CALORIMETRIC DETERMINATION OF PHOSPHOLIPID- MELITTIN INTERACTIONS

Transcription

1 CALORIMETRIC DETERMINATION OF PHOSPHOLIPID- MELITTIN INTERACTIONS Linus N. Okoro Keywords: Pressure erturbation calorimetry, DSC, coefficient of thermal exansion, volume change, hosholiids, Melittin. The effect of melittin incororated into the hosholiid bilayer of 1,2-dialmitoyl-sn-glycero-3-hoshocholine (DPPC) at mole fractions u to 3.75 mol% have been investigated. The study reveals a considerable influence of melittin on the hase transition rofile and volumetric roerties of the DPPC bilayer. The temerature deendence of the coefficient of exansion,, and the volume fluctuations of DPPC melittin bilayer membranes in their different transition hases were determined by using ressure erturbation calorimetry (PPC), a relatively new and efficient technique, and differential scanning calorimetry (DSC). The exeriments were carried out in the temerature range from 10 to 85 C. Incororating melittin u to 1.25 mol% into the liid bilayer abolishes the retransition. Remarkably, there is no shift in the transition temerature u to 2.5 mol% melittin, while more than one shoulder is observed at the low-temerature side of the DSC eak for 3.75 mol%, and a slight shift in melting temerature,tm to 32 o C. This could be linked to the lytic roerty of melittin at these high etide concentrations. * Corresonding Authors linus.okoro@aun.edu.ng [a] American University of Nigeria, Lamido Zubairu Way, Yola, Adamawa State, Nigeria Introduction Melittin Melittin constitutes 50% of the dry weight of the honeybee venom. The active etide melittin is released from its recursor, romelittin, during its biosynthesis in honey bee and later gets formylated. 1 It is a etide comosed of 26 amino acids (NH2-G-I-G-A-V-L-K-V-L-T-T-G-L-P-A-L-I- S-W-I-K-R-K-R-Q-Q-CONH2) with no disulhide bridge, the N-terminal region (residues 1 20) is redominantly hydrohobic and the C-terminal region (residues 21 26) is hydrohilic due to the resence of a stretch of ositively charged amino acids. Melittin is water soluble due to its amhihilic roerty and yet it sontaneously associates with both natural and artificial membranes. 2 The crystal structures of melittin have been resolved by X-ray crystallograhy. 3 It has been observed that in an aqueous solution of high etide concentration, high H value, or high ionic strength, tetrameric melittin of high symmetry is formed readily. 2 Liid Melittin Interaction Melittin causes bilayer micellization and membrane fusion and has also been observed to form voltage-deendent ion channels across lanar liid bilayers. 4,5 The characteristic action of melittin is its hemolytic activity. 6,7 It is commonly believed and acceted that multimeric ore formation is the mode of action of many naturally roduced etides such as antimicrobial etides and toxins. 8,9 The structure and function of such ores and their results show that melittin forms ores that have a rather wide distribution of sizes. For examle, the sizes of the melittin ores that are characterized by the inner ore diameter have been reorted to be in the range of A, A and A from vesicle leakage exeriments. 8 The diameter of these ores is exected to increase when the etide concentration is increased. Under certain conditions, melittin molecules insert into the liid bilayer and form multile aggregated forms that are controlled by temerature, H, ionic strength, liid comosition and the liid-to-etide ratio. It has been observed that liid comosition and hase searation aears to lay a critical role in melittin-induced ore formation. The action of melittin on membrane roteins has been studied and aart from its ability to disrut liid bilayers, melittin affects the dynamics of membrane roteins. For instance, it has been reorted that lytic concentrations of melittin dramatically reduce the rotational mobility of band 3 rotein in human erythrocyte membranes. 10,11 Further DSC study has revealed membrane fusion between liosomes comosed of acidic hosholiids and neutral hosholiids induced by melittin, in which the roles of hydrohobic and electrostatic interactions were investigated in membrane fusion induced by melittin. 12 In this study, we investigated the influence of melittin incororation on the thermodynamic and equilibrium volumetric roerties of the 1,2-Dialmitoyl-sn-glycero-3- hoshocholine (DPPC) bilayer membrane. To accomlish this, ressure erturbation calorimetry (PPC) has been used to determine the temerature deendence of the coefficient of exansion coefficient,, as well as volume changes accomanying liid hase transitions. DSC thermogram was also recorded to show the temerature-deendent hase changes. Materials and methods Samle Prearation Multilamellar vesicles rearation for calorimetric studies 1,2-Dialmitoyl-sn-glycero-3-hoshocholine (DPPC) was obtained from Avanti Polar Liids (Alabaster, AL). Melittin Eur. Chem. Bull. 2013, 2(3), DOI: /ECB

