Thermal Aggregation of Bovine Serum Albumin in Trehalose and

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Thermal Aggregation of Bovine Serum Albumin in Trehalose and Sucrose Aqueous Solutions Massimo Panzica, Antonio Emanuele, and Lorenzo Cordone J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/jp3054197 • Publication Date (Web): 30 Jul 2012 Downloaded from http://pubs.acs.org on August 24, 2012

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The Journal of Physical Chemistry

Thermal Aggregation of Bovine Serum Albumin in Trehalose and Sucrose Aqueous Solutions Massimo Panzica, Antonio Emanuele*, Lorenzo Cordone Dipartimento di Fisica, Università degli Studi di Palermo, Via Archirafi 36, I-90123 Palermo, Italy *corresponding author: [email protected] RECEIVED DATE (to be automatically inserted after your manuscript is accepted if required according to the journal that you are submitting your paper to)

ABSTRACT.

We report results of static and dynamic light scattering measurements performed on bovine serum albumin (BSA) in saccharide (trehalose and sucrose) solutions. Our aim is to study the effects of the two disaccharides on the first steps of thermal aggregation of BSA in aqueous solutions at two protein concentrations (1 mg/ml and 30 mg/ml) at increasing sugar/water ratio. Results show that sugars modify early stages of aggregation mainly by perturbing the thermodynamic behaviour of the solvent (i. e. general solvent effects) without involving direct, specific sugar-protein interactions. This agrees with current hypotheses on sugar action in protein solutions1-3. The linear correlation detected between the characteristic temperature of the aggregation process and the glass transition temperature of the watersugar solvent strengthens the above suggestions.

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Keywords: BSA, serum bovine albumin, trehalose, sucrose, thermal aggregation, glass transition, dynamic light scattering, bioprotection.

DEDICATION This work is dedicated to the memory of Prof.s M.B. Palma-Vittorelli and M.U. Palma.

1. Introduction It is well known that alterations of external condition such as increasing temperature or additions of cosolvents, which destabilize the protein structure may accelerate the proteins aggregation processes4, while addition of cosolvents as e.g. saccharides, which stabilize the protein’s structure against thermal denaturation, leads to a decreased rate of protein’s aggregation5,6. Among saccharides, trehalose, a non-reducing disaccharide composed of two linked units of glucose, resulted the best stabilizer of biostructures against thermal denaturation7,8. Trehalose is found in large quantities in organisms that can survive adverse environmental conditions such as extreme dryness and high temperatures9-12 under a condition, known as anhydrobiosis, in which all metabolic processes are blocked. These organisms can be kept dry and apparently dead for several years, and upon rehydration, their vegetative cycle restarts; it is accepted, by general consensus, that trehalose plays the stabilizing role; furthermore, isolated structures, such as enzymes or liposomes, are preserved against stressing conditions when embedded in trehalose matrixes13,14. Other saccharides accomplish analogous bioprotective effect; however, trehalose has the best structural and functional recovery7,8. Several experimental and simulative studies have been performed on sugar-water-biomolecule systems13,15-32, and several hypotheses have been proposed to rationalize the origin of trehalose peculiarity at molecular level. According to the Water Replacement hypothesis9, trehalose stabilizes biostructures, in the dry ACS Paragon Plus Environment

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state, via the formation of hydrogen bonds (HB) between the disaccharide and the biostructure surface. This hypothesis is considered at the basis of membranes bioprotection by trehalose33,34. At variance, the Water Entrapment hypothesis35 proposes that trehalose keeps the biomolecules native structure by preserving the native solvation by glass formation. This hypothesis is an extrapolation of a model based on thermodynamic data36, which suggest that, in solution, saccharides are preferentially excluded from the protein domain; this extrapolation is valid under the assumption that the stabilization mechanism acting in aqueous solution, does also work under very low-water conditions. A further hypothesis suggests that the fragility degree of the water/disaccharides mixtures plays a role in the bioprotectant effectiveness37. Decreasing the traces of residual water, in low-water trehalose matrixes, progressively inhibits the internal dynamics of encompassed proteins27: such inhibition is thought to be related to the increasing “solvent” viscosity, which hampers the large-scale motions eventually leading to denaturation. Furthermore, Raman and neutron scattering experiments37-39 suggested that trehalose induces a local order of water molecules in its neighbours, so disrupting the bulk tetrahedral network and avoiding ice formation. This, in turn, implies that trehalose alters the inherent structures of the solvent40 (i.e. water structures with overwhelming probabilities) and, as a consequence, the Solvent Induced Forces (SIF)41, which act on solute molecules. MD simulations in ternary carboxymyoglobin/saccharide/water systems42,43 showed that in low-water trehalose matrixes, the protein is confined within a HB network (whose rigidity increases on drying), which includes sugar and water molecules, which connect groups at the protein surface to the surroundings. This network, which starts setting up at very low sugar/water ratios, was thought to anchor the protein surface to the stiff matrix under very low water conditions (dry state). This couples the internal dynamics of the protein to the dynamics of the low-water matrix44,45 and causes the inhibition of the internal protein dynamics. Indeed when strong, extended HB network is formed, as in glassy trehalose–water-protein systems, the large energy required for the rearrangements of the matrix, which accompany large-scale protein internal motions, sizably increases, thus preserving biostructures against denaturation. The existence of such network is also supported by FTIR measurements, which ACS Paragon Plus Environment

