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Spin-labelling study of interactions of ovalbumin with multilamellar liposomes and specific anti-ovalbumin antibodies
International Journal of Biological Macromolecules 40 (2007) 312–318
Spin-labelling study of interactions of ovalbumin with multilamellar liposomes and specific anti-ovalbumin antibodies Marija Brgles b , Krunoslav Mirosavljevi´c a , Vesna Noethig-Laslo a,∗ , Ruˇza Frkanec b , Jelka Tomaˇsi´c b a
“Rudjer Boˇskovi´c” Institute, Bijeniˇcka cesta 54, 10002 Zagreb, Croatia b Institute of Immunology Inc., P.O. Box 266, 10001 Zagreb, Croatia
Received 5 July 2006; received in revised form 18 August 2006; accepted 22 August 2006 Available online 30 August 2006
Abstract Ovalbumin (OVA) has been used continuously as the model antigen in numerous studies of immune reactions and antigen processing, very often encapsulated into liposomes. The purpose of this work was to study the possible interactions of spin-labelled OVA and lipids in liposomal membranes using electron spin resonance (ESR) spectroscopy. OVA was covalently spin-labelled with 4-maleimido-2,2,6,6-tetramethylpiperidine1-oxyl (TEMPO-maleimide), characterized and encapsulated into multilamellar, negatively charged liposomes. ESR spectra of this liposomal preparation gave evidence for the interaction of OVA with the lipid bilayers. Such an interaction was also evidenced by the ESR spectra of liposomal preparation containing OVA, where liposomes were spin-labelled with n-doxyl stearic acids. The spin-labelled OVA retains its property to bind specific anti-OVA antibodies, as shown by ESR spectroscopy, but also in ELISA for specific anti-OVA IgG. © 2006 Elsevier B.V. All rights reserved. Keywords: Spin-labelled ovalbumin; Multilamellar liposomes; Anti-ovalbumin antibodies
1. Introduction Liposomes are microscopic vesicles consisting of one or more lipid bilayers surrounding aqueous compartments. The physicochemical properties of liposomes depend mainly on the lipid composition that determines the liposomal charge as well as the fluidity of the liposomal membrane. A wide variety of different compounds can be encapsulated in liposomes and the resulting liposomal formulations are often more convenient for experimental and therapeutic purposes [1]. Liposomes have also been considered and tested as carriers of antigens, vaccines and immunomodulatory compounds and tested in different experimental models [2,3]. In numerous studies of immune reactions and effects of immunostimulators ovalbumin (OVA) has been used as a model antigen of low immunogenicity according to previously published recommendations [4]. OVA is a glycoprotein from avian egg white. It has a well-defined structure [5] and belongs
∗
Corresponding author. Tel.: +385 1 4561 136; fax: +385 1 4680 245. E-mail address: [email protected] (V. Noethig-Laslo).
0141-8130/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ijbiomac.2006.08.012
structurally to the serpin (serine protease inhibitor) superfamily but shows no protease inhibitor activity [6]. In our previous studies on immunostimulating properties of peptidoglycan monomer, GlcNAc-MurNAc-l-Ala-d-isoGln-mesoDAP(-NH2 )-d-Ala-d-Ala (PGM) and adamantyltripeptides, d(adamant-2-il)-Gly-l-Ala-d-isoGln (AdTP1) and l-(adamant2-il)-Gly-l-Ala-d-isoGln (AdTP-2) [7,8], OVA was used as an antigen. The encapsulation of PGM and AdTPs into liposomes [9] and possible interactions of these compounds with lipids in liposomal membrane were studied using ESR [10], but the possible interaction of OVA and lipids was not addressed. However, our recent results [11] on immune reaction to OVA encapsulated into liposomes demonstrated that the direction of immune reaction was shifted towards a specific T-helper phenotype (Th1) and this finding prompted us to study the interaction of OVA with liposomes using ESR. Only two reports published previously concern the spinlabelling of OVA. In one report [12], the label different from TEMPO-maleimide was mixed with OVA and the analysis of ESR spectra of not covalently labelled OVA described. In the other [13] the spin-label was covalently linked to OVA and used for immunizations of rabbits with the aim of eliciting specific
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antibodies to spin-label, but the ESR spectra were neither studied nor described. The interaction of OVA with liposomes was studied and reported previously in several papers, using different methods [14–16]. However, ESR studies on this subject have not been reported so far. The aim of the present work was to study if there are any interactions of OVA encapsulated into negatively charged, multilamellar liposomes with lipids in the liposomal membrane using either spin-labelled OVA or spin-labelled fatty acids incorporated into lipid bilayers. 2. Materials and methods 2.1. Materials Albumin, from chicken egg; grade VII; min 98%, l-␣phosphatidylcholine, type XI-E: from fresh egg yolk (eggPC), cholesterol from porcine liver (CHL), dicetyl phosphate (DCPh) and 4-maleimido-2,2,6,6,-tetramethylpiperidine-1-oxyl (TEMPO-maleimide) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals used for buffers were of analytical grade and purchased from Sigma (St. Louis, MO, USA) and Kemika (Zagreb, Croatia). Centricon Centrifugal Filter Devices YM-30 were from Millipore (Bedford, Ma, USA).
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2.3. The preparation of liposomes Multilamellar, negatively charged liposomes were prepared as described previously [10]. Briefly, 6.8 mg (8.92 × 10−6 mol) egg-PC; 2.5 mg (6.46 × 10−6 mol) CHL and 0.7 mg (1.28 × 10−6 mol) DCPh, giving a molar ratio of 7:5:1, were dissolved in 2 ml of CHCl3 . After rotary evaporation of the solvent the remaining lipid film was dried in vacuum for an hour and then dispersed by gentle hand shaking in 1 ml OVA solution in phosphate buffered saline (5 mg/ml in PBS). The liposome suspension was left overnight at 4 ◦ C to swell and stabilize. Liposomes were separated from non-encapsulated material by centrifugation at 300,000 × g (Optima XL-80K, Beckman Coulter) at 4 ◦ C for 1 h. The amount of OVA encapsulated into liposomes was determined by the HPLC method [11]. 2.4. Anti-ovalbumin antiserum Rabbits were immunized s.c. with 100 g of OVA. ISA 720 (Seppic, France) and PGM were used as adjuvants. Boosters were administered after 3-week intervals and bleeding performed 7 days after the third dose (unpublished results). AntiOVA IgG was determined by ELISA as described earlier [7,8]. 2.5. Enzyme-linked immunosorbent assay (ELISA)
2.2. Spin-labelling of ovalbumin 6.77 × 10−7 mol)
Ovalbumin (OVA) (30 mg, and TEMPOmaleimide (0.85 mg, 3.39 × 10−6 mol) were dissolved in 8 ml 0.1 M phosphate buffer, pH 7.4. The reaction mixture was stirred either for 4 or 24 h. Separation of the spin-labelled protein from the free maleimide derivative was achieved by simple gel chromatography on Sephadex G-25 (2.5 cm × 60 cm) in phosphate buffer pH 7.4 (Fig. 1). Fractions of 2 ml were collected and absorbance monitored spectrophotometrically at 250 and 280 nm. The pooled fractions of spin-labelled OVA were then concentrated by ultrafiltration on Centricon Centrifugal Filter Devices YM-30. No free spin label was found in the filtrate.
