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Physicochemical Behavior of Polylysine-[HAV-VP3 Peptide] Constructs at the Air-Water Interface P. Sospedra,† I. B. Nagy,§ I. Haro,‡ C. Mestres,† F. Hudecz,§ and F. Reig*,‡ Faculty of Pharmacy, Department of Physicochemistry, Av. Joan XXIII s.n., 08028 Barcelona, Spain, Department of Peptide & Protein Chemistry, CID-CSIC, Jordi Girona 18, 08034 Barcelona, Spain, and Research Group of Peptide Chemistry, Hungarian Academy of Sciences, Eo¨ tvo¨ s L. University, P.O. Box 32, Budapest 112, Hungary H-1518 Received September 22, 1998. In Final Form: April 21, 1999 Branched-chain polypeptides based on a polylysine backbone were synthesized for use as delivery systems for biologically active molecules. To develop a new immunoadjuvant system for hepatitis A virus (HAV) antigens, a peptide fragment (110-121) of the VP3 hepatitis A protein has been linked to cationic and amphoteric polymeric polypeptides. In the present paper, we describe the physicochemical characterization of three branched polypeptide-[HAV-VP3(110-121) peptide] constructs, where the peptide is attached to the polymer side chains by disulfide linkage. The surface activity of these three conjugates has been studied as a function of time and concentration in the subphase. Moreover, its insertion into phospholipid DPPC (dipalmitoylphosphatidylcholine) monolayers has also been determined. The results show that these constructs are surface active and can insert into the lipid monolayers. AK construct is slightly more active than EAK and SAK constructs both in the presence and absence of DPPC monolayers. This behavior suggests that construct disposition at interfaces is mainly dependent on nonsubstituted side chains. Fluorescence polarization studies, performed with DPPC vesicles saturated with either DPH (1,6-diphenyl1,3,5-hexatriene) or ANS (1-anilino-8-naphthalenesulfonic acid), indicate a strong rigidifying effect of the three constructs on the polar heads and alkyl chains of bilayers.
Introduction Synthetic oligopeptides used as immunogens for vaccine design generally induce weak immune response compared to that of intact proteins. To overcome this problem, several carriers and immunoadjuvants have been introduced. Freund’s adjuvant gives the best results, but its inflammatory side effects make it unacceptable for human use. Although alum is also frequently used and it is completely free of side effects, the efficacy of this adjuvant is not optimal. Thus, it still remains a scientific challenge to develop suitable protein/carriers to induce high and specific antibody titers without eliciting adverse effects. Particulate systems such as iscoms, liposomes, and capsules or nanospheres have been assayed as carriers for antigens with different results, but their fast clearance from circulation due to macrophage action reduces its efficacy in most cases. Soluble polymers represent possible alternatives to these short-half-life particles. Immunogenic peptides covalently attached to macromolecular carriers have extensively been used in experimental animals to elicit antibody responses or to induce protection against microbial or parasitic pathogens.1-3 Recently, we have prepared and applied successfully new bioconjugates for constructing synthetic immunogens/ antigens,4 B-cell epitope peptides of epithelial mucin,5 herpes simplex virus (type I) glycoprotein D,6 or T-cell epitope peptides from a 38 kDa protein of Mycobacterium †
Faculty of Pharmacy. CID-CSIC. § Eo ¨ tvo¨s L. University. ‡
(1) Del Giudice, G. Curr. Op. Immunol. 1992, 4, 454-459. (2) Hudecz, F.; To´th, G. K. In Synthetic peptides in the search for Band T-cell epitopes; Rajnavo¨lgy, E Ä ., Ed.; R. G. Landes Company: Austin, TX, 1994; Vol. 4, pp 97-119. (3) Delpierre, C.; Martin, C.; Gequierre, J. C.; Tartar, A.; Cachera, C.; Purchois, P.; Fievet, C.; Fruchart, J. C. Int. J. Rad. Appl. 1987, 14, 281-288.
