Langmuir 1998, 14, 1861-1869
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Analysis of the Interaction with Biomembrane Models of the HAV-VP3(101-121) Sequence Conjugated to Synthetic Branched Chain Polypeptide Carriers with a Poly[L-Lysine] Backbone M. Garcı´a,† I. B. Nagy,‡ M. A. Alsina,§ G. Mezo¨,‡ F. Reig,† F. Hudecz,‡ and I. Haro*,† Departament de Quimica de Pe` ptids i Proteı¨nes, CID, CSIC, Jordi Girona, 18-26, 08034-Barcelona, Spain, Unitat de Fisicoquı´mica, Facultat de Farma` cia, Pius XII s/n, 08028-Barcelona, Spain, and Research Group of Peptide Chemistry, Hungarian Academy of Sciences, Eo¨ tvo¨ s L. University, Budapest 112, POB 32, H-1518, Hungary Received April 29, 1997. In Final Form: January 15, 1998 The conjugation of the [Abu105,109 ] VP3(101-121) peptide sequence from the VP3 capsid protein of hepatitis A virus to synthetic branched polypeptide carriers is described. The establishment of either a disulfide or an amide bond between the peptide and the carrier molecules is reported. The interaction of VP3 conjugates with dipalmitoylphosphatidylcholine (DPPC) mono- and bilayers was studied, and the influence of the different hydrophilicities and peptide orientations on the conjugates is discussed. Results showed an increased interaction with biomembrane models due to the conjugation of VP3 peptide to the described macromolecular structures.
Introduction The covalent conjugation of small molecules possessing specific properties to a protein or a synthetic carrier can lead to the development of diverse bioactive compounds. According to this approach, some different strategies have been extensively used for the induction and analysis of protective immune responses against infectious diseases. One of them is the use of synthetic macromolecules named multiple antigen peptides (MAPs)1 containing defined B and/or T cell epitopes of one2,3 or more4 antigens. Unlike the case for peptide carrier-protein (i.e. KLH, BSA) conjugates, the size, number, ratio, and relative position of T and B epitopes in MAPs can be well-defined.5 Another possibility that was first suggested by Sela6 is the covalent attachment of epitopes to synthetic linear or branched polymeric peptides of controlled chemical composition. New generations of branched chain poly-R-amino acids with a poly(L-lysine) backbone have been introduced by Hudecz and Szekerke for the rational design of polymeric polypeptides for construction of synthetic antigens7 and for drug delivery.8 Recently, a new class of sequential oligopeptide carriers (SOCn), namely (Lys-Aib-Gly)2-7, has * To whom correspondence should be addressed. † CID, CSIC. ‡ Hungarian Academy of Sciences. § Unitat de Fisicoquı´mica. (1) Tam, J. P. J. Immunol. Methods 1996, 196, 17-32. (2) Mota, F. M.; Busquets, M. A.; Reig, F.; Alsina, M. A.; Haro, I. J. Colloid Interface Sci. 1997, 188, 81-93. (3) Pe´rez, J. A.; Gonza´lez-Dankaart, J. F.; Reig, F.; Pinto´, R. M.; Bosch, A.; Haro, I. Biomed. Pept. Proteins Nucleic Acids 1995, 1, 93100. (4) Firsova, T.; Pe´rez, J. A.; Reig, F.; Kruglov, I. V.; Haro, I. Peptides 1996, in press. (5) Nardin, E. H.; Oliveira, G. A.; Calvo-Calle, M.; Nussenzweig, R. S. Adv. Immunol. 1995, 60, 105-127. (6) Sela M. In Molecules, cells and parasites in immunology; Larralde, C., Wills, K., Ortiz-Ortiz, L., Sela, M., Eds.; Academic Press: New York, 1980; pp 215-228. (7) Hudecz, F. Biomed. Pep., Proteins Nucleic Acids 1995, 1, 213220. (8) Hudecz, F. Anti-cancer Drugs 1995, 6, 171-193.
