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Surface Behavior of BSA/Water/Carbohydrate Systems from Molecular Polarizability Measurements Ysaias J. Alvarado, Atilio Ferrebuz, Jose Luis Paz, Patricia Rodriguez-Lugo, Jelem Restrepo, Freddy Romero, Jaqueline Fernández-Acuña, Yhan Williams, and Jhoan Toro-Mendoza J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b11998 • Publication Date (Web): 27 Mar 2018 Downloaded from http://pubs.acs.org on March 27, 2018

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The Journal of Physical Chemistry

Surface Behavior of BSA/Water/Carbohydrate Systems from Molecular Polarizability Measurements Ysaías J. Alvarado, Jelem Restrepo,

§

∗,†

†

Atilio Ferrebuz,

Freddy Romero, Williams,

⊥

k

‡

Jose Luis Paz,

¶

Patricia Rodríguez-Lugo,

Jaqueline Fernández-Acuña,

and Jhoan Toro-Mendoza

⊥

Yhan O'Neil

∗∗,⊥

†Centro de Investigación y Tecnología de los Materiales, Laboratorio de Caracterización

Molecular y Biomolecular, Instituto Venezolano de Investigaciones Cientícas (IVIC-Zulia) ‡Departamento de Física, Escuela Politécnica Nacional, Ladron de Guevara, Quito, Ecuador. ¶Laboratorio de Electrónica Molecular, Facultad Experimental de Ciencias, Departamento de Química, Universidad del Zulia, Maracaibo, Venezuela. §Centro de Investigación y Tecnología de los Materiales, Laboratorio de Investigación y Síntesis Sustentable de Nuevos Materiales. Instituto Venezolano de Investigaciones Cientícas (IVIC-Zulia). Maracaibo, Venezuela kCenter for Translational Medicine and Korman Lung Center, Thomas Jeerson University, Philadelphia, PA 19107, USA ⊥Centro de Estudios Interdisciplinarios de la Física. Instituto Venezolano de Investigaciones Cientícas (IVIC), Caracas, Venezuela. E-mail: *[email protected]; **[email protected]

Abstract

with the concentration is observed in presence of glucose. These results advocate the inuence of the electronic polarization on the repulsive and attractive protein-carbohydrate interactions. An analysis using the scaled particle theory indicates that the accumulation of glucose on the protein surface promotes dehydration. Inversely, hydration and preferential exclusion occur in the vicinity of the protein surface for sucrose enriched systems.

The eect of the presence of glucose and sucrose on the non-intrinsic contribution to partial molar volume hΘini of Bovine Serum Albumin (BSA) is determined by means of static and dynamic electronic polarizability measurements. For that aim, a combined strategy based on high-resolution refractometry, high exactitude densitometry, and synchronous uorescence spectroscopy is applied. Both static and dynamic mean electronic molecular polarizability values are found to be sensitive to the presence of glucose. In the case of sucrose, the polarizability of BSA is not appreciably affected. In fact, our results revealed that the electronic changes observed occurred without a modication of the native conformation of BSA. On the contrary, a nonmonotonous behavior

Introduction and background Inside cells and biological uids, the presence of ions, small molecules, and macromolecules collectively occupy approximately 40% of the total volume fraction. 1 This condition strongly aects the kinetic and thermodynamic behav-