tuberculosis.7 In these constructs, poly[Lys-(Xi-DL-Alam)] (XAK) type branched-chain poly(R-amino acids) were used as synthetic carriers.8,9 For the present investigation, a novel set of conjugates was produced10,11 in which peptide 110-121 representing a B-cell epitope region of the HAV VP3 protein was attached to XAK polypeptides, where X is either glutamic acid (EAK)12 or serine (SAK).13 In this paper, we describe the interaction of peptide conjugates with mono- or bilayers of a neutral phospholipid, DPPC. The binding of three peptide conjugates (AK[C-VP3(110-121)], EAK-[C-VP3(110-121)], SAK-[C-VP3(110-121)] to these membrane models was studied by surface activity measurements and insertion analysis using fluorophore-labeled liposomes. The results presented here demonstrate that the hydrophobic character of the branched-chain polypeptide has a pronounced influence on the membrane-disturbing capacity of the conjugate. Materials and Methods Materials. Branched Polypeptide-Peptide Conjugates. Abbreviations for amino acids and their derivatives follow the revised recommendation of the IUPAC-IUB Committee on Biochemical (4) Hudecz, F. Biomed. Peptides, Proteins Nucl. Acids 1995, 1, 213220. (5) Hudecz, F.; Price, M. R. J. Immunol. Meth. 1992, 147, 201-210. (6) Hudecz, F.; Hilbert, A.; Mezo¨, G.; Mucsi, J.; Kajta´r, J.; Bo¨sze, S.; Kuruez, I.; Rajnavo¨lgy, E Ä . Peptide Res. 1993, 6, 263-271. (7) Wilkinson, K. A.; Vordermeier, M. H.; Wilkinson, R.; Iva´nyi, J.; Hudecz, F. Bioconjugate Chem. 1998, 9, 539-547. (8) Hudecz, F.; Votavova, H.; Gaa´l, D.; Sponar, J.; Kajta´r, J.; Blaha, K.; Szekerke, M. In Polymeric Materials in Medication; Gebelein, Ch. G., Carraher, Ch. E., Eds.; Plenum Press: New York, 1985; pp 265289. (9) Hudecz, F. Anticancer Drugs 1995, 6, 171-193. (10) Nagy, I. B.; Alsina, A.; Haro, I.; Reig, F.; Hudecz, F. Biopolymers 1998, 46, 169-179. (11) Nagy, I. B.; Alsina, A.; Haro, I.; Reig, F.; Hudecz, F. Bioconjugate Chem., submitted. (12) Hudecz, F.; Szekerke, M. Collect. Czech. Chem. Commun. 1980, 45, 933-940. (13) Mezo¨, G.; Kajta´r, J.; Nagy, I. B.; Szekerke, M.; Hudecz, F. Biopolymers 1997, 42, 719-730.