ELISA for determination of anti-OVA IgG was carried out with two OVA preparations (used for coating the plates), native OVA and spin-labelled OVA, respectively, and two assays run in parallel, using the same sera samples in both assays. The basic ELISA, applied for the assay of anti-OVA IgG [7,8], was slightly modified. Briefly, Costar (Cambridge, MA, USA) E.I.A./R.I.A. flat bottom, high binding, 96-well plates were coated with 100 l OVA or spin-labelled OVA (15 g ml−1 ) coating solution in 0.05 M bicarbonate buffer, pH 9.6, incubated at room temperature overnight and washed. After blocking and washing, the rabbit sera samples to be tested were added in serial twofold dilutions and incubated at room temperature for 20 h. After washing, the horseradish peroxidase-conjugated (goat) anti-rabbit IgG (Bio-Rad, Hercules, CA, USA) were added (100 l per well) and incubated at 37 ◦ C for 2 h. After washing, 100 l per well of o-phenylendiamine (OPD) solution was added and incubated for 30 min, in the dark at room temperature. The reaction was stopped by the addition of 50 l of 1 M H2 SO4 . Absorbance was read at 492 nm. The incubation buffer used throughout was phosphate buffered saline, pH 7.3, containing 0.05% Tween-20. All samples were assayed in duplicate. Sera from the rabbit were used as negative controls and the buffer alone as a blank on each plate. 2.6. The assay of sulphydryls
Fig. 1. An example of the chromatographic separation of spin-labelled OVA from free spin-label on Sephadex G 25 column (2.5 cm × 60 cm) in 0.1 M phosphate buffer, pH 7.4. The absorbance of the fractions at 250 and 280 nm was measured. The fractions were pooled as indicated: I spin-labelled OVA; II free spin-label.
The sulphydryl groups of OVA and of spin-labelled OVA were determined by the Ellman method [17], following denaturation with SDS. All solutions were prepared in 0.1 M
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phosphate buffer, pH 8.0 containing 0.2 mM EDTA. Calibration curve was done with cystein in the concentration range (0.8–5)× 10−5 mol dm−3 (r2 = 0.999). Concentration of 5,5 dithiobis(2-nitrobenzoic acid) was 6 × 10−4 M and the final volumes of standard and sample solutions were 3 mL. Absorbance at 412 nm was read after 5 min. Number of free sulphydryls was determined in native and SDS denaturated (2.5 %) OVA. Three separate experiments were carried out and the mean value was taken into account. 2.7. ESR spectra measurements ESR measurements were performed on a Varian E-9 spectrometer (10 GHz) equipped with a Bruker variable temperature control unit. The spectra were recorded with digital acquisition, EW-ESR Ware [18]. Sample capillaries were inserted into the standard 4 mm diameter quartz tubes and centred in a TE102 ESR cavity. To quantitatively determine the number of the maleimide spin-labels covalently bound per one OVA molecule, markers Cu(DMA)2 (aquabis(N,N-dimethyl-l-␣-alaninato)copper(II)) and MnCl2 of known concentration dissolved in water were used. The intensities of the spectra were calculated by double integration with the Bruker WIN-EPR program and ESR Ware. Simulations of ESR spectra of the spin-labelled ovalbumin were performed with the programs “Garlic” and “Chili” (EasySpin 2.5.1. software package [19,20]). Spectra were simulated and rotational correlation times calculated with the error, for sharp lines 0.2 ns and for broad lines 10 ns. Liposomes were spin-labelled with n-doxyl stearic acids (n = 5, 7, 12, 16) by the thin film method, with 1% of spin-labels added to the organic solvent before evaporation as described earlier [21]. ESR spectra of doxyl-stearic acids in liposomes reflect motional properties of the lipid bilayer. One important motional component of the lipid bilayer is wobbling of the long molecular axis about a direction perpendicular to the bilayer surface. This motional effect was measured by the ESR parameters, Amax (mT) and Amin (mT), from which the apparent order parameter S was calculated [21] S=
3(Amax − Amin ) × 0.5396 Amax + 2Amin
3. Results 3.1. Characterization of the spin-labelled ovalbumin Ovalbumin was covalently spin-labelled with a maleimide derivative using the procedure for the unspecific spin-labelling of proteins [22,23]. The ESR spectrum of the maleimide spinlabelled OVA is shown in Fig. 2a, full line. The spectrum is characterized by five resonant transitions, denoted by numbers from 1 to 5. Well-defined outer hyperfine lines denoted by 1 and 5 separated by the hyperfine splitting, 2Amax = 6.5 mT, indicate the highly restricted motion of the labels. Sharp lines denoted
Fig. 2. ESR spectra of spin-labelled ovalbumin: spectrum (a); full line is compared with the dotted line spectrum taken in the presence of 0.5 M Cr-oxalate. The spectra were taken under same conditions, microwave power 10 mW, modulation amplitude 1.3 G under the same receiver gain. Spectrum (b) is denaturated sample in the presence of SDS. Spectra were taken at room temperature (20 ◦ C).
