.28 mmol, 47 pL) was finally added. The mixture was set aside at room temperature and swirledoccasionally. The reaction was complete within 30 min as judged by the ninhydrin color test. The resin was filtered and washed sequentially with DMF, tertamyl alcohol, acetic acid, tett-amyl alcohol, and diethyl ether and dried in vacuo. Surface Activity Measurements. Compression isotherm were recorded on a Langmuir film balance equipped with a Wilhelmy platinum plate as described by Verger and De Haas.lo The output of the pressure pickup (Sartorius microbalance) was calibrated by recording the well-known isotherm of stearic acid. The Teflon trough (surface area 495 om2,volume 330 mL) and platinum plate were regularly cleaned with hot chromic acid. Films composedof lipids,peptides, or their mixtures (dissolved in chloroform)were spread on aqueous surfacesusing a Hamilton microspinge, and at least 10 min was allowed for solvent evaporation. Monolayers were compressed at a constant rate of 4.2 cm/min. Changes in the compression rates did not alter the shape of the isotherms. All the isotherms were run at least three times in the direction of increasingpressure with freshlyprepared films. The accuracy of the system under the conditions in which the bulk of the reported measurements were made was 0.5 mN/m for surface pressure. All the measurements were made at 21 1 OC. The surface activity and insertion of the peptides into monolayers were studied using a minicuvette of 70 mL. To measure the equilibrium spreading pressures,increasingvolumes of concentrated solutions of each peptide were injected beneath the aqueous surface (PBS), and pressure increases after 30 min were recorded. Vesicle Preparation. Stock solutions of DPPC or DPPC/ DPPG (1:l)in chloroform were dried to a f i i using a rotary evaporator, and the residue was further dried under reduced pressure for 3-6 h. Lipids were hydrated with acetate buffer, by vortexing at 55 OC for 30 min, followed by sonication under nitrogen at a temperature above the T,, until no further decrease of turbidity could be detected. These samples were centrifuged to eliminate the remaining MLV and Ti particles and the
*
~~
(7) Atherton, E.; Benoitin, L.; Brown, E.; Sheppard,R. C.; Williams, B. J. J. Chem. Soc., Chem. Commun. 1981,336. (8) Stewart,J. M.; Young,J. D. Solid Phase Peptide Synthesis, 2nd ed.; 1983; pp 105-106. (9) Coste, J.; Lenguyen, D.; Castro, B. Tetrahedron Lett. 1990, 31, 205-208. (10) Verger, R.; De Haas, G. H.Chem. Phys. Lipids
1973,10,127-135.
peptide I
Table 1. Analytical Data of Peptides HAV-HAV-VPl(11-25)(11-25)(I)and St-HAV-HAV-VP1(11-25)(11-25) (11) amino acid analysis FAB-MS
HPLC
Asx, 2.30(2) Thr,2.20(2) [MI+, 1583 k f a = 2.74 Ser, 1.05(1) Glx, 3.30(3) [M+ HI+, 1584 Gly, 1.15(1) Val, 3.15(3)
Ile, l.OO(1) Pro, 1.98(2) Asx, 2.30(2) Thr,2.19(2) k f b = 1.60 Ser, 1.06(1) Glx, 3.16(3) Gly, l.OO(1) Gly, 3.20(3) Ile, l.OO(1) Pro, 1.90(2) a HPLCconditions: SpherieorbC-18(lOpm),1mL/min,detection 226 nm, eluted with HzO (0.05% TFA)/ACN (0% ACN to 100% ACN in 30 min).b HPLC conditions: Spherisorb C-18 (10 pm),1 ml/min,detection226nm,elutedwithACN(O.l% TFA)/H& (0.1% TFA) (60:40). I1