also been developed9 in order to predetermine structural motifs exhibiting defined spatial orientation of the functional sites. In this study the conjugation of the LASI(Abu)QMF(Abu)FWRGDLVFDFQV peptide sequence from a hepatitis A virus capsid to synthetic branched polypeptides with the general formula poly[Lys-(Xi-DL-Alam)] (XAK) (where X ) Glu10 or Ser,11 m ∼ 3, and i < 1) is reported. This sequence was selected in order to link collinearly a previously defined continuous epitope of HAV, VP3(110121),3 to the maximum amphipathicity criteria T epitope VP3(101-109). The anchoring of this antigenic peptide to the above-mentioned carriers allows two different presentations for the predicted epitope, depending on the used conjugation strategy. Thus, a BT orientation is obtained with the carrier-peptide amide bond while a TB one results when the conjugation is done through disulfide bond. To use these structures as immunogens, it is convenient to know how they interact with biological membranes. But, due to its complexity, lipid mono- and bilayers can provide a good model to study these interactions. In the present work the interaction with phospholipid mono- and bilayers, used as biomembrane models, of the VP3(101121) branched polypeptide conjugates is reported. The influence of the different hydrophilicities as well as the peptide orientation in the conjugates is discussed. For this purpose, the ability of the HAV VP3(101-121) conjugates to insert into dipalmitoylphosphatidylcholine monolayers has been determined. Moreover, changes in the fluidity of bilayers induced by these macromolecules by means of polarizable probes such as 8-anilino-1-naphthalenesulfonic acid and 1,6-diphenyl-1,3,5-hexatriene were measured. (9) Tsikaris, V.; Sakarellos, C.; Sakarellos-Daitsiotis, M.; Orlewski, P.; Marraud, M.; Thong Cung, M.; Vatzaki, E.; Tzartos, S. Intl. J. Biol. Macromol. 1996, 19, 195-205. (10) Hudecz, F.; Szekerke, M. Collect. Czech. Chem. Commun. 1980, 45, 933-940. (11) Mezo¨, G.; Katja´r, J.; Nagy, I.; Szekerke, M.; Hudecz, F. Biopolymers, 1997, 42 (6), 719-730.
S0743-7463(97)00443-5 CCC: $15.00 © 1998 American Chemical Society Published on Web 03/13/1998
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Experimental Section Materials. N-R-Fluorenylmethoxycarbonyl amino acids, HMPB linker (4-(4-(hydroxymethyl)-3-methoxyphenoxy)butyric acid), and methylbenzhydrylamine resin (MBHA resin) were obtained from Novabiochem, England. Dimethylformamide (DMF), dicloromethane (DCM), and piperidine/DMF 20% were from Milligen. Washing solvents such as isopropyl alcohol, acetic acid, and diethyl ether were obtained from Merck. Trifluoroacetic acid (TFA) was supplied by Fluka. 2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), N-hydroxybenzotriazole (HOBt), benzotriazol-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate (BOP), and N,N′-diisopropylcarbodiimide (DIPCDI) coupling reagents were obtained from Novabiochem. Dipalmitoylphosphatidylcholine (DPPC), anilinonaphthalene sulfonate (ANS), and diphenylhexatriene (DPH) were from Sigma. Chloroform and methanol were from Merck. Water was double distilled. All the experiments were carried out with the conjutates dissolved in dimethyl sulfoxide (DMSO), sodium acetate solutions, pH ) 7.4, or 10 mM HEPES buffer, pH ) 7.4. Peptide Synthesis. Solid-phase synthesis of the [Abu105,109] VP3(101-121) protected sequence was carried out on a MBHA resin in a manual way following the Fmoc/tBut strategy. MBHA resin (1 g, 0.57 mequiv/g) was kept in DMF for 2 h with 1.5 equiv of HMPB,12 HOBt, and DIPCDI. This step was repeated twice until obtaining a negative Kaiser’s test. The first amino acid of the sequence (Fmoc-Val) was esterified to the handle twice using HBTU reagent, achieving anchoring yields higher than 95%. Thus, the residual benzyl hydroxy groups were left uncapped, as they do not interfere in subsequent amino acid couplings. The following amino acids were used as carboxyl free derivatives and activated with DIPCDI/HOBt. Side chain protection was effected by the following: 2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc) for Arg, tert-butoxycarbonyl (Boc) for Trp, and tert-butyl for Asp and Ser. Three-fold molar excesses of Fmoc-amino acids were used throughout the synthesis. 