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studied the internal dynamics of MbCO together with the dynamics of the surrounding water-sugar matrix, in trehalose systems of decreasing hydration46. Analogous MD simulations42,47 and FTIR measurements44, which compared different disaccharides, evidenced that in low water sucrose or maltose matrixes MbCO has less tight coupling than in trehalose matrixes (see also section below). Recent FTIR and SAXS measurements48,49 on binary saccharide-water and on ternary MbCOsaccharide-water systems, containing trehalose, sucrose, maltose or lactose, showed that, conversely, the embedded protein has different effects on the structure and properties of different water/sugar surroundings. Aggregation of BSA It is well known that thermal aggregation of BSA at low concentrations and at neutral to low acidic pH is the result of many intertwining processes50, such as conformational and structural changes, crosslinking and phase separation. Also, at basic pH thermal aggregation of BSA leads to the formation of amyloid fibrils51. All these pathways are known to have conformational and structural changes as first and necessary step52, directly related to solvent exposure of Cys34 residue53, which is followed by crosslinking. Sugar addition in high concentrated BSA solution appears to increase protein stability against thermal denaturation54, mainly altering the thermodynamic properties of water. More in general, it is well known that thermodynamic and kinetic competition leading to protein aggregation or crystallization can be modulated by changing temperature, pH (i.e. protein charge) 55-57, or by adding cosolutes58-60. Such studies are of high interests spanning from biotechnologies to aggregation associate pathologies, to food technologies to fundamental physics61-63. In this paper, with the aim of separating effects arising from direct sugar-protein interaction, from those related to solvent perturbation on BSA aggregation, we studied the effects of trehalose and sucrose on the first steps of thermal aggregation at low and intermediate concentration of BSA in aqueous solutions at increasing sugar/water ratio. Furthermore, based on a calorimetric study on ternary proteinsugar-water systems, which showed a linear correlation between the denaturation temperature (Tden) of protein embedded in water sugar solutions and the glass transition temperature (TG) of the solvent ACS Paragon Plus Environment

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matrix64, we explored on the correlation between the characteristic temperature of aggregation process and the glass transition temperature of the water-sugar solvent. 2. Materials and Methods BSA (typeA-0281) purchased from Sigma was used without further treatments. Aqueous solutions of 1mg/ml (0.1% wt/wt 0.073% vol/vol) were prepared in phosphate buffer pH 6.2, 0.1 M, and ﬁltered through 0.22µm ﬁlters. Protein concentration value was determined measuring absorption intensity at 279 nm. To ensure the full dissolution of lyophilized protein, addition of sugars were done using the following procedure. Solutions of protein in buffer were prepared at appropriate concentration values. Sugar were added in due amounts to obtain the required protein and sugar concentration values. Solutions were gently stirred for 30 min to assure complete dissolution of sugar. Protein concentration was spectroscopically checked. Sugar have been added to the samples substituting approximately 20 molecules of water with 1 of sugar, in order to alter only the solvent part of the solution; so doing the ratio of the protein mass over total sample mass remained unaltered. This choice appears to be in agreement with previous results1-3, showing that the protein stabilization by sugars is mainly due to solvent modifications, and not to direct, specific sugar-protein interactions. In particular, sugar molecules are preferentially excluded from the protein domain and entrap the residual water at the interface by glass formation, thus preserving the native solvation. Charged sites of the protein with high binding energy are essentially occupied only by water molecules65, so that sugars can weakly bind the protein in low-energy sites. Other consequences of keeping the protein mass fraction constant will be discussed in the Result and Discussion section. Non-equilibrium light scattering measurements were performed on a Brookhaven goniometer BISM200, at fixed 90° scattering angle and a duplicated Nd-doped solid state laser, λ = 532 nm . Intensity of scattered light (SLS) were measured using an APD detector (Perkin Elmer Optoelectronics SPCMAQR-14). Measurements were performed as a function of time, while the sample temperature was increasing at a constant rate of 1,25 °C/h; our independent variable is therefore T(t). Intensity autocorrelation function (DLS) was obtained by using a 1024 channels correlator (Correlator.com FlexACS Paragon Plus Environment