phospholipid content of SUV liposomes was quantified by the Barlettll method. Losses after sonication ranged between 15% and 20%, so that the final phospholipid concentration was 27 mM. For vesicle leakage measurements, 6-CF (70 mM) was encapsulated in SUV,followed by gel fiitration on Sephadex G-50 and dialysis, to attain latencies higher than 90%. Measurements of Membrane Fluidity. Fluorescence polarization studies were carried out in a Perkin-Elmer spectrofluorimeterLS 50, equipped with a thermostatable cuvette holder and polarizers. Small unilamellar vesicles prepared as described before (6 = 105nm) were incubated with ANS or DPH, at several phospholipid/probe relationships, to fiid the saturation concentration of vesicles. Liposomes saturated with fluorescent probes were incubated with the peptides and their fluorescene polarizations measured as a function of the temperature. Excitation and monitoring wavelengths were 398 and 480 nm for ANS and 357 and 450 nm for DPH. The observed fluorescence intensities of the mixtures were corrected for the individual fluoresence of the liposomes, free marker, and peptides. Membrane fluidity was estimated on the basis of the reciprocal of polarization (l/P). The range of temperatures was 25-60 ‘C. Peptide-Induced Vesicle Leakage. The stability of l i p somes after incubation with the peptides was measured by the increase of fluorescence intensity of CF caused by ita dilution upon leakage from aself-quenching concentration (70mM),inside the vesicles. Results are reported as a percentage, taking as total fluorescence the values obtained after disruption of the vesicles with Triton X-100. Excitation and emission wavelengths were 470 and 620 nm.
Results Peptide Synthesis. The overall yield of the synthesis (HAV-VP1( 11-25): Thr-Val-Ser-Thr-Glu-Gln-Asn-Val-
Pro-Asp-Pro-Gln-Val-Gly-Ile) based on the first amino acid attached to the resin was 855%. The purity (HPLC) of the peptides after deprotection was higher than 98%; for this reason no further purification process was necessary. As regards acylationwith stearic acid it was successfully coupled to peptide HAV-VPl(l1-25) resin with pyBop. In less than 30 min as monitored by the ninhydrin test the lipopeptide was afforded in high yield (80%). Cleavage of peptide and lipopeptide resins was carried out with TFA/H20 (955). To ensure optimum cleavage conditions (lengthof reaction, etc.), a preliminary small-scalecleavage was carried out. Peptidesand lipopeptides of HAV capsid virus were successfully cleaved in 1-1.5 h with TFA/H20 (95:5).
The peptides were characterized as shown in Table 1by analytical HPLC, amino acid analysis, and fast-atom (11)Barlett, G. R. J. Phosphorous assay in column chromatography. J. Biol. Chem. 19S9,234,466-468.
Martin et al.
786 Langmuir, Vol. 10, No. 3, 1994 25 I
1
16 1 3E 4 ;12
0
1
2
3
4
5
Peptide concentration ( p M )
Figure 1. Surface pressure increases of HAV-VPl(l1-25) ( 0 ) and HAV-St-VPl(11-25) ( 0 )peptides presented as afunction of
the peptide concentration in the subphase.