10.1021/la981305i CCC: $18.00 © 1999 American Chemical Society Published on Web 06/19/1999
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Figure 1. Molecular structures of conjugates. Nomenclature, entitled “Nomenclature and Symbolism for Amino Acids and Peptides”.14 The nomenclature of branched polypeptides is used in accordance with the recommended nomenclature of graft polymers.15 For the sake of brevity, codes of branched polypeptides were constructed by using the one-letter symbols of amino acids. All amino acids are of the L configuration unless otherwise stated. The average degree of polymerization (DPn) of the polylysine was estimated by sedimentation analysis. The average relative molar mass of the branched polypeptides (AK, EAK, and SAK) was calculated from the DPn of the poly[Lys] backbone and from the amino acid ratios Glu:Ala:Lys as described previously.10 The coupling was performed by derivatization of the amino terminal group in the side chain with N-succinimidylpyridyl dithiopropionate (SPDP) followed by disulfide substitution by the free tiol group of Cys peptide.16 This reaction has been already described.17 The solution was then dialyzed against deionized water, and the product was isolated by freeze-drying. The average molar substitution ratio was calculated according to the amino acid composition of the conjugate obtained from amino acid analysis (Figure 1). Dipalmitoylphosphatidylcholine (DPPC) was obtained from Sigma; its purity was assessed by thin-layer chromatography. 1-Anilino-8-naphthalenesulfonic acid (ANS) and 1,6-diphenyl1,3,5-hexatriene (DPH) were purchased from Sigma and used without further purification. Dimethyl sulfoxide (DMSO), methanol, and chloroform used for the conjugates, and lipid solutions were from Merck (pro-analysi grade). The water used for buffer and the solutions was double-distilled. Experiments were carried out either in phosphate buffer saline (PBS) (pH ) 7.4, 0.017 M NaH2PO4, 0.081 M Na2HPO4, 0.05 M NaCl) (monolayer experiments) or 0.25 M sodium acetate solution adjusted at pH ) 7.4 (TAC) (polarization studies). Methods. Monolayer Experiments. All experiments were carried out in a PTFE (Teflon) trough at room temperature. The (14) IUPAC-IUB Commission on Biochemical Nomenclature. Biochem. J. 1972, 127, 753-756. (15) IUPAC-IUB Commission on Biochemical Nomenclature. Eur. J. Biochem. 1984, 138, 9-37. (16) Carlsson, J.; Drevin, H.; Axen, R. Biochem. J. 1978, 173, 723728. (17) Garcı´a, M.; Nagy, I. B.; Alsina, A.; Mezo¨, G.; Reig, F.; Hudecz, F.; Haro, I. Langmuir 1998, 14, 1861-1869.
Sospedra et al. surface pressure was measured by the Wilhelmy method using a platinum plate and recorded in a Langmuir balance as described elsewhere.18 The PTFE cuvette was regularly cleaned with hot chromic acid and, before each experiment, with ethanol and rinsed with double-distilled water; the platinum plate was cleaned with chromic acid before each experiment. Surface Activity Measurements. These experiments were carried out in a cylindrical trough (3.1-cm radius and 2.3-cm depth) with mechanical stirring. The trough was filled to full capacity (70 mL) with PBS, prepared with double-distilled water, and then different volumes (from 10 to 125 µL) of a stock solution (3 × 10-5 M) of the conjugates were injected directly underneath through a lateral hole. Changes in surface pressure were recorded for 120 min. Subphase purity was controlled by previous measurements of its surface pressure. The accuracy in the measurements of surface pressure was (0.1 mN/m. Insertion of the Constructs into Lipid Monolayers. Monolayers of DPPC were formed spreading the lipid from a 1 mg/mL stock solution in chloroform (using a microsyringe) directly to the air/water interface until the desired initial pressure was achieved. Once the target pressure was stable for 10 min, a fixed volume of the conjugate stock solution was injected in the aqueous phase to give a final concentration of 7.0 nM for AK-VP3, 7.97 nM for SAK-VP3, and 7.62 nM for EAK-VP3. These concentrations were chosen because they are slightly lower than the concentration that promotes maximum surface pressure in the surface activity assays (spreading pressure). The subphase was continuously stirred by a small magnet, to ensure a homogeneous distribution of the conjugate. Variations in surface pressure were recorded as a function of time for 120 min. Small Unilamellar Vesicles (SUV) Preparation. Vesicles of DPPC were prepared as described elsewhere.19 Briefly, a solution of DPPC in chloroform/methanol (2:1 v/v) was evaporated to dryness. The dried film was