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. Repeated couplings were needed for the incorporation of Phe119, Leu115, Asp114, Glu113, Trp111, and Abu109. At the completion of the introduction of Ala102, the peptide resin was removed from the reaction column, washed with DMF, isopropyl alcohol, and ether, dried in vacuo, and divided in two parts. Half of the peptide resin was elongated with Fmoc-Leu and Fmoc-Cys(Trt) in order to obtain a thiol terminal group that allowed formation of a disulfide bond with the branched polypeptide carrier. The remaining half of the peptide-resin was elongated with Boc-Leu to obtain the protected sequence to be attached through an amide bond to the carrier. For the detachment of protected peptides from the resin, 500 mg of each peptide resin was preswollen in DCM, shaken for 2 min with 5 mL of 1% TFA in DCM, and filtered in vacuo into 1 mL of MeOH/pyridine 9/1. This last step was repeated until a TLC test proved the completeness of cleavage. The combined cleavage portions were concentrated, followed by precipitation of the peptide with diethyl ether, lyophylization, and washing with water in order to remove salts. By this process the crude protected peptides [Abu105,109] VP3(101-121) and [Abu105,109] CysVP3(101-121) were obtained. Side chain deprotection of [Abu105,109] Cys-VP3(101-121) was effected as follows: the lyophylized peptide was treated for 2 h with TFA/EDT/H2O (9/0.8/0.2), the crude peptide was then precipitated with diethyl ether, the sample was sonicated and centrifuged, and the supernatant was decanted off. This last step was repeated until the total removal of scavengers. Finally, the peptide was dissolved in water and lyophylized. The synthesized peptides were characterized by analytical HPLC, amino acid analysis, and electrospray mass spectrometry. HPLC analyses were performed on a Spherisorb ODS (10 µm) column eluted with acetonitrile/water (0.05% TFA) mixtures. Conditions used for the deprotected sequence [Abu105,109] Cys(12) Flo¨rsheimer, A.; Riniker, B. In Peptides, 1990; Giralt, E., Andreu, D., Eds.; ESCOM: Leiden, The Netherlands, 1991, pp 131-133.
Garcı´a et al. VP3(101-121) were 5 min isocratic of 60% acetonitrile/H2O (80/ 20) followed by a gradient from 60% to 100% during 30 min. Eluted substances were detected spectrophotometrically at 215 nm. A single peak with a retention time of 24 min was obtained. In the case of the protected sequence [Abu105,109 ] VP3(101-121) the conditions used were 5 min isocratic of 70% acetonitrile followed by a gradient from 70% to 100% during 15 min and a final isocratic step of 100% during 10 min. A single peak with a retention time of 20 min was obtained. Satisfactory amino acid analyses were obtained. The analyses were carried out in a Pico-Tag system (Waters). Samples of 2-5 mg of the peptides were hydrolyzed in 6 M HCl at 110 °C over 24 h. Electrospray-MS spectra were obtained by using a Finnigan (San Jose´, CA) TSQ 700 triple quatropole mass spectrometer equipped with an Analytica of Branford (Branford, CT) electrospray interface. Samples were disolved at a concentration of 20 pmol/µL in MeOH/H2O/AcOH 1/1/0.01 and introduced into the electrospray source at a rate of 1 µL/min using a Harvard Apparatus (Southnatick, MA) perfusion pump. Operating conditions were as follows: electrospray voltage, 3500 V (positive ions); drying nitrogen flow, 5 mL/min; scan range, 1000-6000 units; scan rate, 4 s. Synthesis of Branched Polypeptide Conjugates. The branched polypeptide carriers poly[Lys(DL-Alam)] (AK), poly[Lys(Glui-DL-Alam)] (EAK), and poly[Lys(Seri-DL-Alam)] (SAK) were synthesized as previously described10,11,13 by polymerization of NR-carboxy-N-(benzyloxycarbonyl)lysine anhydride, followed by cleavage of the protecting groups and grafting of short oligomeric DL-Ala side chains onto the -amino groups of poly(Lys). Ser or Glu was attached to the end of the side chains of AK by the HOBt-catalyzed active