bombardment mass spectrometry (FAB-MS). The peptide concentration was determined by amino acid analysis. Surface Activity of Peptides. The peptides HAVVPl(l1-25) and HAV-St-VPl(11-25) when injected into an aqueous subphase lowered the surface tension a t the air-water interface. This activity was obviously stronger for the stearoyl derivative. The pressure increases recorded for the different peptide concentrations are given in Figure 1. HAV-VP1( 11-25) shows a very low surface activity that is independent of the concentration of the peptide in the subphase. On the contrary the stearoyl derivative is able to increase the surface pressure of the system as a function of the initial concentration of the peptide in the subphase. A t saturating peptide concentrations (0.98 pM for HAVVPl(l1-25) and 4.5 pM for HAV-St-VP1(11-25), respectively) the corresponding spreading pressures were 0.7 and 23 mN/m. Comparing these values with those in the literature (melittin, 18 mN m-l;12 HIV peptides, 23 and 25 mN m-1 ls), one can appreciate that HAV-St-VPl(1125) behaves as a highly hydrophobic peptide. The ability of these peptides t o insert into monomolecular layers of phospholipids (DPPC, DPPC/DPPG) was determined by injecting the peptides beneath phospholipid monolayers spread a t different initial surface pressures, and recording the consequent changes in the surface pressure. These experiments were carried out a t constant surface area, and the final concentration of peptides into the subphase was micromolar. The results are given in Figure 2. The presence of a monolayer has a dual effect on the surface activity of the peptides. For HAV-VPl(ll-25), there is a positive effect of helping the insertion of this peptide into the monolayer. On the contrary, when the peptide is highly hydrophobic, the monolayer acts as a barrier, hindering its efficient incorporation into the surface. This behavior has already been described for opioid molecules of different degrees of hydr~phobicity.'~ Moreover, the lower the initial pressure of the film, the higher was this increase in film pressure. The observed kinetics reached a plateau within a few minutes for HAVSt-VPl(ll-25), but penetration was slower for HAV-VP1(11-25), in agreement with the differences in hydrophobicity. (12)Fidelio, G. D.;Austen, B. M.; Chapman, D.; Lucy, J. A.Biochem. J. 1986,238,301-304. (13)Rafalski, M.; Lear, J. D.; De Grado, W .F. Biochemistry 1990,29,
7917-7922. (14)b i g , F.; Buequets, M. A.; Haro,I.; &banal, F.; Alsina, M. A. J . Pharm. SCL 1992,81,546-550.
0 1 0
5
10
15
20
Initial surface pressure (mN/m)
Figure 2. Increase in surface pressure as a function of the initial film pressure of phospholipid monolayers: (*) DPPC/HAV-StVP 1(11-25), (m) (DPPC/DPPG)/HAV-St-VP1(11-25), ( 0 )DP-
PC/HAV-VP1(11-25),
(+)
(DPPC/CPPG)/HAV-VP1(11-25).
Compression Isotherms. The ability of HAV-VP1(11-25) and HAV-St-VPl(11-25) to form stable monolayers was checked by spreading these peptides on an aqueous subphase. Then, despite the solubility of HAVVPl(l1-25) in water, a film of peptide became established a t the surface, in both cases, resulting in compression in the corresponding isotherms. As expected HAV-St-VP1(11-25) gave stable monolayers,the maximum compression pressure achieved being 25 mN/m, without collapse. HAVVPl(l1-25) also gave monolayers, but after compression the final surface pressure was only 2.5 mN/m, and this behavior was not dependent on the amount of peptide spread on the subphase (PBS, pH 7.4; peptide concentration 1Por 10-8 M). Higher quantities of HAV-VP1(11-25) increased the initial surface pressure of the monolayer but did not contribute to higher surface pressures. The miscibility of these two peptides with DPPC and DPPC/DPPG in monolayers was determined by carrying out the compression isotherms of mixtures of different compositions. Mixed monolayers showed intermediate characteristics with respect to those of pure components. The maximum compression surface pressure decreased steeply as the peptide content increased. The area/molecule values represented in Figure 3 show that deviations from ideality are small, thus indicating either ideal miscibility or a lack of interactions. The lack of collapse points in these isotherms does not allow any possibility to be discarded. The area/molecule values of HAV-VPl(l1-25) could not be calculated a t compression presures higher than 2.5. Furthermore, to have more accurate information on the miscibility, the interaction parameters and free excess energies of mixing were calculated by applying the mathematical expressions given by Mestres et al.l5 and are summarized in Table 2. For obvious reasons these calculations were made only a t 2.5 mN/m in the case of HAV-VPl(ll-25), but for HAV-St-VPl(11-25) it was possible to extend the mathematical treatment till 25 mN/ m. The excess free energies of mixing involved are in both peptides lower than RT (2474.6J/mol), which corresponds to small interactions, and can be due to a complete miscibility or to a lack of interactions. As far as the conformation of the peptides is concerned, the secondary structure prediction determined applying Chou and Fass(15) Mestres, C.; A h a , M. A.; Eapina, M.; Rodriguez, L.;Reig, F. Langmuir 1992,8,1368-1391.