hydrated and then sonicated under a nitrogen atmosphere at 20 W with a 3/8-in. flat disrupter tip mounted on a Vibracell sonicator (Sonics & Materials Inc.) at a temperature of 55 °C so that the samples were above the transition temperature of DPPC. The SUVs were isolated as a slightly turbid supernatant after a 60-min 50.000g centrifugation process to remove titanium released from the tip of the probe and residual multilamellar structures. Liposomes were annealed by incubation 10 °C above the phase transition temperature of the lipid for 1 h. The phospholipid concentration was determined by phosphorus quantification by the method of McClare.20 The average vesicle size, determined by dynamic light scattering with a Malvern II-C Autosizer, was 105 nm in diameter, with a polydispersity of 0.2. Fluorescence Experiments. Fluorescence measurements were carried out on a Perkin-Elmer LS-50 spectrofluorimeter, equipped with automatic polarizers and temperature controller, in a four-position thermostated cuvette holder. All the experiments were conducted in 0.25 M acetate buffer, pH 7.4, in quartz cuvettes with constant stirring. Vesicle labeling was performed incubating 250 µL of SUV (0.027 M) with either 250 µL of DPH (5 × 10-5 M) or 250 µL of ANS (9 × 10-5 M). This gave a lipid/ probe molar ratio of 540 and 300, respectively. The process was carried out in the dark for 60 min at 50 °C, the samples being continuously stirred. After this, 50 µL of fluorescently labeled SUV was added to 3 mL of either peptide conjugate solutions or sodium acetate buffer (pH ) 7.4). Experiments were carried out in duplicate. Samples were left to equilibrate for 10 min under magnetic stirring and fluorescence spectra and polarization values determined at different temperatures (20-50 °C). Excitation/emission wavelengths were 380/480 and 365/425 nm for ANSand DPH-containing vesicles, respectively, and the slit widths were kept at 4 nm each. Polarization values were calculated according to P ) (Ivv - GIvh)/(Ivv + GIvh), where Ivv and Ivh are the vertical and horizontal components of emission when excited with vertically polarized light. G is the correction factor for the photomultiplier sensitivity. (18) Fonseca, M. J.; Juve´, A.; Lorincz, Z.; Reig, F.; Alsina, M. A. J. Colloid Interface Sci. 1998, 205, 141-148. (19) Cajal, Y.; Rabanal, F.; Alsina, M. A.; Reig, F. Biopolymers 1996, 38, 607. (20) McClare, C. W. F. Anal. Biochem. 1971, 39, 527.
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Samples were sufficiently diluted so that the contribution from light scattering was negligible ( 0.95 in all cases.
In the case of SAK-[C-VP3(110-121)] conjugate, at low concentrations, surface pressure increase/time curves do not fit rectangular hyperbola; for this reason, Table 1b only shows the values corresponding to the four higher concentrations. The pressure increase values (∆π) determined at the equilibrium (120 min) for the different bulk concentrations (c) were used to calculate the surface excess (Γ) and molecular area applying
Γ)
1 ∆π RT ∆ ln c
area/molec. )
1 NΓ
(2) (3)
where Γ is the surface excess in mol/m2, ∆π the pressure increase in mN/m, T the temperature in K, R the gas constant (8.31 J/(K mol)), c the concentration, and N Avogadro’s number. The concentration was calculated on the basis of the molecular weight average. Although the above-written equations can only be correctly applied to a single-component system, we consider that they can also be used with our polymers because of the low polydispersity range calculated in their molecular weights. The values calculated at all bulk concentrations are summarized in Table 2. Assuming that at equilibrium the surface pressure increase is proportional to the number of molecules located at the surface, it can be concluded that AK-[C-VP3(110-121)] is slightly more active, and as a consequence of this, the area available for molecule is the lowest. Also, SAK-[C-VP3(110-121)] conjugate and EAK-[C-VP3(110-121)] gave similar levels of incorporation to the surface, and the area/molecule values calculated applying eq 3 are almost the same. Insertion of Peptide Conjugates into Phospholipid Monolayers. The ability of these peptide conjugates to insert into DPPC monolayers was analyzed by injecting them beneath the DPPC monolayers spread at different initial surface pressures (5, 10, 20, and 32 mN/m) and recording the changes in surface pressure.
Figure 3. Changes in the surface pressure of the DPPC monolayers (initial pressure: 10 mN/m), induced by (O) AKC-VP3(110-121), (2) EAK-C-VP3(110-121), (b) SAK-C-VP3(110-121).
