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Thermal Hysteresis in Ganglioside Micelles Investigated by Differential Scanning Calorimetry and Light-Scattering Laura Cantu´,† Mario Corti,*,† Elena Del Favero,† Elke Muller,‡ Antonio Raudino,§ and Sandro Sonnino† Dipartimento di Chimica e Biochimica Medica, INFM, Universita´ di Milano, LITA Via F.lli Cervi 93, 20090 Segrate, Italy, Physikalische Chemie, Universita¨ t Kaiserslautern, Erwin Schro¨ dinger Strasse, 67663 Kaiserslautern, Germany, and Dipartimento di Scienze Chimiche, Universita´ di Catania, Viale A. Doria 6, 95125 Catania, Italy Received September 28, 1998. In Final Form: April 23, 1999 Both light-scattering and differential scanning calorimetry (DSC) measurements account for the existence of a dramatic thermal hysteresis effect occurring in micellar solutions of gangliosides. Light-scattering measurements reveal that the mean aggregation number of ganglioside micelles assumes different values depending on the sample thermal history, a hysteresis phenomenon that completely disappears above a well-defined temperature. On the other side, DSC measurements assess that the heat-capacity variation versus temperature for the ganglioside micellar system depends as well on the thermal history of the sample. Although rather complicated, the calorigrams show a peak associated with the melting of the micelle hydrophobic core. The position of the peak undergoes changes consistent with the observed hysteresis of the mean aggregation number. The two parallel descriptions, geometric and calorimetric, can be clearly correlated as two aspects of the same phenomenon and give support to each other. The hysteresis behavior below a critical temperature is related to cooperative conformational variations of the oligosaccharide headgroup at the micellar surface. Such surface phenomena are coupled to the well-known order-disorder transition of the hydrophobic chains in the core via the geometric constraints imposed by the confinement of the amphiphiles in the micellar aggregate.
Introduction Gangliosides are amphiphilic molecules of biological origin, being components of the outer leaflet of the plasma membranes of vertebrate cells and particularly abundant in the nervous system. They appear to play a role in the biotransduction of membrane-mediated information. Their double-tailed hydrophobic part, the ceramide, is made up of a long-chain amino alcohol, commonly called sphingosine, coupled to a fatty acid. Quite a variability is displayed by the ceramide belonging to gangliosides, for example, its length or saturation, but by far the higher variability is found in the structure of the headgroup, which is made up of several sugar units.1 The presence of a bulky hydrophilic headgroup, the size of which is comparable with that of the hydrophobic region, constitutes an important difference with respect to most of the investigated double-tailed lipids, such as phospholipids. In fact, this peculiar structure determines two important consequences: (a) For most gangliosides, the packing parameter of the monomer inside the aggregate (defined as P ) v/〈A〉l, with v and l being the molecular hydrophobic volume and length and 〈A〉 the mean surface area per molecule at the interface2) is between 1/3 and 1/2, but closer to 1/2, which is in the region where the transition between micelles and bilayers occurs. In that region, big micelles are formed and even a small variation of physical * Author to whom correspondence should be addressed. Fax: -39-2-26423209. E-mail:
[email protected]. † Universita ´ di Milano. ‡ Universita ¨ t Kaiserslautern. § Universita ´ di Catania. (1) Tettamanti, G.; Sonnino, S.; Ghidoni, R.; Masserini, M.; Venerando, B. Physics of Amphiphiles: Micelles, Vesicles and Microemulsions; Degiorgio, V., Corti, M., Eds.; North-Holland: Amsterdam, 1985; p 607. (2) Israelachvili, J. N. Intermolecular and Surface Forces; Academic Press: New York, 1990.