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01D). Analyses of correlation functions were performed as a sum of single exponential decays. In fact, in the simple case of scattering by a monodisperse spherical specie of Brownian diffusing particles, the intensity correlation function has the mathematical form of a single exponential decay66. Fitting of −1

correlation functions provides the correlation time τ s = ( Dq 2 ) , where q represents the scattering vector and D the diffusion coefficient of the particles. By using Stokes-Einstein’s relation, the hydrodynamic radius Rhyd of such particles can be obtained as:

Rhydr = k BT 6πη D , where kB is the Boltzmann constant, and η is the viscosity of the solvent at the absolute temperature T. When Brownian spherical particles of different radii are present, the photon correlation function can be analyzed in terms of sum of independent exponential decays relative to each particle species. Intensity of scattered light depends on the product of particle mass and mass concentration of the scattering particles. Thus it allows us to monitor the amount of species diffusing in the sample. The fraction of intensity scattered by each species can be determined by the analysis of intensity correlation functions. Also, by using the radius estimation from DLS it is possible to make a log-log plot, where the relative intensity is reported with respect to the measured radius, for the specie with changing radius value. The slope of the curves so obtained gives an estimate of the fractal dimension of the diffusing particles of non constant radius66. For example, values of slopes equal to 3 reveals a formation of aggregates with the closest possible packing. Corrections to intensity values must be applied for large dimension of aggregates (> 30 nm). Anisotropy of aggregates was checked using depolarized scattering intensity.

3. Results and Discussion [Figure 1 about here] Preliminarily we performed experiments during T(t)-scans on BSA solutions without sugar: here we analysed the time autocorrelation function of the scattered intensity as a sum of two exponential decay (one with a large fixed radius to account for dust), as discussed in Material and Methods section. In ACS Paragon Plus Environment

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Figure 1 typical results are reported. In left panel, scattered intensity of the specie with free time decay is reported as a function of temperature, normalized with respect to its initial value. In the right panel we report the corresponding radius. Figure 1 shows an increase of scattering intensity starting at around 58 °C paralleled by the increase of the radius of a diffusing object, the initial size of which equals the one of the protein as measured at 20°C. We ascribe such simultaneous increase of size and intensity to the progressive formation of protein aggregates as already reported in literature52. At the end of experiments samples appear macroscopically liquid. [Figure 2 about here] We then performed light scattering measurements during T(t)-scans on BSA solution of various sugar concentration (from 0 to 40% w/w). As specified in the Introduction, our aim is to investigate the effect on the protein aggregation of two particular saccharides: trehalose and sucrose. For solutions with added sugar, analysis of light scattering data were performed by using three exponential decays: two were chosen as in the analysis of the sample without sugar, while the third one was added to take into account the diffusion of the sugar molecules and its variations with temperature. With such a choice only amplitudes of two exponentials and the size of only one of the diffusion coefficients were free in the fitting procedure. Figure 2 shows the normalized intensity scattered by the specie with free diffusion coefficient: left panel trehalose, right panel sucrose. Data relative to BSA solution without sugar are also reported for comparison. The showed data are limited up to a value of 5, because we are interested only in the initial stages of the aggregation process, which it is well known to depend on the solvent exposure of Cys34 residue53. The curves appear of identical shape but shifted towards higher temperatures as the saccharide concentration is increased. Shifted curves are shown in the inset of each panel, which shows that suitable shifts makes the whole set of curves to overlap, with different shifts for the two saccharides. Note that the scattered intensity due to the aggregating species, reported in Figure 2, is the most relevant contribution to the total intensity of scattered light. A better superposition of curves in figure 1 takes place in trehalose than in sucrose; this can be observed by sizably enlarging the plots in the inset and it is confirmed in the insets of figure 3. ACS Paragon Plus Environment

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As stated in Materials and Methods section, we expect that the sugar influence on the protein thermal aggregation should depend only on the solvent perturbation induced by the saccharide, and not on direct, specific sugar-protein interactions. As reported in literature52, the first steps of BSA aggregation are (a) conformational and structural changes of protein followed by (b) protein-protein crosslinking, in which Cys34 play a key role53. Both steps can be altered by sugar presence. However, the (a) step is reversible52 and seems to depend on the Solvent Induced Forces, which vary by changing temperature and solvent composition67. At variance, such forces can have only mild effect on the initial crosslinking, step (b), since this step depends on local specific interactions and is not reversible52. Results showed in Fig. 2 are in agreement with these suppositions, since the shape of intensity curves in Fig. 2 does not change by the addition of sugar. As an additional check, we performed measurements, where we kept constant the molar protein concentration rather than the weighted protein concentration. Results, not reported here, evidence scattering curves, which are analogous to the ones showed in Figure 2, but not superimposable by means of simple translations, even at early stages of aggregation. This confirms the hypothesis that initial protein crosslinking process (i.e. the second step of BSA aggregation) depends on the solution protein mass content . However, subsequent growth of aggregates is the result of an intertwining of crosslinking, conformational/structural changes and demixing of the solution50. In this respect, superposition of curves of Fig. 2 is not obtained for higher temperatures and later times. [Figure 3 about here] Figure 3 shows the temperature dependence of the hydrodynamics radii of aggregating specie obtained using the Einstein relation. Data are plotted with same color code as in Figure 2. All radii have an initial value which corresponds to the hydrodynamic radius of the protein and their values increase with T(t). This behavior can be attributed to the initial free diffusion of protein in its essentially monomeric state followed by progressive formation of aggregates. This is confirmed by observing that the scattered intensity of the growing specie is the main contribution to the total scattering intensity during all the Tscan showed in Fig. 3. Values of radii appear to be shifted in temperature as the content in sugar increases. Shifted curves are shown in the inset of each panel. Temperature shifts are slightly larger than ACS Paragon Plus Environment