Figure 3 shows the time course for these changes at 10 mN/m of initial surface pressure. AK conjugate is the derivative that inserts in the monolayer at a faster rate and gives the highest pressure increases. The penetration curves of EAK and SAK constructs rise slowly at the beginning of the process, but when approaching saturation (120 min after injection), the differences with reference to AK conjugate curves decrease. Nevertheless, AK construct is significantly more hydrophobic than EAK and SAK conjugates. Relating the pressure increases to peptide concentration in the suphase, the corresponding values for AK, SAK, and EAK constructs were, respectively, 1.8, 1.44, and 1.57 mN‚m-1/nmol. These figures indicate that the AK derivative is the most surface-active conjugate, being in agreement with previous determinations in the absence of monolayer. Moreover, the time needed to achieve half of the maximum pressure was 10, 15, and 30 min for AK, EAK, and SAK conjugates, respectively, reflecting a faster interaction of AK-derived construct with DPPC. This kind of experiment, as stated before, was repeated under different initial surface pressures (pi) of DPPC monolayers, and Figure 4 shows the changes in the surface pressure (∆π) versus pi. The compression isotherm of a DPPC monolayer spread on PBS subphases shows that
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Figure 4. Maximum values of the pressure increase (∆π) versus the initial pressure of the DPPC monolayers.
the molecules are in different ordered states depending on the surface pressure.23 At 5 mN/m, the monolayer is in a liquid-expanded state; around 10 mN/m, there is a phase transition toward a liquid-condensed state, and up to this value, the monolayer approaches the solid state. The decrease of ∆π as pi increased, for all the constructs, was in agreement with the successive degrees of packing of DPPC molecules in the monolayer. The highest initial pressure permitting construct insertion in the DPPC monolayer (critical insertion pressure) could not be estimated by extrapolation, because it was not possible to obtain a linear adjustment. Nevertheless, pressure increases at 32 mN/ m, that is, a similar pressure to that found in biological membranes, are still significant and even higher than those described for membrane-interacting proteins.22 Also, the order of hydrophobicity found here is in agreement with the values previously obtained for the surface activity of AK, SAK, and EAK constructs. These results suggest that instead of its electrical charge, the hydrophobicity of the nonsubstituted lateral chain has an important contribution to the overall surface activity of the construct. In this sense, obviously a branch with three DL-alanine residues is more hydrophobic than those containing Ser or Glu at the end of the side chains and, therefore, can be inserted in a monolayer slightly more efficiently. From the values of Figure 4, it can be seen that the presence of phospholipids acts as a barrier hindering the incorporation of the conjugate to the surface. This behavior has already been described for molecules of branched-chain polymers10 and constructs.11 Comparing our previous results with those described here, experiments performed with constructs dissolved in water resulted in lower surface activity than those carried out with samples dissolved in DMSO. The adsortion process at the air-water interface reduces the interfacial tension, but with macromolecules, it may take a longer time before the equilibrium is reached. According to the description given in ref 24, the kinetics of the whole process is due to the contribution of several processes such as (a) diffusion from the bulk solution to the subsurface region in direct contact with the surface layer, (b) penetration of macromolecules onto the surface, and (c) rearrangement of adsorbed molecules in the surface. Also, there is a fourth important point related to the unfolding rate of the macromolecules that is highly dependent on the solvent media used. In fact, as described in ref 25, the surface activity is primarily a cumulative property in a macro(23) Cajal, Y.; Alsina, M. A.; Reig, F.; Rodriguez, L.; Mestres, C.; Haro, I. J. Colloid Interface Sci. 1998, 198, 78-86. (24) De Feitger and Benjamins Food Emulsions and Foams; Dickinson, E., Ed.; Royal Society of Chemistry: London, 1987; pp 72-85. (25) Horbett, T. A. Protein Eng. 1988, 2, 172-174.