parameters (e.g., surface area, headgroup hindrance) may result in a dramatic change of shape and size of the aggregate. It has been found that the aggregation number is decreased by more than a factor of 2 by increasing the number of sugar units of the headgroup from 4 to 6 and that sensible variations in N are also induced by a different disposition of the units themselves or even by small chemical modifications of the headgroups.3,4 (b) The bulky and flexible saccharidic headgroup may exist in different conformations. NMR measurements5,6 and theoretical calculations7,8 evidenced the existence of at least two main low-lying conformers at room temperature, accompanied by several more conformers with higher energies. Variations of the headgroup conformational equilibrium may induce changes of the surface area that, according to the above discussion, may sensibly change the shape and size of the aggregates. The observation of the variation of the mean aggregation number 〈N〉 of a ganglioside micelle, then, represents a sensitive way to follow geometrical changes of the monomers within the aggregate. In particular, a decrease in the aggregation number reveals an increase in the average surface area or headgroup hindrance. In turn, the packing in an aggregate of given shape and dimensions affects the monomer conformation. In particular, severe constraints are given to the order parameter of the hydrophobic chains by favoring the more ordered all-trans conformation in the case of a small surface area (3) Cantu`, L.; Corti, M.; Sonnino, S.; Tettamanti, G. Chem. Phys. Lipids 1986, 41, 315. (4) Corti, M.; Cantu`, L.; Del Favero, E. Nuovo Cimento 1994, 16D, 1391. (5) Acquotti, D.; Poppe, L.; Dubrowski, J.; von der Lieth, C. W.; Sonnino, S.; Tettamanti, G. J. Am. Chem. Soc. 1990, 112, 7772. (6) Brocca, P.; Berthault, P.; Sonnino, S. Biophys. J. 1998, 74, 309. (7) Wynn, C. H.; Robson, B. J. Theor. Biol. 1986, 123, 221. (8) Bernardi, A.; Raimondi, L. J. Org. Chem. 1995, 60, 3370.
10.1021/la981355n CCC: $18.00 © 1999 American Chemical Society Published on Web 06/19/1999
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〈A〉 and the entropy-driven gauche conformations for large areas. Therefore, properties related to the trans-gauche population equilibrium, such as the molar heat capacity, may be strongly affected by the interfacial modifications. In this work, we investigate the modifications of the interfacial structure of ganglioside micelles induced by their thermal history by probing the following parameters: (i) the size of the micellar aggregate; (ii) the profile of the molar heat capacity as a function of temperature. Experimental Section (a) Sample Preparation. The ganglioside GM1, on which our attention will be focused, has five sugar units in the headgroup. It is found in the nervous system in what will be called the “natural” form, which is a mixture of molecules, all with the same GM1 headgroup, but with some variability in the ceramide portion. In particular, while the fatty acid is stearic acid (more than 90% of the total fatty acid content), the second chain is almost equally distributed between C18- and C20sphingosine. The molecular species containing a sphingosine of given length have been prepared,9 called GM1(C18) and GM1(C20). To reduce the “random noise” which may affect the calorimetric measurements, preventing us from getting unequivocal results, a fixed protocol for the preparation of the dry samples was followed, which is (a) dissolution of the gangliosides in chloroform: methanol 2:1 (v/v), (b) removal of the solvent at 45 °C in a stream of nitrogen, and (c) drying under high vacuum overnight. Then they were dissolved in deionized and redistilled water at 17 °C to a final concentration of around 15 mg/mL, as assessed a posteriori according to the method of Svennerholm.10 Two to four samples at a time were prepared following the described steps, put in sealed cells, and kept at 17 °C in a water bath before use, for at least 24 h. Prior to DSC measurements, samples were incubated at different temperatures for 24 h. Following this procedure, DSC measurements on different samples submitted to the same treatment were nicely reproducible. (b) Light-Scattering and DSC Experiments. Light-scattering experiments were performed on a noncommercial apparatus equipped with an argon ion laser, a digital correlator, and a thermostated cell. The apparatus and the technique are described in great detail elsewhere.11 DSC experiments were performed on a Microcal MC-2 differential scanning calorimeter (Microcal Inc., Amherst, MA). The reference cell contained deionized and redistilled water. Measurements were recorded over the temperature range 2-60 °C at a scan rate of 13 °C/h. Subsequent heating scans were performed with a delay of at least 1.5 h with respect to each other.