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those used in Figure 2. All temperature shift values are reported in Table 1. The temperature shifts at intermediate sugar concentration depend significantly on sugar type. As mentioned above, there is no evidence of direct protein-sugar interactions, thus, the observed behavior should be due to solvent mediated sugar action. Indeed at low protein content conformational and structural changes precede and promote aggregation50,52. In order to investigate the effects of protein concentration, we studied samples with 3% w/w BSA concentration. This value is sufficiently high to evidence significant effects of protein concentration on temperature shifts but sufficiently low to let us deal with liquid solutions. Also, macroscopically self-supporting gels are formed in all solvent (water or water+sugar) environment. [Figure 4 about here] Experimental results show that addition of sugar to protein solutions of 3% BSA causes temperature shifts similar to the ones observed at 0.1%. In Figure 4 normalized light scattering intensity of 3% BSA samples are reported with the same color code as in Figure 2 and 3 and with suitable temperature shifts. At variance with data at 0.1% of BSA, the same temperature shifts used for the intensity curves make also the hydrodynamic radii to overlap. The observed temperature shift values are reported in Table 1. We note that shape and position of the plotted curves are different from the corresponding ones at lower concentration (inset of Fig. 2). Also, at the end of experiment and at variance with 0.1% BSA concentration, samples appear macroscopically as translucent gels. This is in agreement with the expected dependence of aggregation on protein mass content. In fact, it is known that at low concentration (0.1% or less) only small free floating aggregates are formed while firm self-supporting gel results from aggregation at higher concentration68. [Table 1 about here] No significant dependence of temperature shifts on protein concentration have been observed in the case of trehalose, while smaller ones have been observed in the case of sucrose. The different behavior between the two saccharides can be rationalized based on MD simulations (55,56), which showed that two intramolecular hydrogen bonds can be formed in sucrose (at relatively low ~ 0.1 M sugar) at variance with trehalose, where a single intramolecular HB can take place and the ACS Paragon Plus Environment

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hydration number in the range 10% ─ 90% sugar water w/w is about two units lower for sucrose than for trehalose. This makes sucrose, on the average, more rigid than trehalose and less disposable to fit hydrogen bond networks in its surroundings under conditions of low water. Indeed, when (under gelling conditions, i.e. high protein and sugar concentrations) the various components start competing for hydrogen bonding to residual water, the formation of internal HB may, in sucrose, break the continuity of the water sugar network, thus making the HB network in the protein surroundings softer than in trehalose, and allowing aggregation to take place with different temperature shifts at high protein concentrations. The formation of internal HB in sucrose has been also invoked by Francia et al.45 for rationalizing the sizable difference between trehalose and sucrose in hindering, under low water conditions, the internal dynamics of the reaction center of Rhodobacter Sfaeroides. The above argument confirms that, at least in the initial stages of aggregation, the effects of sugar addition can be ascribed to alterations of thermodynamic properties of water-mediated interactions, i.e. to the different thermodynamic behavior of the sugar-water solvent. [Figure 5 about here] Aggregates resulted isotropic particles, as checked by measuring the depolarized scattering intensity. In this respect, addition of sucrose or trehalose does not modify aggregate morphologies as it was observed by changing solution pH in solutions of the same protein51 or in different protein system58. In particular, pH changes appear more effective in altering protein aggregation51,57. Also, as specified in the Material and Methods section, we evaluated packing of aggregates by plotting the scattered intensities vs. the corresponding radius in a log-log scale; indeed, the slopes of such plots can be related to the socalled fractal dimension of the scattering objects66. In Figure 5 data are reported for BSA solution without sugar. Essentially, aggregate growth show four distinct regions. An initial growth with very low compactness (d ≈ 1.5) is followed by gaussian-coil like growth (d ≈ 2). After this growth, a narrow interval of very compact growth (d ≈ 3) precedes another gaussian-coil like growth. All this fractal dimension values but the initial one reveal the formation of relatively compact objects. This is in agreement with previous study50,52,55 evidencing a complex mechanism of BSA aggregation. The low ACS Paragon Plus Environment