by 2 and 4 indicate a fast motion of the covalently bound labels to OVA. Location of the spin-label binding sites on the protein was determined by addition of 0.5 M Cr-oxalate (CrOx ) (Fig. 2a, dotted line). As a result of the dipole–dipole interaction between paramagnetic Cr ions and the nitroxides a decrease in the lineheights (i.e. broadening of the lines) was detected only for nitroxides with the fast motion (lines 2–4). These spin labels are located at/or near the surface of the protein i.e. accessible to the solvent and to the spin exchange broadening. Possible dipole–dipole interaction between Cr and immobilized nitroxides, would produce diminution of the ESR line heights (1 and 5) [24,25]. The spectral lines due to the restricted motion were simulated with the same parameters (same linewidths) with Chili program [20]. This proved that the spin labels with the restricted motion are not accessible to CrOx i.e. they are located in the protein core. Denaturation of the sample with SDS caused increased tumbling of the spin labels relative to the protein and exposal of all the labels to the solvent. In the presence of CrOx in the denatured sample all the spectral lines are severely broadened by spin exchange interactions. The number of spin-labels covalently bound per OVA molecule was determined by double integration of the spectra relative to the spectra of Cu(DMA)2 and/or MnCl2 as external markers. The ratio of the intensity of the spin-label and the intensity of the marker was found to be 0.5 for similar concentrations of the OVA and the marker (5 × 10−4 M). The result of 0.5 obtained in several different spin-labelled OVA preparations indicates that only one spin-label, at most, was covalently bound per OVA molecule. If motion of the spin-label covalently bound to protein is fully restricted with respect to the protein, the rotational correlation time, τ, calculated from the line shapes of the spectra describes Brownian motion of the whole protein molecule. Separation of the outer hyperfine lines,
M. Brgles et al. / International Journal of Biological Macromolecules 40 (2007) 312–318
2Amax = 6.5 mT, suggests fully restricted motion of the label relative to the protein, compared to 2Amax = 6.6 mT in the frozen sample. In all preparations the spin-labels with restricted motion were the predominant population of the spin-labels covalently bound to OVA. Only after denaturation with SDS changes in the line shapes of all the spin labels were observed (Fig. 2b). Addition of CrOx then influenced all spin labels by both dipolar and spin exchange broadening. The number of –SH groups in the native OVA compared with those in the spin-labelled OVA was determined by the Ellman method. Native OVA has no accessible sulphydryl groups but after denaturation with SDS four free groups were determined (in accordance with the tertiary structure) in unlabelled and three in labelled OVA, thus indicating that only one –SH group reacted with the maleimide spin-label in the OVA molecule. The two methods combined suggest that the maleimide spin-label predominantly binds to one cysteine in the OVA molecule, not accessible to the solvent, i.e. located somewhere in the protein core. ESR spectra of SL-OVA were simulated with EasySpin software package, sharp lines (2–4) with Garlic, and broad lines (1, 3, 5) with Chili program. Motional properties of both populations determined by the rotational correlation times, τ, were calculated from the simulated spectra for both spin label populations. The result of the calculation is inserted in Fig. 3b, dotted line is simulation and full line experimental spectrum. Temperature dependence of rotational correlation times of the two nitroxide populations are shown: in Fig. 3a of nitroxides with fast motion, located at the surface of the protein (circles) compared to the rotational correlation times of the spin-labels dissolved in buffer (squares); in Fig. 3b of nitroxides with restricted motion probably bound to one cysteine (Cys) in the protein core. The rotational correlation time (τ) as a function of temperature (103 /T) suggests no change in the protein conformation in the temperature range studied. The effect of pH value on the OVA conformation is shown in Fig. 4. The spectra of spin-labelled OVA dissolved in buffers of different pH values: phosphate buffer, pH 7.4 (spectrum a) and carbonate buffer, pH 9.2 (spectrum b). At pH 9.2 only a decrease of the line widths 2 and 4 (i.e. increased line heights) were recorded. These small changes in the OVA conformation at pH 9.2 are in accordance with the robust structure of OVA. 3.2. Spin-labelled ovalbumin in the presence of peptidoglycan monomer In our previous studies, the effects of liposomal formulations of OVA and of adjuvant, peptidoglycan monomer (PGM), on immune reactions in mice were investigated [11]. In the present report we studied the possible interaction of PGM and OVA and the interaction of OVA with the lipids in lipid bilayers using spin-labelled OVA. According to our results, there is no interaction between PGM and spin-labelled OVA. In the presence of PGM no change in the line widths, i.e. in the motion of the labels located at the surface of OVA, as well as no change in the hyperfine splitting constant
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Fig. 3. Temperature dependence (1/T) of rotational correlation time (τ) of the spin-labelled OVA: (a) spin labels located at the surface of the protein, with motional freedom relative to the protein (black circles) are compared with spin labels dissolved in the buffer (black squares). (b) Spin labels with restricted motion bound to Cys in the protein core describe Brownian motion of the whole protein molecule. The rotational correlation times τ were obtained from the simulated spectra with EasySpin program [20].
2Amax i.e. in the motion of the labels located in the interior of the protein, were detected. 3.3. Interaction of spin-labelled ovalbumin with liposomes In the studies of the interaction of OVA with the lipids in lipid bilayers two different experiments were carried out: (a) with spin-labelled OVA and (b) with spin-labelled fatty acids. (a) In Fig. 5, the ESR spectrum of the spin-labelled OVA incorporated into multilamellar liposomes (full line) is compared with the spectrum of the spin-labelled OVA in the phosphate buffer (dotted line). Broadening of all lines suggests an unspecific interaction between the spin-labelled OVA and the multilamellar liposomes.
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Fig. 4. ESR spectra of spin-labelled ovalbumin taken at: (a) in phosphate buffer at pH 7.4; (b) in carbonate buffer at pH 9.2 (20 ◦ C).
(b) Liposomes, spin-labelled with n-doxyl stearic acids (n = 5, 7, 12, 16) were prepared in the presence of unlabelled OVA. The motional property (order parameter S) of the spinlabelled fatty acids incorporated into the liposomes prepared with OVA (Fig. 6) was compared with the empty liposomes. A decrease of the parameter S was determined for all spinlabelled fatty acids used, demonstrating the interaction with OVA. 3.4. Interactions of spin-labelled ovalbumin with specific antibodies of ovalbumin Since OVA has been customarily used as an antigen in various experimental models, it was also of interest to study the interaction of spin-labelled OVA, as a structurally modified molecule, with specific antibodies to OVA. Fig. 7 shows the ESR spectra of the spin-labelled OVA recorded in the presence of serum containing specific anti-OVA immunoglobulins, taken 4 h after
Fig. 5. ESR spectrum (a) the spin-labelled ovalbumin in phosphate buffer (pH 7.4), compared with spectrum (b) spin-labelled ovalbumin incorporated into multilamellar liposomes. Spectra were taken at room temperature (20 ◦ C).
Fig. 6. Order parameter (S) of spin-labelled n-doxyl stearic acids (n = 5 (squares), 7 (circles), 12 (triangles), 16 (diamonds)) in empty liposomes (black symbols) and in liposomes with incorporated ovalbumin (white symbols), as a function of temperature (T/K).
mixing (spectrum a), compared with the OVA in the serum but without antibody (spectrum b). In the presence of the antibodies, broadening of the sharp lines in the spectra is determined, concomitant with an increase of the rotational correlation time of the nitroxides with the restricted motion. The reaction between anti-OVA antibodies and the spinlabelled OVA was confirmed by ELISA. In an ELISA described previously [7,8] OVA was used for coating the wells on a plastic plate. In the present experiment, in addition to this ELISA, another ELISA was run in parallel, but in this assay the spinlabelled OVA was used for coating of the plates. Several samples of rabbit sera containing anti-OVA were tested. There was no difference in the reactions of serum samples in these two assays indicating that both the spin-labelled and the native OVA reacted
Fig. 7. ESR spectrum (b) (τ = 40 ns) of the spin-labelled ovalbumin in the presence of serum containing specific anti-ovalbumin immunoglobulins taken immediately after mixing antigen and antiserum, compared with the spectrum (a) (τ = 100 ns), taken after four hours at 37 ◦ C. The spectra were taken at 308 K. Rotational correlation time,τ, of the spin labels with restricted motion (broad component in the spectra) was simulated with EasySpin program [20].