Figure 5. Intensity of the fluorescence variation versus temperature for (b) EAK-C-VP3(110-121), (9) SAK-C-VP3(110-121), (2) AK-C-VP3(110-121), with (a, top) ANS and (b, bottom) DPH.
molecule and the large size and mixture of amino acids provides the possibility of formation of noncovalent bonds either inter- and/or intramolecular that contribute to the overall hydrophobicity of the macromolecule. Depending on the hydrophobic-hydrophilic balance in our macromolecular constructs, the conformations adopted in water or in DMSO will be different. The hydrophobic groups will be more exposed in DMSO media, being, on the contrary, hidden in water. After injection of a construct in an aqueous media from water or DMSO mother solutions, the surface properties will be dependent on the greater ease of unfolding at the interface. This process will be obviously more favored if constructs are injected from DMSO solutions and has also been described for several mutants of tryptophan syntase, where changes in amino acid substitution promoted changes in the surface activity that were related to the unfolding process at the interface. This could explain the differences in the surface activity found in our experiments and points to the need for a strict control of solvents in surface activity measurements. Complementary studies are in process to determine more accurately the conformations in both media. Interaction of Peptide Conjugates with DPPC Bilayers. Modifications in the bilayer structure induced by the presence of added molecules in the media can be determined through fluorescence polarization measurements. In this type of studies, the emission polarization or anisotropy of membrane-bound fluorophores (DPH or ANS) is used as a measure of the extent of probe motion that reveals changes in the microviscosity of its environment. DPH molecules when incubated with phospholipid bilayers are localized mainly in the internal core, and their motion is highly dependent on the ordering degree and motional properties of the surrounding hydrocarbon chains. Also, thermotropic phase transitions in the bilayers
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Figure 6. Percentage change in the polarization values at different temperatures.
of pure phospholipids are strongly reflected in dramatic changes in its microviscosity. ANS molecules are localized in the interfacial region of phospholipid bilayers and give information about the rigidity of this zone of the bilayer. Both probes are not fluorescent in aqueous media, but when located in a hydrophobic environment, they become highly fluorescent, giving information about the presence of aggregates. To estimate the contribution of potential aggregates of the peptide-polymer constructs in the presence of fluorescently labeled liposomes, we first carried out the experiments in the absence of vesicles and in the same range of temperatures (20-50 °C). To this end, peptide constructs were mixed with ANS and DPH and the fluorescence and polarization values measured. The results are given in Figure 5. One can appreciate that peptide constructs are able to accommodate small amounts of DPH and ANS molecules, providing a hydrophobic environment that results in low fluorescence values. It is not possible to know if these hydrophobic zones are due to intermolecular aggregates or intramolecular conformational changes, but at least for ANS, they are not temperature dependent. Moreover, with this fluorescent anion and according to the electrical charge of polymerpeptide constructs, the lack of clear differences between EAK and SAK constructs suggests a predominance of hydrophobic interactions. DPH interactions with these samples show a tendency to increase with temperature, but also here, the identical behavior determined for SAK and EAK conjugates allows us to discard any type of electrostatic contribution. The polarization values also determined in these experiments remain almost constant in the range of temperatures studied. These results indicate that when incubating ANS- or DPH-saturated liposomes with our constructs, the changes in fluorescence or polarization detected will be mainly due to the insertion of constructs to lipid bilayers. The possible contribution of constructs-fluorescent probe interactions is negligible. After incubation of ANS-saturated liposomes with the three constructs, the polarization values were always higher than those of the reference. This behavior is indicative of a rigidifying effect due to the interaction of the conjugates with the polar head of DPPC. The increases in polarization are represented as the percentage of increase at each temperature of the samples versus reference (Figure 6). Here one can appreciate that SAK construct behaves as the most active compound as far as the rigidification of bilayers is concerned, with AK and EAK conjugates following a similar trend. These results are different from those described in Figure 3, where the AK derivative was the most active compound. One possible explanation is to consider that at the same surface pressure, the degree of packing in a planar monolayer is different from that of the half external part
Figure 7. Polarization of fluorescence using DPH. (a, top) (2) DPPC + DPH-TAC; (*) EAK-C-VP3(110-121)-DPPC + DPHTAC. (b, middle) (2) DPPC + DPH-TAC; (b) AK-C-VP3(110121)-DPPC + DPH-TAC. (c, bottom) (2) DPPC + DPH-TAC; (9) SAK-C-VP3(110-121)-DPPC + DPH-TAC.
