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Physicochemical properties of hepatitis B peptide fragments at the air-water and lipid-water interfaces
Thin Solid Films, 210/211 (1992) 750 752
750
Physicochemical properties of hepatitis B peptide fragments at the air-water and lipid-water interfaces M. A. Alsina ~, C. Mestres ", F. Rabanal b, M. A. Busquets a and F. Reig b '~Ph.vsicoehemical Department, Faculty of Pharmacy, University of Barcelona, Pla~'a Pius Xll, 08034 Barcelona (Spain) bPeptide Laboratory, Jordi Girona 18-26, 08034 Barcehma (Spain)
Abstract In the present paper the interaction of four lipopeptides derived from the Pre S ( 120 145) sequence of the hepatitis B virus (HBV) with different lipids is described. The parent peptide structure was M Q W N S T A L H Q A L Q D P R V G L Y L P A G G . The peptide derivatives contained a residue of Pam3CSS, cholanoyl and stearoyl residues attached to their amino terminal end in order to modify the hydrophobicity of the peptide and to improve their potential antigenicity. The interactions have been studied using compression isotherms of monolayers and penetration kinetics. All the peptides showed surface activity and formed stable monolayers. The presence of these peptides under a monolayer of DPPC caused an expansion in the area occupied per molecule and modified the phase change of the DPPC.
I. Introduction
2. Experimental
The possibility that small synthetic peptides with the sequence of antigenic determinants of a protein will react with antibodies to the intact native antigen and neutralize its biological activity has been investigated in order to find a new generation of vaccines [1, 2]. Although the development of the immunogenic response is a complex phenomenon, it seems widely accepted that specific interactions between the peptide sequence and the lipid components of the membrane should play an important role, at least in the first steps of the recognition process [3]. The simplest membrane models to study lipid/ molecule interactions are probably the monomolecular layers of phospholipids spread at the air-water interface. This system can be considered as the outer half of the membrane bilayer and has been frequently used to detect and measure specific interactions between lipids and biologically active molecules [4, 5]. In the present paper four synthetic peptides corresponding to the sequence HBV-Pre S (120-145) have been studied as far as their interactions with different phospholipids are concerned. Compression isotherms of the pure peptides and of DPPC monolayers spread on subphases containing these peptides have been carried out. Moreover, the penetration of these peptides in monolayers of the same lipid composition were studied.
2. I. Chemicals
0040-6090/92/$5.00
The HBV-Pre S sequence (MQWNSTALHQALQDP R V R G L Y L P A G G ) and the corresponding lipophilic analogues were synthesized using an automatic peptide synthesizer (LKB Biolynx 4170). After deprotection, peptides were purified by semipreparative HPLC and their purity was checked by analytical HPLC. Final characterization was carried out by amino acid analysis and FAB mass spectrometry [6]. Egg phosphatidylcholine (PC) was supplied from Merck and purified according to Alsina et al. [7]. Bovine phosphatidyl serine (PS), phosphatidyl inositol (PI), phosphatidic acid (PA), phosphatidyl ethanolamine (PE), dipalmitoylphosphatidyl choline (DPPC) and dicetylphosphate (DCP) were purchased from Sigma. Sphingomyelin (SPH) and sulphatides (S) were from Supelco. Lipid purity was checked by TLC using C~8 coated silica gel 60 plates (Merck) and chloroform/methanol/ water (6.5:4.5:0.4) as developing system. Chloroform and methanol used as spreading solvents were from Merck. 2.2. M e t h o d s
The penetration studies were carried out according to Reig et al. [8]. Lipid monolayers were prepared by spreading a chloroform solution of the lipid on the water surface. All experiments were carried out at
,~'~ 1 9 9 2
Elsevier Sequoia. All rights reserved
M. A. Alsina et al. / Physicochemical properties of hepatitis B peptide fragments
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Fig. 1. Compression isotherm of cholanoyl derivative spread on a subphase containing PBS.
Fig. 2. Compression isotherm of Pam3CSS derivative spread on a subphase containing PBS.
21 _+ 1 °C on a subphase of phosphate buffered saline (PBS). The samples were injected into the subphase, constantly stirred, in small volumes from aqueous solutions of the peptides. The concentration of the peptides in the subphase was 2 x 10 -s M. At this level of dilution, the surface activity of the peptides is null. Compression isotherms were carried out by spreading alcoholic solutions of the peptides on the aqueous surface, allowing 10 min to evaporate the solvent before compression [8]. The temperature of the aqueous subphase was maintained at 2 1 + 0 . 1 °C. When DPPC monolayers were spread on aqueous solutions of the peptides, compression started after 15 min of incubation. Penetration kinetics were performed by spreading the necessary amount of lipid to obtain monolayers at initial pressures of 5 and 20 mN/m. The concentration of peptides in the subphase was 3 x 10 s M. Pressure increases were recorded after 60 min.