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in the same way and to the same extent with the specific antiOVA immunoglobulin G. 4. Discussion OVA has been frequently used as an antigen in numerous experimental models. Various liposomal formulations of OVA have also been extensively studied, with emphasis on their effect on immune response [13,26]. In our recent paper [11] we have shown that the encapsulation of OVA into liposomes markedly affected the direction of immune reaction. It was therefore of interest to study the possible interactions of OVA with the lipids in the liposomal membranes. Such interactions have not been addressed so far. In our study we have spin-labelled OVA using TEMPOmaleimide. ESR spectra of purified spin-labelled OVA were subsequently studied in detail. Two motionaly different binding sites for the TEMPOmaleimide were detected, those with restricted motion being the predominant population, and not accessible to the solvent. Only one spin label bound per OVA molecule was determined by quantitative determination of the intensities of the ESR spectra. One spin label bound to Cys per OVA molecule was found by Ellmann reaction. From the crystal structure of the uncleaved ovalbumin six Cys were determined, two in the S–S bridge, and the other four with fully restricted motion relative to the protein, and the solvent accessible surface about zero [27]. This is in accordance with ESR results, i.e. that the spin label with the restricted motion is bound to one Cys per OVA molecule. The spin label with high freedom of motion relative to the protein that is located at the protein surface might be bound to some of the lysines. The spin label bound to Cys, with restricted motion relative to protein, describes the dynamics of the protein molecule by itself. In order to gain information on dynamics of OVA in solutions, ESR spectra simulated with the most recent sophisticated program EasySpin [20] were studied as a function of temperature. The robust conformation of the protein was confirmed (Fig. 3b). Spin-labelled OVA was further used to study the possible interaction with an adjuvant, peptidoglycan monomer. We demonstrated earlier [7,8] that PGM is an effective adjuvant that could induce the increase of anti-OVA IgG levels. The possible direct interaction of the two molecules, OVA and PGM, has not been studied so far. In the present study no changes in the ESR spectra of spin-labelled OVA were detected upon addition of PGM, indicating that there was no interaction of these compounds on the molecular level. Since we found that liposomes could enhance the humoral immune response to OVA [11], the next step was to study the possible interaction of OVA and lipids in liposomal bilayers. Our results on immunostimulating activity of liposomal formulations concern multilamellar, negatively charged liposomes and therefore in the present study all experiments were carried out with such liposomal formulations as well. We have demonstrated the interaction of spin-labelled OVA and lipids in the liposomal bilayers. Broadening of all lines in the spectra of OVA incorporated into liposomes (Fig. 5) suggested an unspecific interaction
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between the spin-labelled OVA and the multilamellar liposomes, i.e. that the protein is squeezed within the lipid bilayers of the multilamellar liposomes. It should be noted that the results described above were obtained with the molecule that differs slightly in structure and conformation from the unlabelled OVA molecule. The introduction of new molecular entities into the protein molecule might affect its properties including its binding to different molecules studied. In our subsequent experiments with specific anti-OVA antibodies we have shown that the spin-labelling of OVA with the maleimide derivative, as described in this study, does not affect its binding to specific antibodies. The binding of the spinlabelled OVA to specific antibodies was confirmed by ELISA as described in Section 3, but also documented by ESR spectroscopy. In additional, we have also demonstrated that the changes of pH have no effect on the structure of spin-labelled ovalbumin. In conclusion, we have presented the novel data on ESR spectra of spin-labelled ovalbumin. The availability of spin-labelled OVA has enabled us to detect the interactions with the lipids in liposomal bilayers, using ESR. Such studies have not been reported so far, although OVA encapsulated into or associated with liposomes, has been continuously used as a model antigen in numerous analyses of immune reactions or processing by immuno-competent cells. Acknowledgements The authors would like to thank the Ministry of Science, Education and Sport for financial support (projects 0021002 and 0098040). The support of the COST-chemistry, action D27 is acknowledged. The authors would like to thank Prof. L. Berliner for the critical discussion of the results during the Regional Biophysics Meeting 2005, Zreˇce, Slovenia. References [1] T. Lian, R.J.Y. Ho, J. Pharm. Sci. 90 (2001) 667–680. [2] G. Gregoriadis, Immunol. Today 11 (1990) 89–97. [3] G. Gregoriadis, B. McCormack, M. Obrenovic, R. Saffie, B. Zadi, Y. Perrie, Methods 19 (1999) 156–162. [4] D.E.S. Stewart-Tull, in: G. Gregoriadis (Ed.), Vaccines, Plenum Press, New York, 1991, pp. 85–92. [5] A.D. Nisbet, R.H. Saundry, A.J.G. Moir, L.A. Fothergill, J.E. Fothergill, Eur. J. Biochem. 115 (1981) 335–345. [6] J.A. Huntington, P.E. Stein, J. Chromatogr. B 756 (2001) 189–198. ˇ ˇ [7] J. Tomaˇsi´c, I. Hanzl-Dujmovi´c, B. Spoljar, B. Vraneˇsi´c, M. Santak, A. Joviˇci´c, Vaccine 18 (2000) 1236–1243. ˇ ˇ [8] B. Halassy Spoljar, T. Cimbora, I. Hanzl-Dujmovi´c, B. Dojnovi´c, A. Sabioncello, M. Krstanovi´c, J. Tomaˇsi´c, Vaccine 20 (2002) 3543– 3550. ˇ [9] R. Frkanec, D. Travaˇs, M. Krstanovi´c, B. Halassy Spoljar, Ð. Ljevakovi´c, B. Vraneˇsi´c, L. Frkanec, J. Tomaˇsi´c, J. Liposome Res. 13 (2003) 279– 294. [10] R. Frkanec, V. N¨othig-Laslo, B. Vraneˇsi´c, K. Mirosavljevi´c, J. Tomaˇsi´c, Biochim. Biophys. Acta 1611 (2003) 187–196. [11] L. Habjanec, R. Frkanec, B. Halassy, J. Tomaˇsi´c, J. Liposome Res. 16 (2006) 1–16. [12] S. Cavalu, G. Damian, Biomacromolecules 4 (2003) 1630–1635. [13] X. Jiang, M.A. Payne, Z. Cao, S.B. Foster, J.B. Feix, S.M.C. Newton, P.E. Klebba, Science 276 (1997) 1261–1264.