Hepatitis A Viral- VP1 Protein Peptides
Langmuir, Vol. 10, No. 3, 1994 787
2 1
.
0,s
1IO
0.8lO.Z
0.610.4
0.410.6
0.210.8
4a
011
Molar fraction Phospholipid/St-VP1
Figure 3. Area of mixed monolayers versus the molar compo( 0 )5, (+) 10, and (*) 15 (DPPC/DPPG)/HAV-St-VP1-
sition:
(11-25); (A) 5, (H) 10, and ( 0 )15 DPPC/HAV-St-VP1(11-25). Table 2. Mixing Excess Free Energies and Interaction Parameters of DPPC and DPPC/DPPG (1:l) Mixtures with St-HAV-VPl(11-25)
AG@
X Pl/pep
DPPC
0.810.2 0.6/0.4 0.4/0.6 0.2/0.8
-51.27 -579.89 -369.67 -1407.78
(J/mol) DPPC/DPPG 143.95 217.27 -1327.7 -1591.11
a
DPPC -0.93 -0.99 -0.63 -3.6
DPPC/DPPG 0.37 0.37 -2.26 -4.07
man parameters, pointed out a high probability of a p turn located around residues 8-11, The area/molecule values calculated from the compression isotherms are consistent with two j3 sheets linked by a p turn (0.7 nm2 for each j3 sheet).12 The hydrophilicity index of Hopp and Woods is 0.397, with the highest peak around residues 2-7; moreover, the hydropathy index (Kyte and Doolittle) shows a maximum for residues 4-9 (1.285). In both cases a change in conformation is suggested around residues 8 and 9, in agreement with the position of the /3 turn already mentioned. The high hydrophilicity values confirm the low surface activity of the parent compound and the need for a hydrophobic anchor to attach the peptide to the surface of liposomes. Membrane Fluidity. Polarization of DPH-Labeled Liposomes. Liposomes saturated with DPH were incubated with either HAV-VPl(l1-25) or HAV-St-VPl(1125) and the fluorescence and polarization values of this probe (as a function of the temperature) determined by applying eq 1,whereIvvandIn are the observed intensities
4 b
\
15
I
I
,
,
,
,
,
,
20
25
30
35
40
45
50
55
4c 60
T *C
Figure 4. Changes in polarization of DPHIliposomea, after incubation with HAV-St-VP1(11-25), measured at different temperatures: (a)DPPC/HAV-St-VP1(11-25) ( S l ) , (b) (DPPC/ DPPG)/HAV-St-VP1(11-25) (5:1), (c) DPPC/HAV-St-VPl(ll25) (301), (W) TAC, (+) HAV-St-VPl(11-25).
measured with polarizers parallel and perptmdicular to the vertically polarized exciting beam, respectively. G is a factor used to correct for the inability of the instrument to transmit differently polarized light equally. Typical experimental results with DPPC vesicles are shown in Figure 4. The depolarization of DPH fluorescence a t the phase transition due to changes in fluidity is quite dramatic; below the T,,the polarization approaches the theoretical value (0.51,while above the phase transition of DPH is almost completely depolarized.
HAV-VPl(l1-25) had no effect on the membrane fluidity under the experimental conditions employed, independently of the electrical charge of the phospholipid (PL) used. On the other hand, HAV-St-VPl(11-25) clearly increased polarization, thus decreasing the membrane fluidity. The transition process, as reflected by changes in the polarization of the system, was differently affected by the presence of the peptide, depending also on the polar head of the phospholipids. HAV-St-VPl(11-25) has a rigidi-
Martln et al.