ester method.13 The coupling of the HAV VP3(101-121) peptide sequence to branched polypeptides was achieved by two different strategies: by the insertion of a disulfide bond between the peptide and the carrier molecule14 or by formation of an amide bond between the carboxy terminus of the protected peptide and the R-amino group of Ala (AK), Ser (SAK), or Glu (EAK) on the N-terminus of the side chain. A heterobifunctional reagent, N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP),15 was used for the formation of a disulfide bond. First, protected and activated thiol groups were introduced to the side chains terminating the R-amino groups of the carrier polypeptides by reacting AK, SAK, or EAK with SPDP in alkaline solution. In the second step, the disulfide bond was established by the thiolysis of PySS-polypeptide using the free thiol group of deprotected [Abu105,109] Cys-VP3(101-121). This reaction was followed by analytical HPLC, measuring the presence of free carriers spectrophotometrically at 215 nm. Coupling through an amide bond was performed with the molar ratio of branched polypeptide/protected Boc-VP3(101-121)/BOP/ HOBt/DIEA (1/0.6/3.6/3.6/7.2). Briefly, 1 mmol of polymer was dissolved in 0.5 mL of deionized water and then this solution was diluted with 5 mL of DMF and added dropwise to 0.6 mmol of protected peptide in 5 mL of DMF. The mixture was stirred and treated with 3.6 mmol of BOP, 3.6 mmol of HOBt, and 7.2 mmol of DIEA. The reaction mixture was stirred overnight at room temperature. A previous carboxyl activation of the protected peptide with BOP and HOBt for 30 min was necessary for conjugation to EAK polypeptide. After conjugation, cleavage of HAV VP3(101-121) protecting groups was performed with TFA/EDT/H2O (9/0.8/0.2). Monolayer Insertion Experiments. To determine the spreading pressure of the samples, increasing volumes of concentrated solution were injected beneath the surface of 10 mM HEPES, pH ) 7.4, and pressure increases were recorded. The capacity of the Teflon trough was 70 mL. To evaluate peptide insertion into monolayers, a slightly lower concentration than that corresponding to the spreading pressure was chosen. The influence of carrier hydrophilicity and peptide orientation was then studied. (13) Mezo¨, G.; Votavova, H.; Hudecz, F.; Kajta´r, J.; Sponar, J.; Szekerke, M. Collect. Czech. Chem. Commun. 1988, 53, 2843-2853. (14) Hudecz, F.; Price, M. R. J. Immunol. Methods 1992, 147, 201210. (15) Carlsson, J.; Drevin, H.; Axen, R. Biochem. J. 1978, 723-728.
Biomembrane Models of the HAV-VP3(101-121) Sequence Table 1. Analytical Data of Synthetic Peptides [Abu105,109] VP3(101-121) (protected sequence)
peptide amino acid analysisa
HPLC tR (retention time)b ESI-MS
[Abu105,109] Cys-VP3(101-121) (deprotected sequence)
D ) 2.10 (2) S ) 0.60 (1) Q ) 2.09 (2) G ) 1.01 (1) A ) 1.04 (1) V ) 2.16 (2) M ) 0.80 (1) I ) 0.93 (1) L ) 1.88 (2) F ) 3.70 (4) R ) 0.96 (1) W not determined 20 min
D ) 2.10 (2) S ) 0.60 (1) Q ) 2.04 (2) G ) 0.98 (1) A ) 0.99 (1) V ) 2.18 (2) M ) 0.74 (1) I ) 0.97 (1) L ) 1.91 (2) F ) 3.87 (4) R ) 0.96 (1) W and C not determined 24 min
[M]+ ) 3124.64
[M]+ ) 2593.2
a Theory values in parentheses. b HPLC conditions are described in the Experimental Section.
Fluorescence Polarization Studies. Fluorescence polarization was determined using a Perkin-Elmer spectrofluorimeter LS 50, equipped with a thermostatable cuvette holder and polarizers. Small unilamellar vesicles were prepared as previously described16 from DPPC after hydration with 0.25 M sodium acetate of pH 7.4. The diameter of the vesicles determined by quasi-elastic light scattering was 80 ( 5 nm. Liposomes saturated with ANS or DPH were mixed with the peptides and polarization fluorescence was determined as a function of temperature. Excitation and emission wavelengths were 380 and 480 nm and 365 and 425 nm, respectively. The observed fluorescence intensities and polarization were compared with those of probe/ liposomes and probe/samples. Membrane fluidity was estimated on the basis of the reciprocal of polarization. The temperature range was 20-50 °C.