Results and Discussion (a) Light-Scattering Measurements. Recently, lightscattering, X-ray, and neutron-scattering measurements from our laboratory clearly evidenced strong temperaturerelated variations of the micellar mean aggregation number, accompanied by dramatic thermal hysteresis effects, for the ganglioside GM1 and for other gangliosides, like GM2 (with four sugar units in the headgroup) and GD1a and GD1b (with six sugar units). A detailed description of the results is reported in refs 12-14. (9) Gazzotti, G.; Sonnino, S.; Ghidoni, R.; Kirschner, G.; Tettamanti, G. J. Neurosc. Res. 1984, 12, 179. (10) Svennerholm, L. Biochim. Biophys. Acta 1957, 24, 604. (11) Corti, M. Physics of Amphiphiles: Micelles, Vesicles and Microemulsions; Degiorgio, V., Corti, M., Eds.; North-Holland: Amsterdam, 1985; p 121. (12) Cantu`, L.; Corti, M.; Del Favero, E.; Digirolamo, E.; Sonnino, S.; Tettamanti, G. Chem. Phys. Lipids 1996, 79, 137. (13) Cantu`, L.; Corti, M.; Del Favero, E.; Digirolamo, E.; Raudino, A. J. Phys. II Fr. 1996, 6, 1067. (14) Corti, M.; Boretta, M.; Cantu`, L.; Del Favero, E.; Lesieur, P. J. Mol. Struct. 1996, 383, 91.
Figure 1. Typical behavior of the average aggregation number of gangliosides micelles as a function of temperature. Absolute numbers are given for GM1. Details are given in the text.
A typical behavior for most ganglioside micelles is reported in Figure 1 where we plot the mean aggregation number 〈N〉, deduced by light-scattering measurements, as a function of the equilibration temperature. A ganglioside micellar solution, prepared by dissolving the dry ganglioside in water at room temperature, is heated to progressively higher temperatures up to 60 °C. At the beginning, the system is at point A, and after each 5 °C step, the system is allowed to equilibrate. It can be seen that the aggregation number decreases progressively and considerably in a well-defined temperature range, between 30 and 55 °C, following path a. The aggregation number does not change with respect to the initial low-temperature value in the range below 30 °C, while in the range above 55 °C a constant lowest value is attained. On cooling the micellar solution, N does not reincrease but, following path b, stays at the lowest value reached at the highest temperature. At room temperature, the aggregation number is now at C. If the heating procedure along path a is stopped at an intermediate temperature between 30 and 55 °C, say 40 °C, only a slighter reduction of N occurs, and if cooled to room temperature, the sample reaches point B, following path b′. Different parallel paths of the b′ type can be drawn, depending on the highest temperature ever reached and kept by the system for a long enough equilibration time; that is, different 〈N〉 values can be assumed by the ganglioside micelles depending on their thermal history. If the system at B is reheated, it follows path b′ (until it crosses path a) and then path a for higher temperatures. When the critical temperature Tc ) 55 °C is reached, the thermal hysteresis disappears; that is, any subsequent heating and cooling procedure follows path b. This behavior is not related to irreversible thermal decomposition of the ganglioside molecule. By drying and redissolving the sample in water, the behavior reported in Figure 1 is reproduced. As a result, the aggregation number of the ganglioside micelles depends on their thermal history, in particular, it depends on the highest equilibration temperature ever reached by the solution. Although the absolute numbers are different for different gangliosides, that is, both the initial value and the extent of variation of N decrease while going through the series from GM2 to GD1b, nevertheless the topology of thermal hysteresis is independent of the ganglioside headgroup structure.12 Following the considerations pointed out in the Introduction regarding (a) the connection between aggregation number and monomer packing properties and (b) the conformational variability of the ganglioside molecule, we interpreted the described behavior in terms of a cooperative conformational transition involving the hydrophilic part of the ganglioside, changing the average surface area
Thermal Hysteresis in Ganglioside Micelles
Figure 2. Time evolution of the scattered intensity (proportional to the average aggregation number 〈N〉) of a GM1 micellar solution submitted to a sudden increase of temperature from 30 to 35 °C, showing that the process of adjustment of 〈N〉 has a long time constant, of the order of many hours.