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value of fractal dimension of initial growth is compatible with partial reversibility at the onset of aggregation as reported in literature52. Furthermore, aggregates formed at high concentration appear less compact than those formed at low protein concentration indicating the formation of large networks, typical of physical gels. [Figure 6 about here] Figure 6 shows data analogous to those reported in figure 5 for samples with and without sugar. All curves have similar shapes; the small differences in the normalized intensity, at fixed radius, indicate a different concentration of scatterers. The strict similarity of curves in Fig. 6 also confirm that sugar do not alter protein crosslinking, (no binding to protein), in line with the suggestion that they are preferentially excluded from the protein domain1,2. We can conclude that effects of sugar addition can be of the same thermodynamic nature as the inhibition of β2-microglobulin aggregation by the addition of crystallin60. Relevance of even subtle thermodynamic changes is also evidenced for protein crystallization67. As mentioned in the Introduction, a calorimetric study on ternary systems of protein, water and sugar has shown that at low water content (high protein concentration) a linear correlation between the protein denaturation temperature and the glass transition temperature of the whole solutions exists64,69. This correlation links two thermodynamic parameters lying into two far apart temperature ranges. This can be rationalized by considering that the same overwhelming structures of the solvent (as modified by sugar) dictate glass transition and protein denaturation. Accordingly, because the observed effects on BSA aggregation appear to depend on sugar addition only via the conformational changes of the protein, we compared the here obtained temperature shifts to the corresponding shifts of glass transition temperature, TG, of the pure solvent, in the absence of protein. Indeed in the present paper the protein concentration is sufficiently low not to affect the TG values extrapolated for binary water-sugar solutions in reference69, by using the Gordon-Taylor relation. This choice appears appropriate in view of the low dependence of TG on protein concentration in our concentration range69. Also, extrapolation was necessary since in our solution TG cannot be experimentally determined due to the low water to sugar ACS Paragon Plus Environment

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ratios used in this work69. Values of TG for sugar-water solutions obtained as described and relevant to this work are reported in Table 2. In Figure 7 temperature shifts of Table 1 are reported as a function of the glass transition temperature TG. A linear correlation among our temperature shifts and TG values is observed for samples containing trehalose; possible departures from linearity are instead observed for samples containing sucrose. The possible differences between sucrose and trehalose can be ascribed to the structural differences of sugar-water solution as outlined above. [Table 2 about here] [Figure 7 about here]

4. Conclusions In this work, we report on static and dynamic light scattering measurements performed at the onset of thermal aggregation of bovine serum albumin (BSA) dissolved in aqueous saccharide (trehalose or sucrose) solutions of various sugar/water ratio; the effects of both the disaccharides have been studied at two protein concentrations (1 mg/ml and 30 mg/ml). Time-temperature non equilibrium plots of the scattered intensity and of the protein aggregates hydrodynamic radius appear of identical shape, but shifted towards higher temperatures with increasing sugar; the shifts depending on the type of sugar. Furthermore, evaluation of the packing of aggregates by means of their fractal dimension evidences that growth mechanisms are not affected by the presence of sugar. In particular, aggregates show an initial growth with very low compactness (d ≈ 1.5), followed by mainly gaussian-coil like growth (d ≈ 2). The former value is compatible with partial reversibility of initial aggregates as observed in literature52, while the latter reveal the formation of irreversible compact objects. This is in agreement with previous study on BSA aggregation50,52, which evidence such complex mechanism of aggregation. We thus conclude, in agreement with the current literature, that sugar addition does not alter protein crosslinking via a direct, specific sugar-protein interactions. It is well known that aggregation depends also on protein concentration. Our results of measurements on samples with BSA concentration of 3% w/w confirm this evidence. However, addition of sugar to protein solutions of 3% BSA causes temperature shifts similar to the ones observed at 0.1%, in ACS Paragon Plus Environment

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trehalose, while significant differences have been observed for sucrose. This confirms that, at least in the initial stages of aggregation, the effects of sugar addition can be ascribed to alterations of statistical thermodynamics of the solvent, namely to the different overwhelming structures of the sugar-water solvent. Differences between trehalose and sucrose are to be linked to the different structure of their aqueous solution as proposed by various Authors. Specifically, we refer to the different ability of the two saccharides to form intra-molecular hydrogen bonds. The measured temperature shifts of the aggregation process have been put into relation to corresponding shifts of glass transition temperature, TG, of the sugar-water solvent. A linear correlation among our temperature shifts and TG values is observed for samples containing trehalose, while departures from linearity are possible for samples containing sucrose. In trehalose samples the observed linear correlation could illustrate that the same overwhelming configurations of the solvent govern the initial steps of protein aggregation in solution as well as the low temperature thermodynamic property (i.e. TG) of the same solvent. This is not the case for sucrose samples as probably due to temperature dependence of intra-molecular hydrogen bonding of sucrose. However, this question deserves further scrutiny. Thus, the second conclusion of our work is that properties and behavior of an aggregating protein solution depends on statistical thermodynamics of the whole solution. Specifically, based on the observation that exposure to the solvent of Cys34 is a prerequisite for BSA initial crosslinking, we ascribe the effects caused by sugar to the larger free energy variation required for Cys 34 to be exposed to the solvent. Of course this explanation can justify the increase of the protein denaturation temperature due to the presence of sugars. This evidence the importance of statistical thermodynamics of the whole solute-solvent system in protein aggregation though the crosslinking is site specific.