Results and Discussion Peptide Synthesis and Conjugation to Branched Polypeptide Carriers. The syntheses of protected HAV[Abu105,109] VP3(101-121) and deprotected HAV-[Abu105,109] Cys-VP3(101-121) sequences were accomplished by using an orthogonal strategy based on the attachment of the Riniker handle (HMPB) to MBHA resin, as described in the Experimental Section. This method allows the specific cleavage of the peptide sequence from the polymeric support without side chain deprotection.17 Semipreparative HPLC was used for purification of crude peptides. The purity achieved was >95%. As shown in Table 1 the synthesized peptides were well-characterized by analytical HPLC, amino acid analysis, and electrospray mass spectrometry. Six different conjugates of the selected VP3 peptide sequence were prepared in order to study the increment in the immunoresponse and thus to obtain two different presentations for the antigenic sequence: “BT orientation” for AK-VP3(101-121), SAK-VP3(101-121), and EAKVP3(101-121) and “TB orientation” for AK-C-VP3(101121), SAK-C-VP3(101-121), and EAK-C-VP3(101-121). These polypeptides are based on a poly[L-Lys] backbone and contain short side chains composed of about three DL-Ala residues and one Ser or Glu residue at the end of the branches in the case of SAK11 and EAK.10 The identity (16) Cajal, Y.; Alsina, M. A.; Garcı´a-Anton, J. M.; Haro, I.; Reig, F. In Microencapsulation of drugs; Whateley, T. L., Ed.; Harwood Academic Publishers: Newark, NJ, 1992; p 139. (17) Riniker, B.; Flo¨rsheimer, A.; Fretz, H.; Sieber, P.; Kamber, B. Tetrahedron 1993, 49 (41), 9307-9320.
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of the branch-terminating amino acid residue was selected to study the influence of carrier hydrophilicity in the interaction of conjugates with biomembrane models.18 In this sense the polarity of conjugates was EAK > SAK > AK. The average degree of polymerization (DPn) of the branched polypeptides was determined by means of sedimentation analysis.19 The amino acid composition of the carriers and the average substitution ratio of the peptide sequence in the polymer were determined by amino acid analysis. As shown in Table 2 the average degree of substitution for conjugates with a disulfide bond was in the range 45-60%, while in the case of the BT orientation for the peptide sequence (amide bond) the desired conjugates yielded an average substitution ratio of 25-30%. Kinetics of Penetration. Monolayer Binding Properties. The ability of free branched chain polypeptides (AK, SAK, and EAK)18 and HAV-[Abu105,109 ] VP3(101121) conjugates to insert into DPPC monolayers was assessed by injecting the samples beneath phospholipid monolayers spread at diferent initial surface pressures, 5, 10, 20, and 32 mN‚m-1, and recording the subsequent changes in the surface pressure while holding the surface area constant.20,21 The concentration of the samples in the 10 mM HEPES, pH ) 7.4 subphase was 3.2 × 10-2 µM; this value was selected according the previously determined surface activity values and corresponds to a point slightly lower than the spreading pressure of the products. The bilayer equivalence pressure of phospholipid monolayers is thought to be 30-34 mN‚m-1; thus the 32 mN‚m-1 DPPC initial surface was selected in order to approximate our results to more physiological ones.22 From this comparison, and as observed in Figure 1b and c, it is clear that the peptide-lipid interactions observed in these monolayer experiments are strong enough for significant incorporation of HAV-[Abu105,109] VP3(101-121) conjugates into phospholipid bilayers.23 As usual, the higher the surface pressure, the lower the pressure increase by the peptide insertion into the monolayer (Figure 1). As shown in Figure 1a, the contribution of the polymeric carriers to the surface pressure increase is low in comparison to the values obtained for conjugates, thus proving that the interaction with DPPC monolayers is mainly due to the peptide sequence attached to the carrier. On the other hand, a certain relationship between the insertion into phospholipid monolayers and the hydrophobicity of the carriers is observed. As shown in Figure 1, the maximum surface pressure achieved corresponded to the more apolar polymer (AK), followed by SAK, EAK being the carrier which showed the lower phospholipid interaction and the higher hydrophilicity. In Tables 3-5 comparisons between of the ability of the six VP3 conjugates to insert into DPPC monolayers and the peptide sequence orientation in the conjugate are described. These tables show the parameters obtained by mathematical treatment of the experimental data from (18) Nagy, I. B.; Haro, I.; Alsina, M. A.; Reig, F.; Hudecz, F. Biopolymers, submitted. (19) Hudecz, F.; Kova´cs, P.; Kutassi-Ko´vacs, S.; Kajta´r, J. Colloid Polym. Sci. 1984, 262, 208-212. (20) Martin, I.; Haro, I.; Reig F.; Alsina, M. A. Langmuir 1994, 10 (3), 784. (21) Bogdam, K.; Alsina, M. A.; Haro, I.; Martı´n, I.; Reig, F. Langmuir 1994, 10, 4618. (22) Demel, R. A.; Geurts van Kessel, W. S. M.; Zwaal, R. F. A.; van Deenen, L. L. M. Biochim. Biophys. Acta 1975, 406, 97-107. (23) Tamm, L. K. Biochemistry 1986, 25, 7470-7476.