by about 4 Å2,13 rather than the hydrophobic region. The combined discussion of light-scattering and DSC results will help in clarifying this point, showing that a melting transition of the hydrocarbon chains also occurs, but at lower temperatures. Prior to the discussion of calorimetric measurements, it is important to define the different time scales involved in these experiments. A change in the aggregation number in the temperature range 30-55 °C is a slow equilibration process, like the one shown in Figure 2. The long equilibration times are not at all unexpected, as monomer rearrangements in micellar aggregates of gangliosides have to face the high-energy barrier between the inside and outside conditions for the molecule.15 Figure 1 has been obtained using long equilibration times, of the order of 20 h for each experimental point, 5 °C apart from one another. Let’s have a look at what happens to the aggregation number of a sample, prepared and equilibrated at 20 °C, during a fast temperature scan (of the order of 13 °C/h), which does not wait for the long equilibration times required by the ganglioside micelles to rearrange. In the range below 30 °C, the behavior is, of course, identical to the one shown in Figure 1, as N does not change. Instead, above 30 °C, N still remains practically constant until the temperatures of 35-40 °C are reached and then rapidly decreases to the lowest value. As far as the aggregation number is concerned, during a fast scan, micelles are photographed in their initial condition at all temperatures below 35-40 °C. (b) Differential Scanning Calorimetry Measurements. It is common in amphiphilic aggregates of the lamellar type to find a cooperative transition from an ordered solidlike arrangement of the hydrophobic tails to a disordered liquidlike structure, as the temperature is raised. The transition, generally referred to as the Lβ f LR transition, is revealed by a sharp pseudo-first-order variation of the heat capacity.16 While particularly intense in lamellar aggregates, this transition is practically absent in the usual micelles because of the liquidlike nature of the micelle interior, usually made up of shorter chains, and because of their small aggregation number (of the order of or less than 102), which prevents the propagation (15) Cantu`, L.; Corti, M.; Salina, P. J. Phys. Chem. 1991, 95, 5981. (16) Cevc, G.; Marsh, D. Phospholipid Bilayers; Wiley: New York, 1987.
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Figure 3. Typical DSC run for GM1(natural) micellar solution.
of cooperative effects.17 By contrast, the bigger micellar aggregates made up of gangliosides have a rather complex thermotropic behavior. Such behavior has already been observed in the literature, together with some “annoying” irreproducibility features that could be eliminated but are not understood.18 As a matter of fact, systems containing gangliosides or ganglioside/phospholipid mixtures exhibit thermal hysteresis and metastability during the heating and cooling cycles in DSC experiments which disappear only after prolonged thermal annealing of the samples.18 In the present DSC study, the GM1-ganglioside micellar system has been observed to show two endothermic peaks, the more intense below 30 °C, the other one lying in a higher temperature range, roughly between 30 and 60 °C (see Figure 3). Both peaks are quite broad, the second being somehow broader than the first. To get an insight into the peak assignment, let us compare the DSC measurements of samples prepared according to the same procedure (dissolution of the dry compound, temperature equilibration) starting from the different molecular species GM1(C18) and GM1(C20), and the natural mixture of them. The micelles formed by the three GM1 species have been observed by light scattering to have practically the same aggregation number. Instead, by increasing the hydrocarbon chain length from 18 to 20 carbon atoms, the enthalpy associated with the first peak increases (from 5 to 15 kJ/mol) and its position is shifted about 10 °C toward higher temperatures, while the second peak at higher temperatures is less affected by chain length variations. The first peak observed for the natural compound, which is a mixture of the two selected molecular species, occurs in an intermediate position for what concerns both the transition temperature and enthalpy. Moreover, DSC data relative to bilayers made up of different small-headgroup glycosphingolipids with different lengths of the hydrophobic moiety,18 indicate that longer chains yield higher Lβ f LR transition temperatures, as happens for more common diacylglycerol lipids.16 According to these observations, we may conclude that the peak displayed at lower temperatures is likely to be associated with a transition from a more ordered to a more disordered condition of the hydrocarbon chains. (17) Binder, K. Annu. Rev. Phys. Chem. 1992, 43, 33. This reference is a review. The shifting and rounding of the molar heat capacity variation in a confined geometry are well-known in many fields of physics. (18) Maggio, B.; Ariga, T.; Sturtevant, J. M.; Yu, R. K. Biochemistry 1985, 24, 1084.