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Spin-labelling study of interactions of ovalbumin with multilamellar liposomes and specific anti-ovalbumin antibodies Marija Brgles b , Krunoslav Mirosavljevi´c a , Vesna Noethig-Laslo a,∗ , Ruˇza Frkanec b , Jelka Tomaˇsi´c b a
“Rudjer Boˇskovi´c” Institute, Bijeniˇcka cesta 54, 10002 Zagreb, Croatia b Institute of Immunology Inc., P.O. Box 266, 10001 Zagreb, Croatia
Received 5 July 2006; received in revised form 18 August 2006; accepted 22 August 2006 Available online 30 August 2006
Abstract Ovalbumin (OVA) has been used continuously as the model antigen in numerous studies of immune reactions and antigen processing, very often encapsulated into liposomes. The purpose of this work was to study the possible interactions of spin-labelled OVA and lipids in liposomal membranes using electron spin resonance (ESR) spectroscopy. OVA was covalently spin-labelled with 4-maleimido-2,2,6,6-tetramethylpiperidine1-oxyl (TEMPO-maleimide), characterized and encapsulated into multilamellar, negatively charged liposomes. ESR spectra of this liposomal preparation gave evidence for the interaction of OVA with the lipid bilayers. Such an interaction was also evidenced by the ESR spectra of liposomal preparation containing OVA, where liposomes were spin-labelled with n-doxyl stearic acids. The spin-labelled OVA retains its property to bind specific anti-OVA antibodies, as shown by ESR spectroscopy, but also in ELISA for specific anti-OVA IgG. © 2006 Elsevier B.V. All rights reserved. Keywords: Spin-labelled ovalbumin; Multilamellar liposomes; Anti-ovalbumin antibodies
1. Introduction Liposomes are microscopic vesicles consisting of one or more lipid bilayers surrounding aqueous compartments. The physicochemical properties of liposomes depend mainly on the lipid composition that determines the liposomal charge as well as the fluidity of the liposomal membrane. A wide variety of different compounds can be encapsulated in liposomes and the resulting liposomal formulations are often more convenient for experimental and therapeutic purposes [1]. Liposomes have also been considered and tested as carriers of antigens, vaccines and immunomodulatory compounds and tested in different experimental models [2,3]. In numerous studies of immune reactions and effects of immunostimulators ovalbumin (OVA) has been used as a model antigen of low immunogenicity according to previously published recommendations [4]. OVA is a glycoprotein from avian egg white. It has a well-defined structure [5] and belongs
∗
Corresponding author. Tel.: +385 1 4561 136; fax: +385 1 4680 245. E-mail address: [email protected] (V. Noethig-Laslo).
0141-8130/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ijbiomac.2006.08.012
structurally to the serpin (serine protease inhibitor) superfamily but shows no protease inhibitor activity [6]. In our previous studies on immunostimulating properties of peptidoglycan monomer, GlcNAc-MurNAc-l-Ala-d-isoGln-mesoDAP(-NH2 )-d-Ala-d-Ala (PGM) and adamantyltripeptides, d(adamant-2-il)-Gly-l-Ala-d-isoGln (AdTP1) and l-(adamant2-il)-Gly-l-Ala-d-isoGln (AdTP-2) [7,8], OVA was used as an antigen. The encapsulation of PGM and AdTPs into liposomes [9] and possible interactions of these compounds with lipids in liposomal membrane were studied using ESR [10], but the possible interaction of OVA and lipids was not addressed. However, our recent results [11] on immune reaction to OVA encapsulated into liposomes demonstrated that the direction of immune reaction was shifted towards a specific T-helper phenotype (Th1) and this finding prompted us to study the interaction of OVA with liposomes using ESR. Only two reports published previously concern the spinlabelling of OVA. In one report [12], the label different from TEMPO-maleimide was mixed with OVA and the analysis of ESR spectra of not covalently labelled OVA described. In the other [13] the spin-label was covalently linked to OVA and used for immunizations of rabbits with the aim of eliciting specific
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antibodies to spin-label, but the ESR spectra were neither studied nor described. The interaction of OVA with liposomes was studied and reported previously in several papers, using different methods [14–16]. However, ESR studies on this subject have not been reported so far. The aim of the present work was to study if there are any interactions of OVA encapsulated into negatively charged, multilamellar liposomes with lipids in the liposomal membrane using either spin-labelled OVA or spin-labelled fatty acids incorporated into lipid bilayers. 2. Materials and methods 2.1. Materials Albumin, from chicken egg; grade VII; min 98%, l-␣phosphatidylcholine, type XI-E: from fresh egg yolk (eggPC), cholesterol from porcine liver (CHL), dicetyl phosphate (DCPh) and 4-maleimido-2,2,6,6,-tetramethylpiperidine-1-oxyl (TEMPO-maleimide) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals used for buffers were of analytical grade and purchased from Sigma (St. Louis, MO, USA) and Kemika (Zagreb, Croatia). Centricon Centrifugal Filter Devices YM-30 were from Millipore (Bedford, Ma, USA).
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2.3. The preparation of liposomes Multilamellar, negatively charged liposomes were prepared as described previously [10]. Briefly, 6.8 mg (8.92 × 10−6 mol) egg-PC; 2.5 mg (6.46 × 10−6 mol) CHL and 0.7 mg (1.28 × 10−6 mol) DCPh, giving a molar ratio of 7:5:1, were dissolved in 2 ml of CHCl3 . After rotary evaporation of the solvent the remaining lipid film was dried in vacuum for an hour and then dispersed by gentle hand shaking in 1 ml OVA solution in phosphate buffered saline (5 mg/ml in PBS). The liposome suspension was left overnight at 4 ◦ C to swell and stabilize. Liposomes were separated from non-encapsulated material by centrifugation at 300,000 × g (Optima XL-80K, Beckman Coulter) at 4 ◦ C for 1 h. The amount of OVA encapsulated into liposomes was determined by the HPLC method [11]. 2.4. Anti-ovalbumin antiserum Rabbits were immunized s.c. with 100 g of OVA. ISA 720 (Seppic, France) and PGM were used as adjuvants. Boosters were administered after 3-week intervals and bleeding performed 7 days after the third dose (unpublished results). AntiOVA IgG was determined by ELISA as described earlier [7,8]. 2.5. Enzyme-linked immunosorbent assay (ELISA)
2.2. Spin-labelling of ovalbumin 6.77 × 10−7 mol)
Ovalbumin (OVA) (30 mg, and TEMPOmaleimide (0.85 mg, 3.39 × 10−6 mol) were dissolved in 8 ml 0.1 M phosphate buffer, pH 7.4. The reaction mixture was stirred either for 4 or 24 h. Separation of the spin-labelled protein from the free maleimide derivative was achieved by simple gel chromatography on Sephadex G-25 (2.5 cm × 60 cm) in phosphate buffer pH 7.4 (Fig. 1). Fractions of 2 ml were collected and absorbance monitored spectrophotometrically at 250 and 280 nm. The pooled fractions of spin-labelled OVA were then concentrated by ultrafiltration on Centricon Centrifugal Filter Devices YM-30. No free spin label was found in the filtrate.