ACKNOWLEDGMENT


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We thank Italian Ministry of University and Research for funding under PRIN 2008 Project “Studi sperimentali e simulativi delle proprietà dinamiche, strutturali e funzionali di biomolecole in sistemi affollati e/o confinati: accoppiamento col mezzo esterno”.


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Tab. 1 – Temperature shifts for 0.1% BSA samples and for 3% BSA samples at a given sugar content obtained from intensity (Fig. 2 and Fig.4) and radius plot (Fig. 3). The error associated to each value is ±0.25 °C.

BSA 0.1%

BSA 3%

∆T trehalose (°C)

∆T sucrose (°C)

10 %

1.40

1.40

0.90

0.90

1.25

1.40

20 %

2.10

2.80

2.80

3.20

2.50

1.80

30 %

3.70

4.40

4.70

5.40

3.75

3.10

40 %

5.40

5.90

5.60

6.50

5.10

5.10

% sugar (w/w)

∆T trehalose (°C) (from Fig.2, (from Fig.3, (from Fig.2, (from Fig.3, (from Fig.4, right) right) left) left) right)

∆T sucrose (°C) ) (from Fig.4, left)

Tab. 2 – Values of glass transition temperature, TG, extrapolated for binary water-sugar solutions in reference58, by using the Gordon-Taylor relation.

TG (K) % sugar (w/w) trehalose

sucrose

10 %

141.43

140.85

20 %

147.94

146.65

30 %

155.81

153.62

40 %

165.63

162.28


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Figure 2

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Figure 4

Figure 5


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Figure 7 ACS Paragon Plus Environment

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Fig. 1 – Scattering intensity (left panel), normalized with respects to its initial value, and hydrodynamic radius (right panel) of BSA in solutions at 0.1% w/w concentration in absence of sugar.

Fig. 2 - Scattering intensity, normalized with respect to its initial value, of BSA in solutions at 0.1% w/w concentration in trehalose (left panel) and sucrose (right panel). The total sugar w/w percent in solution is: 0% (black lines), 10% (red lines), 20% (green lines), 30% (orange lines) and 40 % (blue lines). In the inset temperature-shifted curves are reported, showing a good superposition.

Fig. 3 – Hydrodynamic radius of BSA in solutions at 0.1% w/w concentration in trehalose (left panel) and sucrose (right panel). The total sugar w/w percent in solution is: 0% (black lines), 10% (red lines), 20% (green lines), 30% (orange lines) and 40 % (blue lines). In the inset temperature-shifted curves are reported, showing a good superposition.

Fig. 4 – Temperature-shifted scattering intensities, normalized with respects to their initial value, of BSA in solutions at 3% w/w concentration in trehalose (left panel) and sucrose (right panel). The total sugar w/w percent in solution is: 0% (black lines), 10% (red lines), 20% (green lines), 30% (orange lines) and 40 % (blue lines).

Fig. 5 – Normalized scattering intensity as a function of the hydrodynamic radius of BSA (0.1 % w/w) in log-log scale for a sample, which does not contain any sugar. The slope of the curves can be related to the fractal-dimension55 of the BSA: 1.5 (green line), 2 (pink line) and 3 (cyan line)..

Fig. 6 - Normalized scattering intensity as a function of the hydrodynamic radius of BSA (0.1 % w/w) in log-log scale for samples containing trehalose (left panel) and sucrose (right panel). The slope of the curves can be related to the fractal-dimension of the BSA aggregates. The total sugar w/w percent in solution is: 0% (black lines), 10% (red lines), 20% (green lines), 30% (orange lines) and 40 % (blue lines).

Fig. 7 – Temperature shifts of scattering curves induced by trehalose (left panels) and sucrose (right panels) as a function of the glass transition temperature of the system. The protein is present in the ACS Paragon Plus Environment

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samples at 0.1% w/w (upper panels) and at 3% w/w(lower panels), while the total sugar w/w percent in solution is: 0% (black), 10% (red), 20% (green), 30% (orange) and 40 % (blue). The black dashed lines shows qualitatively a linear correlation among the temperature shifts and the glass transition temperatures in trehalose, while larger departures from linearity are observed in sucrose.