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Let us pause and consider that the meaning of “order or disorder of the hydrophobic chains” in a volume as small as the micelle core is markedly different from the one which can be attributed to the same concept in a bilayer core. In fact, a bilayer, although limited in thickness to at most twice the length of the chains, is extended in volume, a feature which is essential for the onset and propagation of a cooperative behavior like that involved in the chain conformational rearrangement. In addition, a bilayer displays a geometry that favors the parallel disposition of the hydrophobic chains. In general, it can be said that the high number of monomers per aggregate, giving rise to a large hydrophobic volume, together with a favorable geometry should determine the possibility of realizing a more ordered Lβ phase undergoing a cooperative transition to a less ordered LR phase when the temperature is raised. Usually, these conditions are not met in micellar systems, as micelles are often too small and spherical or rodlike in shape. Rather, both the considerable size of the ganglioside micelles (200-300 monomers) and their disklike shape14 (axial ratio of about 2) allow the really unusual appearance of a Lβ f LR type transition in such micellar aggregates, somehow resembling small pieces of a bilayer. The above considerations are easily overlooked, as DSC measurements for molecules of biological interest, including micellar gangliosides, are naturally taken as an extension of those belonging to liposome-lipids and interpreted accordingly. In liposome-type systems, made up of a very large number of lipids per aggregate, the size on the colloidal scale is not so important. As far as the second peak in the calorigram is concerned, it is important to realize that it occurs in the temperature range 35-50 °C and that its position does not depend on the ganglioside hydrophobic chain length. This is an interesting observation since the enthalpy associated with this second peak could be due to the cooperative transition of the headgroups at the micellar surface, which we propose to occur in the same temperature range. Of course, it would have been of great interest to perform a direct quantitative correlation between the geometrical and thermodynamical observations of the headgroup conformational transition. Unfortunately, DSC data are not sufficiently clear to allow a convincing discussion, since the intensity and width of the second peak change with the different preparation protocols of the dry compound before dissolution. Nevertheless, somehow unexpectedly, it is the first peak (which is insensitive to the preparation protocol of the dry compound) that brings direct and indirect information on the thermal hysteresis phenomenon we are interested in, although occurring in a different temperature range with respect to the headgroup conformational transition. Mainly the results on GM1(C18) will be discussed, which are clearest and show the least superposition of the two peaks, although exactly the same behavior is observed for GM1(C20) and the natural GM1, shifted in temperature, as already pointed out. Figure 3 reports the behavior of the specific heat versus temperature obtained for the GM1(C18) sample prepared as described above, kept at an equilibration temperature of 20 °C and submitted to a temperature scan at a rate of 13 °C/h in the range 2-60 °C. A chain-melting peak centered at 17.5 °C is observed. The same sample is then cooled again to 2 °C, without being removed from the measuring cell, and submitted to a second identical scan. A chain-melting peak is again observed, now centered 5 °C lower, at 12.5 °C, a result which is observed for any subsequent identical scan. Let’s now move to another
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Figure 4. DSC “first” runs for GM1(C18) micellar solutions kept at different equilibration temperatures, 17, 30, 35, 40, 45, 50, and 60 °C, displayed in sequence starting from the bottom with an offset. “Second” and subsequent runs are all superimposable to a “final” one, which is the same as the one obtained for the sample kept at an equilibration temperature of 60 °C. Interval between tics corresponds to 200 J/(mol K).