ELISA for determination of anti-OVA IgG was carried out with two OVA preparations (used for coating the plates), native OVA and spin-labelled OVA, respectively, and two assays run in parallel, using the same sera samples in both assays. The basic ELISA, applied for the assay of anti-OVA IgG [7,8], was slightly modified. Briefly, Costar (Cambridge, MA, USA) E.I.A./R.I.A. flat bottom, high binding, 96-well plates were coated with 100 l OVA or spin-labelled OVA (15 g ml−1 ) coating solution in 0.05 M bicarbonate buffer, pH 9.6, incubated at room temperature overnight and washed. After blocking and washing, the rabbit sera samples to be tested were added in serial twofold dilutions and incubated at room temperature for 20 h. After washing, the horseradish peroxidase-conjugated (goat) anti-rabbit IgG (Bio-Rad, Hercules, CA, USA) were added (100 l per well) and incubated at 37 ◦ C for 2 h. After washing, 100 l per well of o-phenylendiamine (OPD) solution was added and incubated for 30 min, in the dark at room temperature. The reaction was stopped by the addition of 50 l of 1 M H2 SO4 . Absorbance was read at 492 nm. The incubation buffer used throughout was phosphate buffered saline, pH 7.3, containing 0.05% Tween-20. All samples were assayed in duplicate. Sera from the rabbit were used as negative controls and the buffer alone as a blank on each plate. 2.6. The assay of sulphydryls
Fig. 1. An example of the chromatographic separation of spin-labelled OVA from free spin-label on Sephadex G 25 column (2.5 cm × 60 cm) in 0.1 M phosphate buffer, pH 7.4. The absorbance of the fractions at 250 and 280 nm was measured. The fractions were pooled as indicated: I spin-labelled OVA; II free spin-label.
The sulphydryl groups of OVA and of spin-labelled OVA were determined by the Ellman method [17], following denaturation with SDS. All solutions were prepared in 0.1 M
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phosphate buffer, pH 8.0 containing 0.2 mM EDTA. Calibration curve was done with cystein in the concentration range (0.8–5)× 10−5 mol dm−3 (r2 = 0.999). Concentration of 5,5 dithiobis(2-nitrobenzoic acid) was 6 × 10−4 M and the final volumes of standard and sample solutions were 3 mL. Absorbance at 412 nm was read after 5 min. Number of free sulphydryls was determined in native and SDS denaturated (2.5 %) OVA. Three separate experiments were carried out and the mean value was taken into account. 2.7. ESR spectra measurements ESR measurements were performed on a Varian E-9 spectrometer (10 GHz) equipped with a Bruker variable temperature control unit. The spectra were recorded with digital acquisition, EW-ESR Ware [18]. Sample capillaries were inserted into the standard 4 mm diameter quartz tubes and centred in a TE102 ESR cavity. To quantitatively determine the number of the maleimide spin-labels covalently bound per one OVA molecule, markers Cu(DMA)2 (aquabis(N,N-dimethyl-l-␣-alaninato)copper(II)) and MnCl2 of known concentration dissolved in water were used. The intensities of the spectra were calculated by double integration with the Bruker WIN-EPR program and ESR Ware. Simulations of ESR spectra of the spin-labelled ovalbumin were performed with the programs “Garlic” and “Chili” (EasySpin 2.5.1. software package [19,20]). Spectra were simulated and rotational correlation times calculated with the error, for sharp lines 0.2 ns and for broad lines 10 ns. Liposomes were spin-labelled with n-doxyl stearic acids (n = 5, 7, 12, 16) by the thin film method, with 1% of spin-labels added to the organic solvent before evaporation as described earlier [21]. ESR spectra of doxyl-stearic acids in liposomes reflect motional properties of the lipid bilayer. One important motional component of the lipid bilayer is wobbling of the long molecular axis about a direction perpendicular to the bilayer surface. This motional effect was measured by the ESR parameters, Amax (mT) and Amin (mT), from which the apparent order parameter S was calculated [21] S=
3(Amax − Amin ) × 0.5396 Amax + 2Amin
3. Results 3.1. Characterization of the spin-labelled ovalbumin Ovalbumin was covalently spin-labelled with a maleimide derivative using the procedure for the unspecific spin-labelling of proteins [22,23]. The ESR spectrum of the maleimide spinlabelled OVA is shown in Fig. 2a, full line. The spectrum is characterized by five resonant transitions, denoted by numbers from 1 to 5. Well-defined outer hyperfine lines denoted by 1 and 5 separated by the hyperfine splitting, 2Amax = 6.5 mT, indicate the highly restricted motion of the labels. Sharp lines denoted
Fig. 2. ESR spectra of spin-labelled ovalbumin: spectrum (a); full line is compared with the dotted line spectrum taken in the presence of 0.5 M Cr-oxalate. The spectra were taken under same conditions, microwave power 10 mW, modulation amplitude 1.3 G under the same receiver gain. Spectrum (b) is denaturated sample in the presence of SDS. Spectra were taken at room temperature (20 ◦ C).