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theoretically shown by Xi et al. 20 However, the use of electronic polarizability to evaluate this thermodynamic property in globular proteins in presence of crowders has not been reported yet. Instead, several studies about preferential hydration of protein in sugar solutions appear in literature using dialysis technique and refractometric and/or densitometric methods in presence of salt and buer at constant chemical potential. 2123 Chalikian has proposed that the mechanism of action of any particular additive on the stability of the protein is determined by the interplay between the excluded volume eect and the attractive protein-additive interactions. 24 On one hand, Graziano has highlighted that the magnitude of solvent-excluded volume effect plays the fundamental role. 16 Also, Guzey et al. have proposed that the concentration of sugar inuences the surface properties of globular proteins in two ways: at low concentrations, the specic binding of sugars to patches of the protein surface, and at high concentrations, preferential interactions between sugar and protein surface occur. 25 One the other hand, Anton et al. suggested that the attractive or repulsive interactions between the osmolyte and the peptide backbone or the amide groups are responsible for the stability of the proteins. 26 An interesting mechanism relating the static and dynamic polarizability of protein+water+additive to the Lifshitz theory of van der Waals dispersion forces was proposed by Damoradan. 8,27 He estimated molecular polarizability empirically from an additive model of the molar polarization values of amino acid residues. In his model, the high anity of water to protein surface and the thermodynamic stability of BSA structure in presence of additives, such as sucrose, is attributed to the repulsive Lifshitz-van der Waals interactions, and also to the electrodynamic pressure generated on the protein surface by the additive. Unfortunately, there are few reports of the optical dispersion of electronic polarizability for globular proteins. Marenich et al. have theoretically demonstrated that the electronic polarizabilities of interior atoms and functional

ior of macromolecules of interest which are immersed in these crowded environments with limited free space. 2,3 To reproduce these conditions in vitro, the degree of crowding of the medium is tuned by using synthetic polymers and proteins. It was demonstrated that the excluded volume largely aects protein folding, stability, aggregation, diusion, structural conformations, and binding of ligands. 4,5 These eects are commonly interpreted in terms of steric repulsions between hard spheres due to the mutual impenetrability of species. 6 However, when large deviations to this ideal model are observed, electrostatic, specic (e.g., hydrogen bonding), and chemical interactions are accounted for explaining the observed behavior. 5,7,8 Curiously, recent studies advocate that small molecules might induce even stronger crowding eects than those observed with larger crowders. 4,911 In particular, the presence of carbohydrates have important eects on the conformational stability of proteins. 1117 For example, the stabilization of Ovalbumin protein (OVA) in presence of small-sized additives such as glucose is mainly due to changes in the hydrophobic interactions assisted by other non-specic interactions as well as the steric exclusion effect of glucose molecules. 18 Indeed, the addition of some carbohydrates induces an increase in volume packing density and, consequently, in the magnitude of the solvent-excluded volume eects. This leads to stabilization of the native state of globular proteins by reducing the translational entropy of water. 2,7,11,12,1619 Here, our interest is to assess the behavior of protein-in-water systems in presence of carbohydrates used as crowding agents. For that, we use Bovine Serum Albumin (BSA) as a model globular protein to compare with previous reports where the interactions between additives and BSA were evaluated. Besides, we verify the eect of glucose and sucrose on the static and dynamic mean electronic polarizability of BSA and determine the non intrinsic contribution to limiting partial molar volume hΘini of BSA. The importance of electronic polarizability as a mean to characterize water density uctuations in the hydration shell of protein was


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groups in small molecules, nanoparticules, and peptides are highly quenched, thus reduced in comparison with the same atoms or functional groups exposed. 28 For this reason, models based on the additive contribution of atoms or functional polarizability are not adequate to fully predict the behavior of proteins under the action of electric elds.

at 298.15 K and atmospheric pressure using a variable-temperature Anton-Paar DSA-5000 acoustic densitometer calibrated before each series of measurements following the procedure previously described. 31,32 Each density value was determined by measuring the oscillation of a U -tube sample cell with a sample volume of 3.5 cm3 . For water, the values of density ρ1 (0.997069 g·cm−3 ), and refraction index n1(ν) at 589.9 nm (1.331538) obtained at 298.15 K are in agreement with previously reported values. 31 The values of refractive index in the visible regime (o-resonance regime) and density of medium (water and sugar+water), as well as binary (protein+water) and ternary solutions (protein+water+sugar) obtained are collected and shown in the Tables S1 and S2 of the supplementary material. Linear relationships were found for ρ and n(ν) at each frequency ν of the applied electric eld of the solutions against the respective protein concentration C2 . For simplicity, we represented the values of frequencies in terms of its complementary wavenumber. The dynamic mean electronic molecular polarizability of BSA e α2(ν) in water and in each carbohydrate-water solution was estimated using the data and the refractometric equation of Proutiere 33,34 which reads " 6n1(ν) M2 ∂n(ν) 3M 2 Φ1(ν) e + 2 α2(ν) = 4πN ρ1 n1(ν) + 2 ∂C2 c2 →0 # Φ1(ν) ∂ρ(ν) − , (1) ρ1 ∂C2 C2 →0