sample, equilibrated at a different temperature, namely, 40 °C, and submitted to the same temperature scan for the first time: a chain-melting transition is observed, now centered around 14 °C, while a second scan again shows a peak at 12.5 °C, as well as any subsequent one. The same experiment has been performed on different GM1(C18) samples, each of them equilibrated at different temperatures, leading to the following landscape: (a) Samples equilibrated at different increasing temperatures give rise to chain-melting peaks centered at different decreasing temperatures during the first scan up to 60 °C. (b) Samples submitted to a second scan give rise to chain-melting peaks centered around a temperature that is lower than that of the first scan. The peak position is the same for all samples, independently of their equilibration temperature before the first scan; actually, the second and all subsequent scans are superimposable to a “final” reversible spectrum. A small enthalpy reduction (about 10%) is observed on passing from the first to the second scan. (c) Samples equilibrated at 60 °C show no difference between the first and second and subsequent scans, all being of the “final” type. As already mentioned, similar behavior has been also observed for GM1(C20) and the natural GM1. In Figure 4, some calorigrams are shown, while in Figure 5, a summary of the results regarding the position of the chainmelting peak is reported. (c) Light-Scattering and DSC Results, Combined. The combined analysis of the results obtained by laser light scattering and by DSC on ganglioside micelles provides additional interesting points for discussion. We want to underline that the parallel use of the two techniques has allowed the clear experimental observation of a coupling between headgroups and chains conformations and between conformational transitions, coupling mediated and modulated by the aggregated structure. This direct observation has been made possible because, while revealing changes in the heat capacity of the solution by DSC, on one side, the behavior of the mean micellar aggregation number 〈N〉 was followed by light scattering, on the other side. In this way, the packing properties and their variation were watched both from a geometric and a thermodynamic point of view.
Thermal Hysteresis in Ganglioside Micelles
Figure 5. Chain-melting temperature as a function of the sample equilibration temperature for micellar solutions of GM1(C18), natural GM1, and GM1(C20). The black and white circles account for the first and second DSC runs, respectively. The general features are the same, although the temperatures are shifted.
Figure 6. (a) Average aggregation number 〈N〉 of GM1 micelles as a function of the equilibration temperature, provided that this temperature has never been exceeded; (b) first DSC run (heating mode) relative to the three solutions, A, B, and C, indicated in panel a, limited to the part pertaining to the hydrophobic chain transition; (c) second DSC run (heating mode) of the A, B, and C solutions, heated to 60 °C during the first run.
The light-scattering data give the key information necessary to understand why the transition temperature varies along the different DSC runs, as shown in Figures 4 and 5. Let’s look at Figure 6. In panel a, the values of the average aggregation number of GM1 micelles measured at low temperatures are reported as a function of the highest equilibrium temperature ever reached by the solution. Three points are labeled A, B, and C. They
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correspond to the micellar solutions on which DSC measurements will be discussed in the following. The behavior of the aggregation number measured for the three micellar solutions (A, B, and C) during a temperature scan has been shown in Figure 1, in which a vertical dashed line is drawn to separate two temperature regions. In the lower one, below 30 °C, the three distinct paths a, b′, and b keep constant values, and only in the upper region, above 30 °C, do they eventually deviate and finally merge to the same final value (the same as solution C) when a temperature of 60 °C is reached. We recall that any solution appearing in panel a of Figure 6 behaves as solution C, once it has been heated above the critical temperature of 55 °C. Panel b shows the first DSC runs (heating mode) relative to the three solutions (A, B, and C) restricted to the part pertaining to the hydrophobic chain transition, as discussed above. Since the size of the micelle decreases on going from A to C (see panel a), accordingly also the transition temperature must change, as we shall discuss later. On the other hand, during a second upward DSC run (panel c), all the micellar solutions (heated to 60 °C during the first DSC run and cooled again at 0 °C) have the same size, as discussed previously. Therefore, at this stage, the micelles must have identical transition temperatures, as experimentally observed. It can be easily seen that in the three cases, the transition occurs and dies out in the region below 30 °C, where the aggregation number remains constant. As discussed above, the position of the calorimetric peak and the micelle