ordered states, throughout the compression process, depending on the peptide derivative. The area/molecule, the surface pressure corresponding to the phase changes and the maximum pressure achieved were determined and were indicative of the existence of different conformations. The values of surface compressibility at 20 m/ mN were between 1.95 x 10 2 and 2.2 x 10 2m/mN for the three lipopeptides. This indicates that the peptides at the interface are effectively in a liquid state. The parent peptide Pre S (120-145) showed a compressibility value of 2.8 × 10 -2 at the same surface pressure. This implies that the intramolecular motions remain thawed during the compression process. The maximum compression pressures give information about the stability of the monolayer. In this case according to the values given in Table 1, the stearoyl and cholanoyl derivatives are the most stable. At surface pressures that are relevant to biological membranes the lipopeptides have molecular areas between 1.6 and 2.1 nm~/molecule. This value is consistent with an a-helix perpendicular to the interface or with a loop structure in which two antiparallel fl strands are linked by a/3 turn region [9]. The presence of these peptides under a monolayer of DPPC caused
3. Results and discussion
The four peptides formed insoluble surface monolayers (Figs. 1 and 2). The monolayers were in different
M. ,4. Alsina el a/. / Physieochemiea/ properties O/h~Tmlitis B peptide /i'aement.s
752
T A B L E 1. Phase change and m a x i m u m compression pressures of the tbur peptides Peptide
Phase change
Parent peptide Chol-peptide Stearyl-peptide Pam-peptide
M a x i m u m compression
Pressure (mN/m)
Area (nm2/molecule)
Pressure (mN/m)
Area (nm-~/molecule)
9.3 20. I 22.0 12.5
2.37 2.18 2.14 3.36
16.8 52.8 48.0 31.2
1.25 0.9 I 0.85 1.82
an expansion in the area occupied per molecule and modified the phase change of this lipid. This effect is illustrated in Fig. 3 for DPPC and cholanoyl derivative. When these peptides were injected under monolayers of PC, PI, PS, PA, SPH and DCP spread on PBS at 3 x 10 ~M concentration, equilibrium was achieved very slowly. The final pressure increases obtained for the cholanoyl peptide derivative are given in Table 2.
T A B L E 2. Pressure increases of cholanoyl derivative 13 x 10 s M) injected under dicetylphosphate (DCP), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PIL phosphatidic acid (PA) and sphingomyelin (SPH) monolayers (Pi 5 mN/mt, spread on PBS Monolayer
Pressure increase
DCP PC PS PI PA SPH
4.08 1.44 1.44 4.08 2.64 2.88
40 ¸
Although the peptide sequence was the same in all of them, no relationship could be found between the polarity of the lipids in the monolayers and the maximum pressure increases.
3O
References
~7 E
Z
E
Lid [t£
W rr" l:L
LU 0
20-
132 :D CO
10-
0,8
1,2 1,6 2
AREA ( n r n 2 . m o l c c
~)
Fig. 3. Compression isotherms of DPPC monolayers spread on subphases containing PBS ( • ) and cholanoyl derivative (Or).
1 R. A. Neurath, S. B. H. Kent, N. Strick, K. Parker, A. M. Couronce, M. M. Riottot, M. A. Petit, A. Budkowska, M. Girard and J. Pillot, Molecular hnmunol., 24 (1987) 975. 2 M. E. Patarroyo, R. Amador, P. Clavijo, A. Moreno, F. Guzman, P. Romero, R. Tascom A. Franco, L. Murillo, G. Ponton and J. Trujillo, Nature, 332 (1988) 158. 3 R. L. Hunter, in: B. H. T o m and H. R. Six (eds.), Liposomes and lmmunobiology, Elsevier/North-Holland, Amsterdam, 1980, p. 25. 4 F. Reig, M. A. Busquets, J. M. Garcla Ant6n, G. Valencia and M. A. Alsina, Int. J. Pharm., 44 (1988) 257. 5 N. A. Williams and N. D. Weiner, Int. J. Pharm., 50 (1989) 261. 6 F. Rabanal, I. Haro, F. Reig, and J. M. Garcia Ant6n, J. Chem. Soe., Perkin Trans., 1 (1991) 945. 7 M. A. Alsina, C. Mestres, G. Valencia, J. M. Garcia Anton and F. Reig, ColloM Polyrn. Sci., 266 (1988) 832. 8 FI Reig, C. Espigol, J. M. Garcia Anton, G. Valencia and M. A. Alsina, J. Bioenergetics Biomembr., 20 (1988) 533. 9 G. Fidelio, B. M. Austen, D. C h a p m a n and J. A. Lucy, Biochem J., 238 (1986) 301.