of a bilayer in SUV. The last one due to the curvature radius presents empty sites available for amino acid chains to be inserted in. As the differences among them are significant, it is more likely that peptide chains will face the water media and short (Ala)3, (Ala)3Glu, and (Ala)3Ser nonsubstituted chains will insert in the half external part of the bilayer. In this model, the bulkiness and whole steric characteristics of these polylysine core side chains will condition its accommodation between DPPC polar heads. The tendency of SAK and EAK derivatives to give a maximum around Tc seems to point to a deeper insertion
Physicochemical Behavior of Polylysine-[HAV-VP3 Peptide] Table 3. Mathematical Parameters Defining Polarization versus Temperature Adjusted to a Sigmoidal Curve for DPH Experiments sigmoid DPPC + AK-C-VP3 EAK-C-VP3 SAK-C-VP3 parameters DPH/TAC 1 2 3 A B C D r2
0.146 0.410 41.5 -0.401 0.995
0.274 0.437 41.8 -0.5844 0.988
0.309 0.443 42.2 -0.5268 0.992
0.329 0.454 42.5 -0.3943 0.981
in the bilayer that could in part be sensitive to the transition of DPPC alkyl chains from the gel to crystal liquid state. The same type of experiments was carried out with DPH as the membrane probe, and the results obtained are given in Figure 7. Polarization versus temperature values follow a sigmoid curve both for the reference and samples. Using eq 4, it was feasible to calculate the first derivative that is represented in the inset of each figure, and its mathematical parameters are given in Table 3:.
Y)A+
B-A 1 + (10C/10X)D
(4)
where Y represents the polarization, X represents the temperature, A and B are the bottom and top plateau of the polarization curve, and C is the X value at the middle of the curve (C ) Tc). D is the Hill coefficient or slope factor. From these curves and the corresponding insets, one can appreciate that the presence of constructs in the incubation media has no influence in the transition temperature of DPPC. The differences in the Tc values (Table 3) are not significant although SAK construct is
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the more active compound as far as bilayers rigidifying is concerned. By comparing the changes in polarization before and after transition with reference to buffer media, we found 54%, 50%, and 39% for SAK, EAK, and AK constructs, respectively. These values reflect that after transition the constructs remain firmly inserted in the bilayers, stabilizing a more packed structure. In conclusion, we have demonstrated that these three conjugates interact with neutral phospholipid monolayers. This interaction was slightly dependent on the hydrophobicity of the conjugates. This could be related to the amino acid composition of the spacer side chains rather than the net electrical charge of the constructs. Those composed of DL-Ala3, lacking a hydrophilic amino acid at their N-terminal as in Glu or Ser, behave under the conditions studied in planar monolayers as the most hydrophobic. But when using bilayers as membrane models, the differences among the three conjugates are not so clear. In fact, AK, SAK, and EAK constructs are able to mix with phospholipid alkyl chains without changing its transition temperature but rigidifying the bilayers. These studies suggest that these constructs associated with liposomes stabilize vesicles, rendering these preparations able to present peptides to the immune system in order to induce immunoresponse. Acknowledgment. This work was supported by Grants BIO 95-0061-C03-02, BIO 95-0061-C03-03, and SAF 97-0174 from CICYT, Spain, the Hungarian-Spanish Intergovernmental Program (E-13/1997), the Hungarian Research Fund (OTKA; No. T 014964), and a predoctoral CIRIT grant (1997 FI 000073) awarded to P. Sospedra. We acknowledge the technical assistance of H. Carvajal. LA981305I