REFERENCES

(1) Timasheff, S. N. Adv Protein Chem 1998, 51, 355. (2) Timasheff, S. N. Biochemistry 2002, 41, 13473. (3) Shimizu, S.; Matubayasi, N. Chemical Physics Letters 2006, 420, 518. (4) Carrotta, R.; Manno, M.; Giordano, F. M.; Longo, A.; Portale, G.; Martorana, V.; Biagio, P. L. Phys Chem Chem Phys 2009, 11, 4007. (5) Cao, X. M.; Yang, X.; Shi, J. Y.; Liu, Y. W.; Wang, C. X. Journal of Thermal Analysis and Calorimetry 2008, 93, 451. (6) Cao, X.; Wang, Z.; Yang, X.; Liu, Y.; Wang, C. Journal of Thermal Analysis and Calorimetry 2009, 95, 969. (7) Crowe, J. H. Adv Exp Med Biol 2007, 594, 143. (8) Jain, N. K.; Roy, I. Eur J Pharm Biopharm 2008, 69, 824. (9) Crowe, J. H.; Crowe, L. M.; Chapman, D. Science 1984, 223, 701. (10) Bianchi, G.; Gamba, A.; Murelli, C.; Salamini, F.; Bartels, D. Plant Journal 1991, 1, 355. (11) Uritani, M.; Takai, M.; Yoshinaga, K. J Biochem 1995, 117, 774. (12) Panek, A. D. Braz J Med Biol Res 1995, 28, 169. (13) Cordone, L.; Cottone, G.; Giuffrida, S.; Palazzo, G.; Venturoli, G.; Viappiani, C. Biochim Biophys Acta 2005, 1749, 252. (14) Chiantia, S.; Giannola, L. I.; Cordone, L. Langmuir 2005, 21, 4108. (15) Hagen, S. J.; Hofrichter, J.; Eaton, W. A. Science 1995, 269, 959. (16) Sastry, G. M.; Agmon, N. Biochemistry 1997, 36, 7097. (17) Kleinert, T.; Doster, W.; Leyser, H.; Petry, W.; Schwarz, V.; Settles, M. Biochemistry 1998, 37, 717. (18) Lichtenegger, H.; Doster, W.; Kleinert, T.; Birk, A.; Sepiol, B.; Vogl, G. Biophys J 1999, 76, 414. (19) Rector, K. D.; Jiang, J. W.; Berg, M. A.; Fayer, M. D. Journal of Physical Chemistry B 2001, 105, 1081. (20) Schlichter, J.; Friedrich, J.; Herenyi, L.; Fidy, J. Biophys J 2001, 80, 2011. (21) Dantsker, D.; Samuni, U.; Friedman, A. J.; Yang, M.; Ray, A.; Friedman, J. M. J Mol Biol 2002, 315, 239. (22) Ponkratov, V. V.; Friedrich, J.; Vanderkooi, J. M. Journal of Chemical Physics 2002, 117, 4594. (23) Mei, E.; Tang, J.; Vanderkooi, J. M.; Hochstrasser, R. M. J Am Chem Soc 2003, 125, 2730. ACS Paragon Plus Environment