2 (97% urity) as a lyohilized solid was obtained from Calbiochem (Germany). Both samles were used without further urification. Multilamellar vesicles (MLV) of DPPC and melittin with designated mole ratios were mixed in a chloroform-methanol mixture (3:1 v/v) and dried as a thin film under a stream of nitrogen and then freeze-dried in a freeze-dryer (Christ, Osterode, Germany) under high vacuum overnight. The liid films were hydrated in a Tris buffer (10 mm Tris-HCl, 100 mm NaCl, H 7.4), followed by vortexing at ~62 C (above the main hase transition temerature, T m, of DPPC (~41.5 C [Cevc 1987]), and five freeze-thaw cycles, resulting in homogeneous multilamellar vesicles (MLVs). The final DPPC concentration used in the calorimetric measurements was 5 mg/ml. Calorimetric measurements Differential scanning calorimetry Pressure erturbation calorimetry and differential calorimetry measurements were erformed with a MicroCal (Northamton, USA) VP-DSC micro-calorimeter, equied with a ressurizing system from the same manufacturer. The reference cell was filled with the Tris buffer solution. Both buffer and samle solutions were degassed before being injected into the resective cells. Included in the standard VP-DSC instrument is a ressuring ca that allows alication of 1.8 bars to the cells in order to avoid air bubbles at elevated temeratures. The instrument was oerated in the high gain mode at a scanning rate of 40 o C h - 1. Baseline subtraction (ure buffer) and normalization with resect to scan rate and concentration were erformed by the instrument software, yielding the temerature-deendent aarent (excess) molar heat caacity of the vesicles, C, with resect to the buffer solution. Pressure erturbation calorimetry (PPC) Pressure erturbation calorimetry (PPC) measurements were carried out on the VP-DSC calorimeter equied with MicroCal s (Northamton, MA, USA) PPC accessory. A descrition of the technique and its otential alications is laced elsewhere [37-43].This method enables the measurement of heat effects induced by small eriodic changes of gas ressure ( = 5 bar) above the solution The hysical rincile is the same as in a heat-induced thermal exansion, although the measurable is Q - the heat released uon a ressure change of at temerature T. Three control and calibration measurements were erformed, namely water (samle cell) versus water (reference cell), buffer versus water and buffer versus buffer. The results of these control exeriments were fit by second-order olynomials and were used for the evaluation of the coefficient of thermal exansion α (T) of the measured system (fig. 1). From the relative volume changes ΔV/V, the absolute volume changes ΔV can be determined when the secific volume and the molar mass of the samle are known. 17 Knowing the thermal exansion coefficient of the solvent,, mass, m, and artial secific volume of the solute, V s, through a series of reference measurements, one can calculate the aarent thermal exansion coefficient of the dissolved articles. Figure 1. Schematic diagram of PPC describing the measurement curves and the derived thermodynamic arameters (Microcal user note, 2000). Theory The basic theory of ressure erturbation calorimetry is outlined below: The heat of a reversible rocess, dq rev, is related to the entroy change, ds, at the temerature T. dqrev TdS Differentiation with resect to ressure,, yields From dg = Vd SdT, it follows that Eqn. (2) can thus be rewritten as Q rev V T T T The thermal exansion coefficient of volume V is defined as and can thus be determined from an isothermal measurement of the heat consumed or released uon a small ressure change: (1) Q rev S T (2) T T S V (3) T T (4) 1 V v V T Q rev v TV (5) (6) Eur. Chem. Bull. 2013, 2(3), DOI: /ECB