Experimental details General details

BSA (molecular mass M2 =66430 g·mol−1 ) Dglucose and sucrose were purchased from Sigma Aldrich and used without further purication. The solutions were prepared by dissolving the lyophilized powder with bi-distilled, deionized water (18 MΩ resistence). The concentration of protein was determined by UV-Vis spectroscopy using the value of molar absorption coecient reported in literature, 29,30 a spectrophotometer Shimadzu model UV-3101PC and using 1-cm quartz cells. The temperature was kept constant at 298.15 K with a Peltier eect based thermostatic unit Shimadzu CPS260. The concentrations C2 of protein ranged from 0.05 × 10−3 to 1.5 × 10−3 M. The concentration range was based on the solubility of protein in the absence and presence of sugar in pure water. The pH values of the solutions prepared uctuated close to 6.8. Static

and

dynamic

mean

elec-

tronic polarizability of BSA

where Φ1(ν) =

The refractive index n of both aqueous binary solutions (carbohydrate-water), and ternary solutions (BSA-carbohydrate-water) were measured with an Anton Paar Abbemat MW spectroscopic refractometer equipped with a highresolution CCD sensor, Fresnel analysis, and a LED as light source at 298.15 ± 0.03 K (Peltier eect). The wavelength was tuned in the range of 436.5 − 657.7 ±0.2 nm using an interference lter. The densities ρ of binary and ternary solutions, as well as the water solvent density ρ1 , were determined

(n2 −1) 1(ν) n21(ν) +2

and N is Avogadro's

number. The static mean electronic molecular polare izability α2(0) was obtained in each case by extrapolation to zero frequency (innite wavelength) of the plot between the dynamic mean electronic polarizability and the wavenumber. e This Cauchy-type dispersion curve (α2m(ν) = e 2 4 α2m(0) +A1 v +A2 ν , where Ai are the respective Cauchy coecients) only allows for an extrapolation of the electronic part of the polarizability.


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Results and discussions

Fluorescence studies

All uorescence spectra in steady state of pure protein and their mixture with sugar (cosolvent) in water were recorded on a RF-5301PC spectrouorophotometer (Shimadzu), coupled to a PC and equipped with an electronic thermo regulating water-bath for controlling the temperature. 1.0-cm quartz cells were used for the measurements. A solution of BSA (1 × 10−5 M) was titrated by successive addition of carbohydrate. The excitation wavelengths were set to 280 nm, and the emission spectra in each experiment were recorded from 220 to 500 nm. All measurements were performed at constant temperature of 298.15 K. The correction based on the inner lter eect was considered in these measurements due to strong absorbance from both protein and glucose at 280 nm. The excitation and emission slit widths were 5 nm. Synchronous uorescence spectra were recorded from 220 to 500 nm when the values were xed to ∆λ = 15 nm and ∆λ = 60 nm for tyrosine and tryptophan, respectively. 56 spectra were scanned in each case.

Figure 1 shows the dispersion curve of the electronic molar refraction of BSA in the presence of a constant concentration of glucose 6.94 × 10−2 M which acts as co-solvent. As expected, we observe an increment in electronic molar refraction with the increase of frequency. The dynamic behavior of polarizabilities can be tted to the two-term Cauchy dispersion equation due to high uncertainties in the slope A2 (see the corresponding static value and A1 Cauchy coefcients in Table S3). Electronic polarization of BSA.