by 2 and 4 indicate a fast motion of the covalently bound labels to OVA. Location of the spin-label binding sites on the protein was determined by addition of 0.5 M Cr-oxalate (CrOx ) (Fig. 2a, dotted line). As a result of the dipole–dipole interaction between paramagnetic Cr ions and the nitroxides a decrease in the lineheights (i.e. broadening of the lines) was detected only for nitroxides with the fast motion (lines 2–4). These spin labels are located at/or near the surface of the protein i.e. accessible to the solvent and to the spin exchange broadening. Possible dipole–dipole interaction between Cr and immobilized nitroxides, would produce diminution of the ESR line heights (1 and 5) [24,25]. The spectral lines due to the restricted motion were simulated with the same parameters (same linewidths) with Chili program [20]. This proved that the spin labels with the restricted motion are not accessible to CrOx i.e. they are located in the protein core. Denaturation of the sample with SDS caused increased tumbling of the spin labels relative to the protein and exposal of all the labels to the solvent. In the presence of CrOx in the denatured sample all the spectral lines are severely broadened by spin exchange interactions. The number of spin-labels covalently bound per OVA molecule was determined by double integration of the spectra relative to the spectra of Cu(DMA)2 and/or MnCl2 as external markers. The ratio of the intensity of the spin-label and the intensity of the marker was found to be 0.5 for similar concentrations of the OVA and the marker (5 × 10−4 M). The result of 0.5 obtained in several different spin-labelled OVA preparations indicates that only one spin-label, at most, was covalently bound per OVA molecule. If motion of the spin-label covalently bound to protein is fully restricted with respect to the protein, the rotational correlation time, τ, calculated from the line shapes of the spectra describes Brownian motion of the whole protein molecule. Separation of the outer hyperfine lines,
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2Amax = 6.5 mT, suggests fully restricted motion of the label relative to the protein, compared to 2Amax = 6.6 mT in the frozen sample. In all preparations the spin-labels with restricted motion were the predominant population of the spin-labels covalently bound to OVA. Only after denaturation with SDS changes in the line shapes of all the spin labels were observed (Fig. 2b). Addition of CrOx then influenced all spin labels by both dipolar and spin exchange broadening. The number of –SH groups in the native OVA compared with those in the spin-labelled OVA was determined by the Ellman method. Native OVA has no accessible sulphydryl groups but after denaturation with SDS four free groups were determined (in accordance with the tertiary structure) in unlabelled and three in labelled OVA, thus indicating that only one –SH group reacted with the maleimide spin-label in the OVA molecule. The two methods combined suggest that the maleimide spin-label predominantly binds to one cysteine in the OVA molecule, not accessible to the solvent, i.e. located somewhere in the protein core. ESR spectra of SL-OVA were simulated with EasySpin software package, sharp lines (2–4) with Garlic, and broad lines (1, 3, 5) with Chili program. Motional properties of both populations determined by the rotational correlation times, τ, were calculated from the simulated spectra for both spin label populations. The result of the calculation is inserted in Fig. 3b, dotted line is simulation and full line experimental spectrum. Temperature dependence of rotational correlation times of the two nitroxide populations are shown: in Fig. 3a of nitroxides with fast motion, located at the surface of the protein (circles) compared to the rotational correlation times of the spin-labels dissolved in buffer (squares); in Fig. 3b of nitroxides with restricted motion probably bound to one cysteine (Cys) in the protein core. The rotational correlation time (τ) as a function of temperature (103 /T) suggests no change in the protein conformation in the temperature range studied. The effect of pH value on the OVA conformation is shown in Fig. 4. The spectra of spin-labelled OVA dissolved in buffers of different pH values: phosphate buffer, pH 7.4 (spectrum a) and carbonate buffer, pH 9.2 (spectrum b). At pH 9.2 only a decrease of the line widths 2 and 4 (i.e. increased line heights) were recorded. These small changes in the OVA conformation at pH 9.2 are in accordance with the robust structure of OVA. 3.2. Spin-labelled ovalbumin in the presence of peptidoglycan monomer In our previous studies, the effects of liposomal formulations of OVA and of adjuvant, peptidoglycan monomer (PGM), on immune reactions in mice were investigated [11]. In the present report we studied the possible interaction of PGM and OVA and the interaction of OVA with the lipids in lipid bilayers using spin-labelled OVA. According to our results, there is no interaction between PGM and spin-labelled OVA. In the presence of PGM no change in the line widths, i.e. in the motion of the labels located at the surface of OVA, as well as no change in the hyperfine splitting constant
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Fig. 3. Temperature dependence (1/T) of rotational correlation time (τ) of the spin-labelled OVA: (a) spin labels located at the surface of the protein, with motional freedom relative to the protein (black circles) are compared with spin labels dissolved in the buffer (black squares). (b) Spin labels with restricted motion bound to Cys in the protein core describe Brownian motion of the whole protein molecule. The rotational correlation times τ were obtained from the simulated spectra with EasySpin program [20].
2Amax i.e. in the motion of the labels located in the interior of the protein, were detected. 3.3. Interaction of spin-labelled ovalbumin with liposomes In the studies of the interaction of OVA with the lipids in lipid bilayers two different experiments were carried out: (a) with spin-labelled OVA and (b) with spin-labelled fatty acids. (a) In Fig. 5, the ESR spectrum of the spin-labelled OVA incorporated into multilamellar liposomes (full line) is compared with the spectrum of the spin-labelled OVA in the phosphate buffer (dotted line). Broadening of all lines suggests an unspecific interaction between the spin-labelled OVA and the multilamellar liposomes.
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Fig. 4. ESR spectra of spin-labelled ovalbumin taken at: (a) in phosphate buffer at pH 7.4; (b) in carbonate buffer at pH 9.2 (20 ◦ C).
(b) Liposomes, spin-labelled with n-doxyl stearic acids (n = 5, 7, 12, 16) were prepared in the presence of unlabelled OVA. The motional property (order parameter S) of the spinlabelled fatty acids incorporated into the liposomes prepared with OVA (Fig. 6) was compared with the empty liposomes. A decrease of the parameter S was determined for all spinlabelled fatty acids used, demonstrating the interaction with OVA. 3.4. Interactions of spin-labelled ovalbumin with specific antibodies of ovalbumin Since OVA has been customarily used as an antigen in various experimental models, it was also of interest to study the interaction of spin-labelled OVA, as a structurally modified molecule, with specific antibodies to OVA. Fig. 7 shows the ESR spectra of the spin-labelled OVA recorded in the presence of serum containing specific anti-OVA immunoglobulins, taken 4 h after
Fig. 5. ESR spectrum (a) the spin-labelled ovalbumin in phosphate buffer (pH 7.4), compared with spectrum (b) spin-labelled ovalbumin incorporated into multilamellar liposomes. Spectra were taken at room temperature (20 ◦ C).
Fig. 6. Order parameter (S) of spin-labelled n-doxyl stearic acids (n = 5 (squares), 7 (circles), 12 (triangles), 16 (diamonds)) in empty liposomes (black symbols) and in liposomes with incorporated ovalbumin (white symbols), as a function of temperature (T/K).
mixing (spectrum a), compared with the OVA in the serum but without antibody (spectrum b). In the presence of the antibodies, broadening of the sharp lines in the spectra is determined, concomitant with an increase of the rotational correlation time of the nitroxides with the restricted motion. The reaction between anti-OVA antibodies and the spinlabelled OVA was confirmed by ELISA. In an ELISA described previously [7,8] OVA was used for coating the wells on a plastic plate. In the present experiment, in addition to this ELISA, another ELISA was run in parallel, but in this assay the spinlabelled OVA was used for coating of the plates. Several samples of rabbit sera containing anti-OVA were tested. There was no difference in the reactions of serum samples in these two assays indicating that both the spin-labelled and the native OVA reacted
Fig. 7. ESR spectrum (b) (τ = 40 ns) of the spin-labelled ovalbumin in the presence of serum containing specific anti-ovalbumin immunoglobulins taken immediately after mixing antigen and antiserum, compared with the spectrum (a) (τ = 100 ns), taken after four hours at 37 ◦ C. The spectra were taken at 308 K. Rotational correlation time,τ, of the spin labels with restricted motion (broad component in the spectra) was simulated with EasySpin program [20].