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Figure 1. Pressure increases recorded after injection of (a) AK, SAK, and EAK, (b) AK-C-VP3(101-121), SAK-C-VP3(101-121), and EAK-C-VP3(101-121), and (c) AK-VP3(101-121), SAK-VP3(101-121), and EAK-VP3(101-121) solutions under DPPC monolayers spread at 5, 10, 20, and 32 mN‚m-1 of initial surface pressure.
conjugate penetration kinetic curves, where ∆πm corresponds to the maximum pressure achieved at saturation
in mN‚m-1 and K is the time to achieve ∆πm/2. Curves were adjusted to rectangular hyperbola (eq 1) with Inplot4
Biomembrane Models of the HAV-VP3(101-121) Sequence
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Figure 2. Monolayer surface pressure increase for the AK carrier and its conjugates at 5 mN‚m-1 DPPC monolayer initial surface pressure versus time. Table 2. Analytical Data of the Conjugates HAV-VP3(101-121) with Branched Chain Polypeptide Carriers with a Poly[L-lysine] Backbone conjugate
DPn
MWmonomer
carrier amino acid composition
carrier/peptide
AK-VP3(101-121) AK-C-VP3(101-121) SAK-VP3(101-121) SAK-C-VP3(101-121) EAK-VP3(101-121) EAK-C-VP3(101-121)
58 58 58 58 58 58
348 348 436 436 478 478
(Lys/Ala) ) (1/3.1) (Lys/Ala) ) (1/3.1) (Lys/Ala/Ser) ) (1/3.1/1) (Lys/Ala/Ser) ) (1/3.1/1) (Lys/Ala/Glu) ) (1/3.1/0.9) (Lys/Ala/Glu) ) (1/3.1/0.9)
1/0.27 1/0.60 1/0.28 1/0.54 1/0.30 1/0.47
Table 3. Parameters Obtained by the Mathematical Treatment of Experimental Data from Penetration Kinetic Curves for AK-C-VP3(101-121) and AK-VP3(101-121)a AK-C-VP3(101-121) DPPC π0 (mN‚m-1)
∆πm b (mN‚m-1)
5 10 20 32
30.21 ( 1.41 24.28 ( 0.31 15.83 ( 0.19 5.61 ( 0.17
Table 5. Parameters Obtained by the Mathematical Treatment of Experimental Data from Penetration Kinetic Curves for EAK-C-VP3(101-121) and EAK-VP3(101-121)a
AK-VP3(101-121)
Kc (min)
∆πmb (mN‚m-1)
12.46 ( 1.99 4.29 ( 0.35 6.15 ( 0.38 15.76 ( 1.45
22.92 ( 0.25 16.76 ( 0.07 12.08 ( 0.29 7.46 ( 0.23
EAK-C-VP3(101-121)
Kc (min)
DPPC π0 (mN‚m-1)
∆πmb (mN‚m-1)
4.23 ( 0.29 2.73 ( 0.09 7.33 ( 0.81 16.34 ( 1.52
5 10 20 32
23.56 ( 0.46 20.68 ( 0.36 11.74 ( 0.17 6.85 ( 0.33
EAK-VP3(101-121)
Kc (min)
∆πmb (mN‚m-1)
Kc (min)
6.73 ( 0.63 6.69 ( 0.57 5.71 ( 0.45 20.82 ( 2.66
20.22 ( 0.20 17.93 ( 0.07 12.25 ( 0.18 9.04 ( 0.53
4.46 ( 0.19 2.32 ( 0.09 7.48 ( 0.51 53.08 ( 5.52
a Curves were adjusted to rectangular hyperbola with r2 > 0.97 in all cases. b Maximum pressure increase achieved at saturation. c Time to achieve ∆π /2. m
a Curves were adjusted to rectangular hyperbola with r2 > 0.97 in all cases. b Maximum pressure increase achieved at saturation. c Time to achieve ∆π /2. m
Table 4. Parameters Obtained by the Mathematical Treatment of Experimental Data from Penetration Kinetics Curves for SAK-C-VP3(101-121) and SAK-VP3(101-121)a
jugates containing the HAV-[Abu105,109] VP3(101-121) with the free carboxyl terminal group (“TB orientation”) would be the most suitable for establishing an increased interaction with biomembrane models.