750
Physicochemical properties of hepatitis B peptide fragments at the air-water and lipid-water interfaces M. A. Alsina ~, C. Mestres ", F. Rabanal b, M. A. Busquets a and F. Reig b '~Ph.vsicoehemical Department, Faculty of Pharmacy, University of Barcelona, Pla~'a Pius Xll, 08034 Barcelona (Spain) bPeptide Laboratory, Jordi Girona 18-26, 08034 Barcehma (Spain)
Abstract In the present paper the interaction of four lipopeptides derived from the Pre S ( 120 145) sequence of the hepatitis B virus (HBV) with different lipids is described. The parent peptide structure was M Q W N S T A L H Q A L Q D P R V G L Y L P A G G . The peptide derivatives contained a residue of Pam3CSS, cholanoyl and stearoyl residues attached to their amino terminal end in order to modify the hydrophobicity of the peptide and to improve their potential antigenicity. The interactions have been studied using compression isotherms of monolayers and penetration kinetics. All the peptides showed surface activity and formed stable monolayers. The presence of these peptides under a monolayer of DPPC caused an expansion in the area occupied per molecule and modified the phase change of the DPPC.
I. Introduction
2. Experimental
The possibility that small synthetic peptides with the sequence of antigenic determinants of a protein will react with antibodies to the intact native antigen and neutralize its biological activity has been investigated in order to find a new generation of vaccines [1, 2]. Although the development of the immunogenic response is a complex phenomenon, it seems widely accepted that specific interactions between the peptide sequence and the lipid components of the membrane should play an important role, at least in the first steps of the recognition process [3]. The simplest membrane models to study lipid/ molecule interactions are probably the monomolecular layers of phospholipids spread at the air-water interface. This system can be considered as the outer half of the membrane bilayer and has been frequently used to detect and measure specific interactions between lipids and biologically active molecules [4, 5]. In the present paper four synthetic peptides corresponding to the sequence HBV-Pre S (120-145) have been studied as far as their interactions with different phospholipids are concerned. Compression isotherms of the pure peptides and of DPPC monolayers spread on subphases containing these peptides have been carried out. Moreover, the penetration of these peptides in monolayers of the same lipid composition were studied.
2. I. Chemicals
0040-6090/92/$5.00
The HBV-Pre S sequence (MQWNSTALHQALQDP R V R G L Y L P A G G ) and the corresponding lipophilic analogues were synthesized using an automatic peptide synthesizer (LKB Biolynx 4170). After deprotection, peptides were purified by semipreparative HPLC and their purity was checked by analytical HPLC. Final characterization was carried out by amino acid analysis and FAB mass spectrometry [6]. Egg phosphatidylcholine (PC) was supplied from Merck and purified according to Alsina et al. [7]. Bovine phosphatidyl serine (PS), phosphatidyl inositol (PI), phosphatidic acid (PA), phosphatidyl ethanolamine (PE), dipalmitoylphosphatidyl choline (DPPC) and dicetylphosphate (DCP) were purchased from Sigma. Sphingomyelin (SPH) and sulphatides (S) were from Supelco. Lipid purity was checked by TLC using C~8 coated silica gel 60 plates (Merck) and chloroform/methanol/ water (6.5:4.5:0.4) as developing system. Chloroform and methanol used as spreading solvents were from Merck. 2.2. M e t h o d s
The penetration studies were carried out according to Reig et al. [8]. Lipid monolayers were prepared by spreading a chloroform solution of the lipid on the water surface. All experiments were carried out at
,~'~ 1 9 9 2
Elsevier Sequoia. All rights reserved
M. A. Alsina et al. / Physicochemical properties of hepatitis B peptide fragments
50-
50-
40-
40-
i
E
E
E UJ tv"
E 30
W rr
tt) tt)
30-
tt) W O~ ,7
LII n W
751
20-
UJ
20-
o
U_ n,"
n,'
10-
10-
2 AREA (nrn 2. molec -1)
4
6
AREA (nm2. rnolec -~)
Fig. 1. Compression isotherm of cholanoyl derivative spread on a subphase containing PBS.
Fig. 2. Compression isotherm of Pam3CSS derivative spread on a subphase containing PBS.