788 Langmuir, Vol. 10, No. 3, 1994 fying effect on DPPC bilayers from temperatures slightly lower than T, all over the transition process, and the residual polarization remains constant after the phase change, thus suggesting that the peptide remains firmly incorporated into the bilayers. When liposomes prepared withDPPC/DPPG, containing DPH, were incubated with HAV-St-VP1(11-25), the behavior as far as the polarization was concerned was similar to the description given in the previous paragraph; nevertheless, the transition temperature was not so strongly affected, and only a t T > T,the rigidifying effect began to be clear. Moreover, differences in fluidity between the reference and samples are lower in these liposomes than with DPPC vesicles. The results described were obtained a t a molar ratio of lipid to peptide of 5:l. This is a very low rate compared with similar studies described in the literature.l6 A new set of experiments carried out with a PL/peptide (pep) (301) showed no interaction (Figure 4c). This fact suggests that the interactions detected are mainly located a t the external half of the bilayer, and only when there is an excess of peptide in the incubation media, these interactions affect the motion of the alkyl chain core in the bilayer. In these experimental conditions, thus, the attachment of an alkyl chain to the amino terminal group of a highly hydrophilic peptide acts as an anchor to link the peptide to the surface of the liposome in a permanent way. Nevertheless, as the amount of peptide necessary to promote a detectable effect is unusually high compared to the values cited in the literature,ll7 one cannot exclude the possibility that HAV-St-VPl(11-25) forms micelles or some type of aggregates that would be stable in solution and compete with the inclusion of the peptide into the bilayers or form mixed micelles with the phospholipids. These possibilities were checked by studying the polarization and fluorescence intensity values of HAV-StVPl(ll-25)/DPH mixtures, and changes induced in the turbidity of liposome/HAV-St-VP1(11-25) mixtures. Solutions of HAV-St-VPl(11-25) prepared a t the same peptide concentration (5.9 = lo3 M) as used in the studies with liposomes (PL/pep, 51)were mixed with DPH, and after 1h of incubation the fluorescence polarization and intensity were recorded as a function of the temperature. Polarization values were high (0.44) and constant in the range of temperatures under study (19-50 OC), suggesting the presence of an ordered structure of HAV-St-VPl(1125) molecules in solution; able to accommodate DPH molecules. Moreover, the high values of polarization and the lack of change when increasing the temperature suggest that DPH should more likely be located in the hydrophobic core of micelles and not between the alkyl chains. The fluorescence intensity of HAV-St-VP1(11-25)/DPH mixturesincreased with the temperature, but on the contrary, the system liposome/DPH or liposome/DPH/HAV-StVPl(l1-25) showed a constant decrease a t high temperatures. Nevertheless, the presence of HAV-St-VPl(1125) smoothed this tendency. The formation of mixed micelles between phospholipids and hydrophobic peptides has already been described in several papers.le As the interactions between HAV-StVPl(l1-25) and liposomes had been detected a t high peptide concentrations, the possibility of a micellization
5 a
1
15
1
20
1
25
1
30
1
35
40
1
45
1
50
Jb
'
55
60
T OC
Figure 5. Polarization-temperature dependence of ANSsaturated liposomes in the presence of HAV-St-VPl(11-26): (a) DPPC/HAV-St-VP1(11-25) ( 51);(b) (DPPC/DPPG)/HAV-StVPl(11-25) (51), (m)TAC, (+)HAV-St-VP1(11-25).