SAK-C-VP3(101-121) DPPC π0 (mN‚m-1)
∆πmb (mN‚m-1)
5 10 20 32
28.64 ( 1.31 24.35 ( 0.30 17.16 ( 0.08 10.28 ( 0.47
SAK-VP3(101-121)
Kc (min)
∆πmb (mN‚m-1)
Kc (min)
11.65 ( 1.61 7.80 ( 0.43 6.26 ( 0.14 30.35 ( 3.12
21.29 ( 0.11 22.67 ( 0.85 8.99 ( 0.23 9.66 ( 0.36
3.16 ( 0.13 9.68 ( 1.42 7.14 ( 0.87 38.99 ( 2.94
a Curves were adjusted to rectangular hyperbola with r2 > 0.97 in all cases. b Maximum pressure increase achieved at saturation. c Time to achieve ∆π /2. m
GraphPad software, with r2 > 0.97 in all cases. The same treatment was applied to the free polypeptide carriers AK, SAK, and EAK, but in this case the mathematical model was confusing, although, as shown as an example in Figure 2 for AK carrier and its conjugates at 5 mN‚m-1 DPPC initial surface pressure, the experimental curves for free polymers were always under the experimental conjugate ones. The obtained results suggest that con-
∆π )
(∆πmt) (K + t)
(1)
Comparing the present results to those obtained for the HAV-VP3(101-121) free sequence at the same conditions,24,25 an increased insertion into DPPC monolayers at lower subphase peptide concentrations was recorded for the described conjugates with respect to free peptide. These data suggest the suitability of the polypeptide carriers described to improve the interaction of the peptide sequence with biomembranes. The best results were obtained with the conjugates synthesized by means of a disulfide bond. (24) Garcı´a, M.; Pujol, M.; Reig, F.; Alsina, M. A.; Haro I. Analyst 1996, 121, 1583-1588. (25) Garcı´a, M.; Pujol, M.; Reig, F.; Alsina, M. A.; Haro, I. Biomed. Chromatogr. 1996, 11, 121-123.
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Figure 3. Fluorescence polarization of DPH-saturated liposomes after incubation with (a) AK, SAK, and EAK. (b) AK-C-VP3(101-121), SAK-C-VP3(101-121), and EAK-C-VP3(101-121), and (c) AK-VP3(101-121), SAK-VP3(101-121), and EAK-VP3(101-121), measured at different temperatures.
Effect of Branched Chain Polypeptide Carriers and Their Conjugates with HAV-[Abu105,109] VP3(101-121) on Membrane Fluidity. Two fluorescent
probes, ANS and DPH, were used to determine structural or conformational changes induced by the polymeric carriers18 and their conjugates with the peptide sequence
Biomembrane Models of the HAV-VP3(101-121) Sequence
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Figure 4. Fluorescence polarization of ANS-saturated liposomes after incubation with (a) AK, SAK, and EAK, (b) AK-C-VP3(101-121), SAK-C-VP3(101-121), and EAK-C-VP3(101-121), and (c) AK-VP3(101-121), SAK-VP3(101-121), and EAK-VP3(101-121), measured at different temperatures.
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Figure 5. Fluorescence polarization of ANS molecules in solution after incubation with AK-C-VP3(101-121) and AK-VP3(101121) measured at different temperatures.
in bilayers composed of DPPC. ANS26 is noncovalently incorporated into the polar heads of the phospholipids and its fluorescence is highly sensitive to changes in the molecular environment of the bilayer, increasing when the molecule moves from a polar medium toward a lesspolar one. Moreover, a hydrophobic fluorescent probe, DPH,27 was selected to get information about the fluidity in the internal core of the bilayers. The first step in these studies was the incorporation of the probe into the bilayers. Liposomes were incubated with increasing amounts of ANS or DPH, and fluorescence intensity was recorded in order to determine the saturating probe concentrations. DPPC bilayers were saturated at an ANS/PL molar relationship of 0.35 × 10-3 and a DPH/ PL molar relationship of 5.7 × 10-3. Polarization of DPH-Labeled Liposomes. Liposomes saturated with DPH were incubated with each of the following synthetic products: AK, SAK, EAK, AKVP3(101-121), SAK-VP3(101-121), EAK-VP3(101-121), AK-C-VP3(101-121), SAK-C-VP3(101-121) and EAKC-VP3(101-121). The fluorescence and polarization values of this probe as a function of the temperature were determined by applying eq 2, where IVv and IVh are the observed intensities measured with polarizers parallel and perpendicular to the vertically polarized exciting beam, respectively. G is a factor used to correct for the inability of the instrument to transmit equally differently polarized light.