3 Moreover, the relative volume change ΔV/V at a hase or structural transition, taking lace in the temerature range from T o to T e can be obtained by ΔV V Te ( T) d T (7) To For two-comonent systems, such as bioolymer solutions, one has to extend these equations [Lin 2002, Kujawa 2001]. If the sufficiently dilute solution is comosed of m s grams of a solute dissolved in m 0 grams of solvent, the total solution volume V may be exressed as In a differential PPC exeriment, with samle solution in the samle cell and buffer in the reference cell, both cells are subjected to the same Δ so that the net heat change ΔQ rev will be equal to the difference between Eqn. (11) for the samle cell and that for the reference cell. If the cells have an identical volume, then ΔQ rev arises because the volume occuied by the solute in the samle cell, is relaced by solvent in the reference cell, i.e., Q T ( m s V s s m s V s 0 ) (12) rev which then rearranges to where V 0 is the secific volume of the ure solvent, and V s is the artial secific volume of the solute in the solution. The artial volume of the solute includes not just its intrinsic volume, but also any volume changes induced as a result of interactions with the solvent. Differentiating Eqn. (8) with resect to temerature at constant ressure yields and after substituting the right hand side of Eq. (9) into Eq. (4) and (5), we obtain (10) where V m 0 V 0 m s V s V total V 0 Vs m 0 m T T s T Q rev V 0 V T m s 0 m T s T T (1/ V )( V / T ) 0 0 is the of the solvent volume and (1/ V )( V T ) s s s is the of the solute artial volume. The heat arising from ressure erturbation of the solution can thus be viewed as the sum of that arising from the erturbation of the solvent and from the erturbation of the solute in solution. Integration of Eqn. (10) over a small ressure range Δ T m 0V00 m s V s s (8) (9) (10) Q rev s 0 TmsVs where the total mass of solute, m s, is obtained by multilying its concentration, c s [g/ml] with the cell volume, V cell [ml]. Results and discussion (13) The excess heat caacity (C -rofiles) of multilamellar vesicle (MLV) susensions of DPPC with different concentrations of melittin added is dislayed in figure 2. The heat caacity trace of ure DPPC dislays a maximum at 41 o C, referred to as the main hase transition temerature, T m. It is also observed that ure DPPC dislays a small endothermic eak due to the L -P retransition which aears around 35 o C. Overall, the heat caacity increase in the transition regime is mainly due to cooerative fluctuations of a large number of liid molecules. When the DPPC liosomes are reared in the resence of melittin concentrations of 1 to 2.5 %, there is no change in the temeratures of the main transition. Addition of 1 mol% melittin leads to a reduction of the liid re-transition eak. The eak is not comletely abolished; however the temerature of the re-transition shifts slightly to a lower temerature (32 o C). The retransition is comletely abolished at a melittin concentration of 1.25 mol% and at this articular concentration there aears a noticeable base eak broadening, a small asymmetry at the low-temerature wing and a reduction in transition rofile which continues to increase with increasing melittin concentration. There is a concomitant reduction in the cooerativity of the chain melting transition, as a result of melittin incororation, which is evidenced by the broadening of the transition rofile. A shoulder (a small eak) aears at the low-temerature side (at 39 o C) of the main transition eak for 2.5 % melittin, with a further reduction of the transition rofile and cooerativity. Q rev T m 0 V 00 m s V s s leads to (11) Additionally, we observe no shift in the main transition temerature u to 2.5 mol% melittin. More than one shoulder is observed at the low-temerature side of the eak for 3.75 mol%, and a slight shift in T m to 32 o C. Oliynyk et al. observed a similar low-temerature shoulder in the C - Eur. Chem. Bull. 2013, 2(3), DOI: /ECB