Estimation of non-intrinsic contribution to limiting partial molar volume

Figure 1: Electronic molar refraction dependence on the frequency of the applied electrical eld using a xed concentration of glucose of 6.94×10−2 M.

hΘini of BSA at innite dilution was determined using a method recently reported, which is based on refractometric measurements and a linear electronic molecular polarization model. A value of intrinsic molar volume of 42326.95 cm3 ·mol−1 of BSA is used. 31 In this strategy, hΘini is determined as e R2m(ν) − Γ(ν) − Vi , (2) hΘini = Φ1(ν) e e where, R2m(ν) = 4π/3 N α2(ν) , Γ(ν) = 2 ∂n(ν) 2 6n1(ν) / n1(ν) + 2 and Vi is the ∂C2

Figure 2a represents the behavior of electronic e molar refraction R2m(ν) of BSA as a function of carbohydrate concentration and variable free quency. We observe that R2m(ν) decreases as the carbohydrate concentration increases. It is noteworthy that the magnitude of this reduction at each concentration of glucose or sucrose can be considered to be signicant compared to the values found for this property in absence of carbohydrate. In fact, a strong diminution of the dynamic electronic molar refraction of BSA was observed at the highest molar concentration of glucose studied here (2.8 × 10−1 M). These results underline that charge separation, or their induced uctuation by the applied elec-

C2 →0

intrinsic molar volume of the protein.


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viewed crudely as a measure of electrical deformability of protein. The previous suggests that the electronic polarization of protein increases as carbohydrate concentration rises. In accordance, Ferreira et al. 35 have reported that the dipolarity/polarizability π ∗ of water is modied by the concentration of osmolyte. In particular, this empirical electronic parameter increased with the glucose concentration. Based on the relation between electronic polarizability and molar volume, 31,36 we consider that the changes observed in the molecular polarizability of BSA in presence of carbohydrate is the result of alterations in the electronic environment in the vicinity of the protein and/or carbohydrate-protein surface. However, the latter contribution involves possible structural or conformational changes of the proteins in solution. 3740 Conformational changes of BSA? To detect any possible changes in the protein conformation, we evaluated the synchronous uorescence spectra for shifts in the maximum emission wavelength of the intrinsic uorescence of protein in the presence of dierent concentrations of glucose and sucrose used as additives. Figure 3 displays the spectra of BSA with ∆λ = 60 nm in the presence of dierent concentrations of glucose. Noticeably, the absence of a shift in the spectra maximum indicates that there is no change in the tryptophan microenvironment, inuential after the addition of glucose. Besides, similar results were obtained in the synchronous spectra at ∆λ = 15 nm for tyrosine (see Fig. S1 in the supplementary material). The spectra are nearly equal in shape and maximum of the band. Similar tendencies were obtained for this protein in presence of sucrose (see Fig. S2 in supplementary material), in agreement with the reported in the literature which highlight that direct strong interactions with globular proteins are considered unlikely due to no induced change in the circular dichroism spectra of these proteins. 22,41 We suggest that the perturbations in the conformation or shape of BSA by possible specic interactions of carbohydrates, and the corresponding formation of a complex under the experimental conditions of this study may be disregarded. In

Figure 2: (a) Electronic molar refraction of BSA vs. carbohydrate concentration for dierent frequencies of an applied electric eld. (b) A1 Cauchy coecients as a function of carbohydrate concentration. tric eld on the protein is very sensitive to the local concentration of molecules of glucose. Likewise, we notice that for a given concentrae proportionally intion of carbohydrate, R2m(ν) creases as the applied frequency rises (see Table S2 of supplementary material). However, in the e case of sucrose, the values of R2m(ν) vary around a constant value with the increase in concentration. The value obtained for a concentration of 2.08×10−1 M of sucrose is close to the obtained for electronic molar refraction at the same molar concentration of glucose. The corresponding values of the coecient A1 , which is related to the oscillator force of the protein at each carbohydrate concentration, is shown in Fig. 2b. Here, similar trends are found for both glucose and sucrose, though relatively greater values of the Cauchy coecients are acquired for glucose. The magnitude of A1 Cauchy-coecient can be


5


this sense, this behavior indicates that the inner structure of the protein is not appreciably altered by an interaction with glucose or sucrose molecules, and, only changes in the hydration shell around the surface of BSA possibly occur in presence of carbohydrate.