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in the same way and to the same extent with the specific antiOVA immunoglobulin G. 4. Discussion OVA has been frequently used as an antigen in numerous experimental models. Various liposomal formulations of OVA have also been extensively studied, with emphasis on their effect on immune response [13,26]. In our recent paper [11] we have shown that the encapsulation of OVA into liposomes markedly affected the direction of immune reaction. It was therefore of interest to study the possible interactions of OVA with the lipids in the liposomal membranes. Such interactions have not been addressed so far. In our study we have spin-labelled OVA using TEMPOmaleimide. ESR spectra of purified spin-labelled OVA were subsequently studied in detail. Two motionaly different binding sites for the TEMPOmaleimide were detected, those with restricted motion being the predominant population, and not accessible to the solvent. Only one spin label bound per OVA molecule was determined by quantitative determination of the intensities of the ESR spectra. One spin label bound to Cys per OVA molecule was found by Ellmann reaction. From the crystal structure of the uncleaved ovalbumin six Cys were determined, two in the S–S bridge, and the other four with fully restricted motion relative to the protein, and the solvent accessible surface about zero [27]. This is in accordance with ESR results, i.e. that the spin label with the restricted motion is bound to one Cys per OVA molecule. The spin label with high freedom of motion relative to the protein that is located at the protein surface might be bound to some of the lysines. The spin label bound to Cys, with restricted motion relative to protein, describes the dynamics of the protein molecule by itself. In order to gain information on dynamics of OVA in solutions, ESR spectra simulated with the most recent sophisticated program EasySpin [20] were studied as a function of temperature. The robust conformation of the protein was confirmed (Fig. 3b). Spin-labelled OVA was further used to study the possible interaction with an adjuvant, peptidoglycan monomer. We demonstrated earlier [7,8] that PGM is an effective adjuvant that could induce the increase of anti-OVA IgG levels. The possible direct interaction of the two molecules, OVA and PGM, has not been studied so far. In the present study no changes in the ESR spectra of spin-labelled OVA were detected upon addition of PGM, indicating that there was no interaction of these compounds on the molecular level. Since we found that liposomes could enhance the humoral immune response to OVA [11], the next step was to study the possible interaction of OVA and lipids in liposomal bilayers. Our results on immunostimulating activity of liposomal formulations concern multilamellar, negatively charged liposomes and therefore in the present study all experiments were carried out with such liposomal formulations as well. We have demonstrated the interaction of spin-labelled OVA and lipids in the liposomal bilayers. Broadening of all lines in the spectra of OVA incorporated into liposomes (Fig. 5) suggested an unspecific interaction
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between the spin-labelled OVA and the multilamellar liposomes, i.e. that the protein is squeezed within the lipid bilayers of the multilamellar liposomes. It should be noted that the results described above were obtained with the molecule that differs slightly in structure and conformation from the unlabelled OVA molecule. The introduction of new molecular entities into the protein molecule might affect its properties including its binding to different molecules studied. In our subsequent experiments with specific anti-OVA antibodies we have shown that the spin-labelling of OVA with the maleimide derivative, as described in this study, does not affect its binding to specific antibodies. The binding of the spinlabelled OVA to specific antibodies was confirmed by ELISA as described in Section 3, but also documented by ESR spectroscopy. In additional, we have also demonstrated that the changes of pH have no effect on the structure of spin-labelled ovalbumin. In conclusion, we have presented the novel data on ESR spectra of spin-labelled ovalbumin. The availability of spin-labelled OVA has enabled us to detect the interactions with the lipids in liposomal bilayers, using ESR. Such studies have not been reported so far, although OVA encapsulated into or associated with liposomes, has been continuously used as a model antigen in numerous analyses of immune reactions or processing by immuno-competent cells. Acknowledgements The authors would like to thank the Ministry of Science, Education and Sport for financial support (projects 0021002 and 0098040). The support of the COST-chemistry, action D27 is acknowledged. The authors would like to thank Prof. L. Berliner for the critical discussion of the results during the Regional Biophysics Meeting 2005, Zreˇce, Slovenia. References [1] T. Lian, R.J.Y. Ho, J. Pharm. Sci. 90 (2001) 667–680. [2] G. Gregoriadis, Immunol. Today 11 (1990) 89–97. [3] G. Gregoriadis, B. McCormack, M. Obrenovic, R. Saffie, B. Zadi, Y. Perrie, Methods 19 (1999) 156–162. [4] D.E.S. Stewart-Tull, in: G. Gregoriadis (Ed.), Vaccines, Plenum Press, New York, 1991, pp. 85–92. [5] A.D. Nisbet, R.H. Saundry, A.J.G. Moir, L.A. Fothergill, J.E. Fothergill, Eur. J. Biochem. 115 (1981) 335–345. [6] J.A. Huntington, P.E. Stein, J. Chromatogr. B 756 (2001) 189–198. ˇ ˇ [7] J. Tomaˇsi´c, I. Hanzl-Dujmovi´c, B. Spoljar, B. Vraneˇsi´c, M. Santak, A. Joviˇci´c, Vaccine 18 (2000) 1236–1243. ˇ ˇ [8] B. Halassy Spoljar, T. Cimbora, I. Hanzl-Dujmovi´c, B. Dojnovi´c, A. Sabioncello, M. Krstanovi´c, J. Tomaˇsi´c, Vaccine 20 (2002) 3543– 3550. ˇ [9] R. Frkanec, D. Travaˇs, M. Krstanovi´c, B. Halassy Spoljar, Ð. Ljevakovi´c, B. Vraneˇsi´c, L. Frkanec, J. Tomaˇsi´c, J. Liposome Res. 13 (2003) 279– 294. [10] R. Frkanec, V. N¨othig-Laslo, B. Vraneˇsi´c, K. Mirosavljevi´c, J. Tomaˇsi´c, Biochim. Biophys. Acta 1611 (2003) 187–196. [11] L. Habjanec, R. Frkanec, B. Halassy, J. Tomaˇsi´c, J. Liposome Res. 16 (2006) 1–16. [12] S. Cavalu, G. Damian, Biomacromolecules 4 (2003) 1630–1635. [13] X. Jiang, M.A. Payne, Z. Cao, S.B. Foster, J.B. Feix, S.M.C. Newton, P.E. Klebba, Science 276 (1997) 1261–1264.
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