P)
IVv - GIVh IVv + GIVh
(2)
Typical experimental results with DPPC vesicles are shown in Figure 3. The depolarization of DPH fluorescence at the phase transition due to changes in fluidity is quite dramatic. Below the transition temperature (Tc), depolarization approaches the maximum value (0.5), while, above it, the phase transition of DPH is almost completely depolarized. (26) Joshi, U. M.; Kodavanti, P. R. S.; Lockard, V. G.;Mehendale, H. M. Biochem. Biophys. Acta 1989, 1004, 309-320. (27) Schwarz, S. M.; Lambert, A. S.; Medow, M. S. Biochim. Biophys. Acta 1992, 1107, 70-76.
As shown in Figure 3a no significant differences were detected between the free polymeric carriers AK, SAK and EAK and phospholipid bilayers. When the [Abu105,109] VP3(101-121) peptide was conjugated to the carrier, the polarization did not decrease strongly with temperature, thus proving that the conjugates have a rigidifying effect on the alkyl chains of the bilayer (Figure 3b and c). Interaction was improved for conjugates in which the peptide was linked to free carrier by a disulfide bond. This result would probably be related with a different peptide orientation that allows a different conformation in comparison to that of the conjugates formed by an amide bond. As shown in Figure 3, no significant differences in the fluorescence polarization behavior were observed for the three polypeptide carriers with different amino acid compositions. Polarization of ANS-Labeled Liposomes. Although polarization values of ANS do not change as drastically as those of DPH due to the location of this probe near the headgroups, a temperature dependence was also clear. In this case, a clear gradation in the interaction of the different polymeric carrier compositions with DPPC bilayers was observed. Similarly as obtained at kinetics of penetration studies, the better interaction corresponded to conjugates which have AK in their composition (Figure 4). On the other hand, as observed in fluorescence polarization of DPH-labeled liposomes, the type of bond between peptide and polymeric carrier has some relevance in the influence of the peptide in membrane fluidity. In this sense, disulfide bond conjugates increase the polarization response and thus the interaction with phospholipid polar heads of the HAV-[Abu105,109] VP3(101-121) peptide. As observed in Figure 4b and c, the hydrophobic conjugates, AK-VP3(101-121) and AK-C-VP3(101121), induce a slight increase of polarization with temperature over the Tc, achieving the values P > 0.5. These experimental artifact data could be explained as a result of two different phenomena: the interaction of the ANS probe with conjugate molecules which cover the liposome surface, and the interaction between ANS and lipidic molecules. As conjugates are macromolecular structures, they could “chelate” the probe from the vesicles. These
Biomembrane Models of the HAV-VP3(101-121) Sequence
results are consistent with other studies28 using an ANS fluorescence probe to determine the exposed hydrophobic surfaces of proteins. In our case, this possibility was also checked by studying the polarization and fluorescence intensity values of AK-C-VP3(101-121) and AK-VP3(101-121)/ANS mixtures. Solutions of the above-mentioned conjugates prepared at the same concentration as used in the studies with liposomes were mixed with ANS, and after 1 h of incubation the fluorescence polarization and intensity were recorded as a function of the temper(28) Islam, T. A.; Miller-Martini, D. H.; Horowitz, P. H. J. Biol. Chem. 1994, 269 (11), 7908-7913.
Langmuir, Vol. 14, No. 7, 1998 1869
ature (Figure 5). Polarization values were high and nearly constant in the range of temperatures under study (2350 °C), suggesting the presence of an ordered structure of the AK conjugate molecules in solution, which could be able to accommodate ANS molecules. Acknowledgment. This work was supported by Grants BIO95-0061-CO3-02 and BIO95-0061-CO3-03 from CICYT, Spain and by Hungarian Research Fund (OTKA) No. T014964. A predoctoral CIRIT grant (FI/958109) awarded to M.G. is also acknowledged. LA970443P