dimensions are directly correlated: the smaller the micelle size, the lower the transition temperature. This result is consistent with the physics of firstorder phase transitions in confined systems, because the packing inside the aggregate considerably affects the trans-gauche population equilibrium of the chains, at any given temperature. In fact, since the micelle size depends on forces acting on the monomers, any change in aggregation number can be viewed as revealing a change of the lateral pressure to which the monomers themselves are subjected in the micelle. According to the Clapeyron equation, a first-order transition temperature, Tm, is related to an applied pressure, Π, through the relationship ∂Tm/∂Π ) Tm∆ν/∆H,19,20 where ∆H and ∆ν are the melting enthalpy and volume variation upon the transition. Since ∆H is small but positive, about 5 kJ/mol for GM1(C18), and ∆ν has been measured to be positive, the melting transition should occur at lower temperatures for aggregates with larger surface areas (or smaller aggregation number), as observed in the present DSC data. The comparison between the geometric and thermodynamic results provides some important pieces of information: (a) It confirms that the irreversible reduction of the micellar aggregation number of gangliosides, which is observed to occur in a temperature range above 30 °C, is connected to the existence of a cooperative conformational transition of the hydrophilic headgroups on the micellar surface, as discussed in a previous paper.13 This transition (above 30 °C) is distinct from the hydrophobic chain transition, which takes place in a different temperature range (below 30 °C). Then, ganglioside monomers can adopt different conformations both in the hydrophobic and in the hydrophilic moieties, developing correlated domains both in the core and surface regions of the aggregate. (19) Guggenheim, E. A. Thermodynamics; North-Holland: Amsterdam, 1952. (20) Raudino, A.; Zuccarello, F.; La Rosa, C.; Buemi, G. J. Phys. Chem. 1990, 94, 4217 and references therein.
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(b) Although distinct, the behavior of such domains is seen to be coupled by the aggregated structure and made visible through the hysteretic behavior of the headgroup transition. At a given temperature, the conformation of the chains in the core depends on the conformation of the headgroups on the surface. This has been clearly and unequivocally seen in these experiments, as nothing has been added or chemically modified in the two domains, just the property of ganglioside headgroups has been exploited to adopt different conformations at the same temperature, depending on their thermal history. Finally, we recall that the correlation between the geometric and thermodynamic results has been particularly significant due to the peculiar features of the ganglioside micelles core, in which the rather small micellar size (unfavoring the chain ordering) and the flat geometry (favoring the chain ordering) compete in a delicate interplay which amplifies even small variations. Conclusions The present work gives experimental evidence of some unexpected phenomena. First, it points out that the orderdisorder transition usually observed in the hydrophobic domain of membrane-type aggregates also exists in ganglioside micelles and that the hydrophobic chain transition is affected by the aggregate geometry, in the direction of reducing the transition temperature when the aggregate is smaller. Second, this work reveals the existence of an interesting interplay between surface and core conformational cooperative transitions in ganglioside micelles. The con-
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formation of the headgroups at the micellar surface is clearly seen to be coupled to the conformation of the hydrophobic chains in the core via the geometric constraints imposed by the aggregated structure. The confinement of monomers in aggregates of the micellar type, constituted by a rather small number of units, makes this coupling particularly effective. This feature induces metastabilities in the melting temperature of the hydrophobic chains, a transition which is known to be reversible. Finally, such hysteresis effects, connected to cooperative and coupling behavior occurring in aggregates containing gangliosides, may be relevant in biological events. The cooperative conformational transition of the ganglioside headgroups has been observed to be triggered not only by temperature but also by other agents, like interparticle forces.21 The irreversibility of the transition makes the system a potentially regulated one, in a biological environment. Then, the fact that a morphological modification induced in the hydrophilic domain causes a direct morphological response in the hydrophobic one could be of importance for the implication of gangliosides in biotranduction processes. Acknowledgment. This work has been partially supported by the Italian MURST (Cofinanziamento 1997), by the Italian Consiglio Nazionale delle Ricerche (CNR), and by the ECC HC&M Project ERBCHRXCT920019. LA981355N (21) Boretta, M.; Cantu`, L.; Corti, M.; Del Favero, E. Phys. A 1997, 236, 162.