21 _+ 1 °C on a subphase of phosphate buffered saline (PBS). The samples were injected into the subphase, constantly stirred, in small volumes from aqueous solutions of the peptides. The concentration of the peptides in the subphase was 2 x 10 -s M. At this level of dilution, the surface activity of the peptides is null. Compression isotherms were carried out by spreading alcoholic solutions of the peptides on the aqueous surface, allowing 10 min to evaporate the solvent before compression [8]. The temperature of the aqueous subphase was maintained at 2 1 + 0 . 1 °C. When DPPC monolayers were spread on aqueous solutions of the peptides, compression started after 15 min of incubation. Penetration kinetics were performed by spreading the necessary amount of lipid to obtain monolayers at initial pressures of 5 and 20 mN/m. The concentration of peptides in the subphase was 3 x 10 s M. Pressure increases were recorded after 60 min.
ordered states, throughout the compression process, depending on the peptide derivative. The area/molecule, the surface pressure corresponding to the phase changes and the maximum pressure achieved were determined and were indicative of the existence of different conformations. The values of surface compressibility at 20 m/ mN were between 1.95 x 10 2 and 2.2 x 10 2m/mN for the three lipopeptides. This indicates that the peptides at the interface are effectively in a liquid state. The parent peptide Pre S (120-145) showed a compressibility value of 2.8 × 10 -2 at the same surface pressure. This implies that the intramolecular motions remain thawed during the compression process. The maximum compression pressures give information about the stability of the monolayer. In this case according to the values given in Table 1, the stearoyl and cholanoyl derivatives are the most stable. At surface pressures that are relevant to biological membranes the lipopeptides have molecular areas between 1.6 and 2.1 nm~/molecule. This value is consistent with an a-helix perpendicular to the interface or with a loop structure in which two antiparallel fl strands are linked by a/3 turn region [9]. The presence of these peptides under a monolayer of DPPC caused
3. Results and discussion
The four peptides formed insoluble surface monolayers (Figs. 1 and 2). The monolayers were in different
M. ,4. Alsina el a/. / Physieochemiea/ properties O/h~Tmlitis B peptide /i'aement.s
752
T A B L E 1. Phase change and m a x i m u m compression pressures of the tbur peptides Peptide
Phase change
Parent peptide Chol-peptide Stearyl-peptide Pam-peptide
M a x i m u m compression
Pressure (mN/m)
Area (nm2/molecule)
Pressure (mN/m)
Area (nm-~/molecule)
9.3 20. I 22.0 12.5
2.37 2.18 2.14 3.36
16.8 52.8 48.0 31.2
1.25 0.9 I 0.85 1.82
an expansion in the area occupied per molecule and modified the phase change of this lipid. This effect is illustrated in Fig. 3 for DPPC and cholanoyl derivative. When these peptides were injected under monolayers of PC, PI, PS, PA, SPH and DCP spread on PBS at 3 x 10 ~M concentration, equilibrium was achieved very slowly. The final pressure increases obtained for the cholanoyl peptide derivative are given in Table 2.
T A B L E 2. Pressure increases of cholanoyl derivative 13 x 10 s M) injected under dicetylphosphate (DCP), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PIL phosphatidic acid (PA) and sphingomyelin (SPH) monolayers (Pi 5 mN/mt, spread on PBS Monolayer
Pressure increase
DCP PC PS PI PA SPH
4.08 1.44 1.44 4.08 2.64 2.88
40 ¸
Although the peptide sequence was the same in all of them, no relationship could be found between the polarity of the lipids in the monolayers and the maximum pressure increases.
3O
References
~7 E
Z
E
Lid [t£
W rr" l:L
LU 0
20-
132 :D CO
10-
0,8
1,2 1,6 2
AREA ( n r n 2 . m o l c c
~)
Fig. 3. Compression isotherms of DPPC monolayers spread on subphases containing PBS ( • ) and cholanoyl derivative (Or).
1 R. A. Neurath, S. B. H. Kent, N. Strick, K. Parker, A. M. Couronce, M. M. Riottot, M. A. Petit, A. Budkowska, M. Girard and J. Pillot, Molecular hnmunol., 24 (1987) 975. 2 M. E. Patarroyo, R. Amador, P. Clavijo, A. Moreno, F. Guzman, P. Romero, R. Tascom A. Franco, L. Murillo, G. Ponton and J. Trujillo, Nature, 332 (1988) 158. 3 R. L. Hunter, in: B. H. T o m and H. R. Six (eds.), Liposomes and lmmunobiology, Elsevier/North-Holland, Amsterdam, 1980, p. 25. 4 F. Reig, M. A. Busquets, J. M. Garcla Ant6n, G. Valencia and M. A. Alsina, Int. J. Pharm., 44 (1988) 257. 5 N. A. Williams and N. D. Weiner, Int. J. Pharm., 50 (1989) 261. 6 F. Rabanal, I. Haro, F. Reig, and J. M. Garcia Ant6n, J. Chem. Soe., Perkin Trans., 1 (1991) 945. 7 M. A. Alsina, C. Mestres, G. Valencia, J. M. Garcia Anton and F. Reig, ColloM Polyrn. Sci., 266 (1988) 832. 8 FI Reig, C. Espigol, J. M. Garcia Anton, G. Valencia and M. A. Alsina, J. Bioenergetics Biomembr., 20 (1988) 533. 9 G. Fidelio, B. M. Austen, D. C h a p m a n and J. A. Lucy, Biochem J., 238 (1986) 301.