4 Excess Heat Caacity Calorimetric determination of hosholiid-melittin interactions rofile at higher melittin concentrations, in good agreement with our data. 18 DPPC / Melittin reach a value close to that obtained below the transition temerature (α is K -1 below the retransition (20 o C) and K -1 above the main transition (65 o C)). Similar α values for melittin concentrations 0% and 1% was observed. α = 1.01 x 10-3 K -1 for 2.5 mol%, and α = 1.12 x 10-3 K -1 for 3.75 mol% at 20 o C. Above T m (65 o C), Just below the retransition, a very slight increase in α, with increase in melittin concentration (as an examle, = 0.92 x 10-3 K -1 and = 1.02 x 10-3 K -1 at 65 o C for 1.25 % and 2.5 % melittin resectively), whereas the main eak height of in the transition region decreases and broadens markedly with increase in melittin concentration. 0% 1% 1.25% 2.5% 3.75% T / o C Figure 2. DSC thermograms of melittin in DPPC multilamellar vessicles The PPC stacked lot of the coefficient of thermal exansivity versus temerature curves of the DPPC melittin mixtures is revealed in figure 3. Figure 4. Concentration deendence of the absolute volume change, ΔV, of the DPPC/melittin mixtures / K DPPC / Melittin 0% 1% From the integration of α over the temerature range where the main transitions occur, we obtain the volume volume changes, ΔV,as shown in figure 4, for the ure DPPC and for each of the DPPC / melittin concentrations. ΔV/V = (4 %), which corresonds to an absolute volume change of 29 ml/mol, for ure DPPC, ΔV/V is for 1.25 mol%, for 2.5 mol%, and for 3.75 mol% melittin. The corresonding width of the overall transition, T 1/2, increases from about 1 o C for the main transition of ure DPPC to about 3 o C for the DPPC / 3.75 mol% melittin mixture % T / o C Figure 3., of DPPC/Melittin mixture 1.25% 2.5% The PPC scans were carried out with solutions of DPPC/melittin in Tris buffer yielding the changes with temerature of the thermal exansion coefficient, α. The lots can be divided into three temerature regimes. We observe a slight increase in α below the retransition with increase in melittin concentration, but not in any articular order. For instance, as reorted in our revious ublication 14, below the main hase transition temerature of ure DPPC, α increases slightly with increasing temerature. At the main hase transition temerature, α undergoes a rather shar increase, reaches a maximum of about 0.15 K -1 at T m (41 o C), which then raidly decreases with increasing temerature, to Conclusion The study has rovided further insights into the disrutive effects of melittin on membrane bilayers having exlored the use of calorimetry (ressure erturbation calorimetry and differential scanning calorimetry) to determine the absolute and relative volume change of DPPC melittin bilayer membranes in their different hases. We have shown that as the concentration of membranebound melittin increases, the relative volume change at the main transition of the liid bilayer decreases. From the calorimetric measurements, uon incororation of melittin into the liid bilayer u to 1.25 %, it is observed that the retransition is comletely abolished. At this articular concentration, we observe a noticeable base eak broadening and a reduction in transition rofile which continues with increase in melittin concentration. The aearance of a small endothermic eak at 39 o C for 2.5 % melittin could ossibly reflect morhological changes of free liid vesicles in solution, the onset of the disrutive effect of melittin in DPPC bilayers. Remarkably, there is no Eur. Chem. Bull. 2013, 2(3), DOI: /ECB

5 shift in the transition temerature u to 2.5 %, while more than one shoulder was observed at the low-temerature side of the eak for 3.75 %, and a slight shift in T m to 32 o C. This imlies a disordering effect, which could be attributed to melittin ore formation (disrutive effect), at this higher melittin concentration. At the main transition region, the eak height of decreases and broadens markedly with increase in melittin concentration. Petides in both model and biological membranes have been observed to strongly affect the local state of the system, and the effect of etide may equally deend on the overall state of the membrane. 18 At the main transition region, the eak height of decreases and broadens markedly with increase in melittin concentration. Acknowledgement Sincere grateful goes to Prof. Dr. R. Winter, Deartment of Physical Chemistry, Dortmund University of Technology, Germany, in whose laboratory this work was carried out. Financial suort from the Deutsche Forschungsgemeinschaft (DFG), Germany and the regional county of Northrhine Westfalia is gratefully acknowledged. 5 Monette, M. and Lafleur, M., Biohys. J., 1995, 68, Raghuraman H. andchattoadhyay, A., Chem. Phys. Liids, 2005, 134, Rudenko S. V. and Patelaros, S. V.. Biochim. Biohys. Acta, 1995., 1235, Rex S., Biohys. Chem. 1996, 58, Allende D., Simon, S. A. and McIntosh, T. J., Biohys. J , 88, Clague M. J. and Cherry, R. J., Biochem. J., 1988, 252, Hui S. W., Stewart, C. M., and Cherry, R. J., Biochim. Biohys. Acta, 1990, 1023, Bourinbaiar, A. S.; Coleman, C., Arch Virol., 1997, 142, Heerklotz, H. and seelig, J., Biohys. J., 2002, 82, Okoro, L. N. and Winter, R., Z. Naturforsch., 2008, 63b, Okoro, L. N.,. Int. J. Chem., 2011, 3(1), Okoro, L. N., Can. J. Pure Al. Sci., 2010, 4(3), Seemann, H. and Winter, R., Z. Phys. Chem. 2003, 217, Oliynyk, V., kaatze, U. and Heimburg, T., Biochim. Biohys. Acta 2007, 1768, REFERENCES 1 Habermann, E., Science,1972, 177, Demsey, C. E., Biochim. Biohys. Acta. 1990, 1031, Terwilliger, T.; Weissman L., and Eisenberg, D., Biohys. J. 1982, 37, Bechinger, B. J. Membr. Biol. 1997, 156, Received: Acceted Eur. Chem. Bull. 2013, 2(3), DOI: /ECB

6 Eur. Chem. Bull. 2013, 2(3), DOI: /ECB