20

Page 21 of 23

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(24) Caliskan, G.; Mechtani, D.; Roh, J. H.; Kisliuk, A.; Sokolov, A. P.; Azzam, S.; Cicerone, M. T.; Lin-Gibson, S.; Peral, I. J Chem Phys 2004, 121, 1978. (25) Massari, A. M.; Finkelstein, I. J.; McClain, B. L.; Goj, A.; Wen, X.; Bren, K. L.; Loring, R. F.; Fayer, M. D. J Am Chem Soc 2005, 127, 14279. (26) Cordone, L.; Ferrand, M.; Vitrano, E.; Zaccai, G. Biophys J 1999, 76, 1043. (27) Librizzi, F.; Viappiani, C.; Abbruzzetti, S.; Cordone, L. Journal of Chemical Physics 2002, 116, 1193. (28) Librizzi, F.; Vitrano, E.; Cordone, L. Biophys J 1999, 76, 2727. (29) Parak, F.; Knapp, E. W.; Kucheida, D. J Mol Biol 1982, 161, 177. (30) Doster, W.; Cusack, S.; Petry, W. Nature 1989, 337, 754. (31) Pal, S. K.; Peon, J.; Zewail, A. H. Proc Natl Acad Sci U S A 2002, 99, 15297. (32) Cottone, G.; Ciccotti, G.; Cordone, L. Journal of Chemical Physics 2002, 117, 9862. (33) Leslie, S. B.; Israeli, E.; Lighthart, B.; Crowe, J. H.; Crowe, L. M. Appl Environ Microbiol 1995, 61, 3592. (34) Lambruschini, C.; Relini, A.; Ridi, A.; Cordone, L.; Gliozzi, A. Langmuir 2000, 16, 5467. (35) Belton, P. S.; Gil, A. M. Biopolymers 1994, 34, 957. (36) Gekko, K.; Timasheff, S. N. Biochemistry 1981, 20, 4677. (37) Magazu, S.; Maisano, G.; Migliardo, F.; Mondelli, C. Biophys J 2004, 86, 3241. (38) Affouard, F.; Bordat P.; L.; Descamps, M.; Lerbret, A.; Magazu, S.; Migliardo, F.; Ramirez-Cuesta A.J.; Telling M.F.T. Chemical Physics 2005, 317, 258. (39) Hedoux, A.; Willart, J. F.; Ionov, R.; Affouard, F.; Guinet, Y.; Paccou, L.; Lerbret, A.; Descamps, M. Journal of Physical Chemistry B 2006, 110, 22886. (40) Weber, T. A.; Stillinger, F. H. J Chem Phys 1984, 80, 2742. (41) Vaiana, S. M.; Manno, M.; Emanuele, A.; Palma-Vittorelli, M. B.; Palma, M. U. Journal of Biological Physics 2001, 27, 133. (42) Cottone, G.; Giuffrida, S.; Ciccotti, G.; Cordone, L. Proteins 2005, 59, 291. (43) Cottone, G.; Cordone, L.; Ciccotti, G. Biophys J 2001, 80, 931. (44) Giuffrida, S.; Cottone, G.; Cordone, L. Biophys J 2006, 91, 968. (45) Francia, F.; Dezi, M.; Mallardi, A.; Palazzo, G.; Cordone, L.; Venturoli, G. J Am Chem Soc 2008, 130, 10240. (46) Giuffrida, S.; Cottone, G.; Librizzi, F.; Cordone, L. Journal of Physical Chemistry B 2003, 107, 13211. (47) Cottone, G. Journal of Physical Chemistry B 2007, 111, 3563. (48) Longo, A.; Giuffrida, S.; Cottone, G.; Cordone, L. Phys Chem Chem Phys 2010, 12, 6852. (49) Giuffrida, S.; Panzica, M.; Giordano, F. M.; Longo, A. Eur Phys J E Soft Matter 2011, 34, 1. (50) San Biagio, P. L.; Martorana, V.; Emanuele, A.; Vaiana, S. M.; Manno, M.; Bulone, D.; Palma-Vittorelli, M. B.; Palma, M. U. Proteins 1999, 37, 116. (51) Vetri, V.; D’Amico, M.; Foderà, V.; Leone, M.; Ponzoni, A.; Sberveglieri, G.; Militello, V. Arch. Biochem. Biophys. 2011, 508, 13. (52) Vaiana, S. M.; Emanuele, A.; Palma-Vittorelli, M. B.; Palma, M. U. Proteins 2004, 55, 1053. (53) Militello, V.; Vetri, V.; Leone, M. Biophys Chem 2003, 105, 133. (54) Hedoux, A.; Willart, J. F.; Paccou, L.; Guinet, Y.; Affouard, F.; Lerbret, A.; Descamps, M. Journal of Physical Chemistry B 2009, 113, 6119. (55) Militello, V.; Casarino, C.; Emanuele, A.; Giostra, A.; Pullara, F.; Leone, M. Biophysical Chemistry 2004, 107, 175. (56) Krebs, M.R.H.; Devlin, G.L.; Donald, A.M. Biophys. J. 2007, 92, 1336. (57) Manno, M.; San Biagio, P.L.; Palma, M.U. Proteins 2004, 55, 196. 21 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 23

(58) Smith, M. I.; Foderà, V.; Sharp, J. S.; Roberts, C. J.; Donald, A. M. Colloid Surf. BBiointerfaces 2012, 89, 216. (59) Pullara, F.; Emanuele, A.; Palma-Vittorelli, M. B.; Palma, M. U. Faraday Disc 2008, 139, 299. (60) Pullara, F.; Emanuele, A. Proteins 2008, 73, 1037. (61) Jahn, T. R.; Parker, M.; Homans, S. W.; Radford, S. E. Nat. Struct. Mol. Biol. 2006, 13, 195. (62) Dobson, C. M.; Nature 2005, 435, 747. (63) Oxtoby, D. W. Philos. Trans. R. Soc. London, Ser. A 2003, 361, 419. (64) Bellavia, G.; Giuffrida, S.; Cottone, G.; Cupane, A.; Cordone, L. Journal of Physical Chemistry B 2011, 115, 6340. (65) Careri, G.; Milotti, E. Phys Rev E Stat Nonlin Soft Matter Phys 2003, 67, 051923. (66) Schärtl, W. Springer 2007. (67) Pullara, F.; Emanuele, A.; Palma-Vittorelli, M. B.; Palma, M. U. Biophys J 2007, 93, 3271. (68) San Biagio, P. L.; Bulone, D.; Emanuele, A.; Palma, M. U. Biophys J 1996, 70, 494. (69) Bellavia, G.; Cottone, G.; Giuffrida, S.; Cupane, A.; Cordone, L. Journal of Physical Chemistry B 2009, 113, 11543.


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Thermal Aggregation of Bovine Serum Albumin in Trehalose and

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