of our system due to the partial solubilization of liposomes could not be discarded. To check this point, the absorbance of liposomes and liposome/HAV-St-VPl(11-25) mixtures was determined in the range of temperatures under study (20-50 OC). Obviously, absorbance decreases during heating, but no differences between both preparations were found. These results seem to point out that when mixing liposome/DPH with St-HAV-VPl(11-25) an equilibrium is established between HAV-St-VPl(11-25) micelles and liposomes with the peptide inserted into the bilayer, and this state can be displaced using a higher PL/peptide relationship, but in no case is there a aolubilization of phospholipid by the peptide. Polarizationof ANS-LabeledLiposomes. A similar type of experience was encountered using ANS as the fluorescent probe. This molecule is able to interact with the polar heads of phospholipids, giving in this case information about changes in fluidity in this zone. Although polarization values of ANS do not change as drastically as those of DPH, a temperature dependence was also clear. Nevertheless, differences between the reference and peptide samples were lower. HAV-St-W1(16)Lakey,J.H.;Maesotte,D.;Heitz,F.;Dasseux,J.-L.;Faucon,J.-F.; (11-25) increased polarization of ANS-bound liposomes; this behavior is essentially due to the immobilization of Parker, M.W.; Pattua, F. Eur. J. Biochem. 1991,196, 599-607. (17)Mc Lean, L. R.; Hagaman,K. A.; Oven, T. J.; Payne, M. H.; the marker upon peptide binding. There is an apparent Davidson, W.S.; Krstenanaky,J. L. Biochim. Biophys. Acta 1991,1086, paradox in the results above described because as ANS 106-114. fluorescence is related to changes in the polar environment (18)McLean, L. R.;Hagaman, D. A.; Owen, T. J.; Krstenansky, J. L. Biochemistry 1991,30, 31-37. of the membrane; both peptides HAV-VP1(11-25) and
Hepatitis A Viral-VPl Protein Peptides HAV-St-VPl(11-25) should give the same level of interaction, or at least a similar behavior. Mixtures of HAV-St-VPl(11-25) with ANS in similar concentrations used in the presence of liposomes showed the values for the fluorescence intensity slightly higher than those of the buffer, and the polarization of these peaks was not significant. This behavior can also be explained by the presence of micelles whose hydrophilic moiety is negatively charged, thus giving a low interaction with the anionic marker (ANS), Figure 5. This fact has also been described for anionic surfactanta.le Moreover, according to D. Tsernoglou,20the mechanism for the plausible insertion of a protein into a lipid bilayer can be assumed as the result of two consecutive processes: the binding to the membrane surface (electrostatic) and the insertion of the peptide into the hydrophobic core of the bilayer. This second step can be driven by the partitioning of hydrophobic residues into the aliphatic environment of the bilayer. As HAV-VPl(l1-25) is highly hydrophilic and has two net negative charges, electrostatic interaction with the lipids would be low. But the insertion of the stearoylchain into the bilayer will render the peptide chain in close proximity to the polar heads of the phospholipids and as a consequence alter the motion of ANS molecules in ita close vicinity. Stability of Bilayers. The time course of carboxyfluorescein leakage from DPPC and CPPC/DPPG lipo(19) Vendittiee, D. E.; Palumbo, G.; Parlato, G.; Bocchini, V. Anal.
Biochem. 1981,115,278-286. (20) Parker. M. W.: Pattua,. F.:. Tucker. A. D.: Twmoalou, - D.Nutwe 1989, &7,93-k. .
Langmuir, Vol. 10, No. 3, 1994 789 some8 was determined by incubation with HAV-VPl(1125) and HAV-St-VPl(11-25). Neither of these peptides was able to disturb the bilayers strongly enough to induce the leakage of the entrapped dye. These resulta are in agreement with the preceding ones and c o n f i i that the HAV-VP1(11-25)/phospholipid interaction is of electrostatic nature and only detectable when using monomolecular layers. In this model changes in solvation around the polar heads of phospholipids can slightly modify their packing and change the surface pressure of the monolayer. Furthermore, HAV-St-VPl(11-25) interaction is due to the stearoyl residue that exerts ita effect mainly at the external half of the bilayer. All these data indicate that liposomes incubated with HAV-St-VPl(11-25) can be good carriers for this peptide in immunization experiments. Moreover, the derivatization of peptides with hydrophobic residues allows them to form some type of aggregate that can act as a carrier for small molecules by themselves. Furthermore, the incorporation of these peptides into the surface of liposomes canbe a good alternative for targeting, sometimes better than the direct linkage to preformed liposomes, when a good characterization of the species involved is required. Acknowledgment. We gratefully acknowledge Mrs. Marla de la Sierra Osuna and Mr. Emili Nogu6s for excellent technical assistance. This work was supported by a grant (N.BI 092-0982-CO 2-02) from CICYT, Spain.