Page 6 of 14

of contributions from two terms: the van der Waals volumes of all the protein constitutive atoms Vw and the volume of cavities within the protein from imperfect atomic packing Vc . Vi is dependent on temperature, proportional to molecular weight M , and is considered as the geometric volume of protein impenetrable to surrounding solvent molecules (see equation 4). From our results obtained via SFS, refractometry, acoustic densitometry, and intrinsic molar volume Vi of BSA, we used equation (2) to calculate hΘini at each applied electric eld frequency in presence of carbohydrate, and the results are shown in Fig. 4. In the supplementary material (Tables S4), it can be noted that, for BSA at a xed concentration of glucose, hΘini is always a positive quantity, independent on the applied electrical eld frequency. This quantity represents a reduction of approximately 14% compared to the non-intrinsic contribution to the partial molar volume at innite dilution of BSA in water.

Figure 3: Synchronous uorescence spectra with inner lter correction at ∆λ = 60 nm of the BSA/water system in the presence of various concentrations of glucose. From these spectroscopic results, we propose that the changes observed in the molecular polarizability of BSA in presence of carbohydrate are dependent on the volume of the hydration shell of proteins because the intrinsic structure is undisturbed. In other words, the volumetric contribution to molar volume from intrinsic structure experiences no change under our experimental conditions. Intrinsic and non-intrinsic contribution to the limiting molar volume. By deni-

tion, the partial molar volume of solute at innite dilution V2∞ in an aqueous medium translates into two volumetric contributions: an intrinsic contribution Vi and a non-intrinsic one hΘini 31,32,42,43 ∞ V2,m = Vi + hΘini ,

(3)

Vi = V w + V c ,

(4)

hΘini = VT + Vint .

(5)

Figure 4: Eect of carbohydrate concentration on the non-intrinsic contribution to limiting partial molar volume of BSA at a xed frequency of an applied electric eld 1.52 µm (= 657.7 nm). Numbers in data-points reprecarb w sent the values of ∆hΘiexp ni = hΘini − hΘini . Also, it can be noticed dierent tendencies in Fig. 4 for the two carbohydrates used. Fluctuations in hΘini are observed with the increase in the concentration of sucrose, while a clear in-

The intrinsic term of protein Vi is composed


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verse linear relation with glucose concentration is observed. It has been widely accepted that sucrose does not exhibit long range strong electrostatic (dipolar) interactions with protein. 17 Glucose is a monosaccharide, while sucrose is a disaccharide composed of the condensation of one glucose molecule and one fructose molecule, binding via − 1, 2−glycosidic bond. Theoretically, a similar water structure around both glucose and sucrose has been reported, indicating that water is not aected by this condensation. 44 However, the hydrophobic attraction (h∆Gigwg = −20.3 mJ/m2 ) between glucose (g) molecules and surrounding water (w) is higher than estimated for the sucrose (s), (h∆Gisws = −11.5 mJ/m2 ). 45,46 With this in mind, it is plausible to assume that the aggregation of glucose close to protein surface is a more favorable process which can aect the structure, dipolar orientation, and electric local eld of water in the vicinity of protein. Our proposed model considers the medium as a dielectric continuum, and advocates that at elevated concentrations of glucose, preferential interaction occurs due to the dominating accumulation of glucose close to protein surface (i.e.Vint is high in magnitude in relation to BSA in water, see equation 5). Hence, the repulsive contribution VT is strongly balanced by the attractive protein-carbohydrate electronic interactions, mediated or not by water molecules in the concentration range of glucose studied, inducing possible dehydration of BSA. 47 Similarly, the interaction of glucose with lysozyme protein occur with dehydration and the number of water molecules expelled from protein's hydration shell to the bulk increases with the concentration of glucose, although the mechanism is not clear. 37 Hence, we infer that dehydration of the protein surface induced by this kind of electronic local interactions promote a diminution in the charge of the protein surface, thus aecting the corresponding magnitude of the dipole induced on the protein surface. The above results indicate that: (1) the structural exibility of protein in presence of carbohydrate is not aected, as can be seen from the invariance of the magnitude of hΘini with the change of frequency of the applied electric eld

at constant carbohydrate concentration, and (2) the solvation shell shall be governed by the interplay between repulsive interactions and attractive protein-cosolvent interactions (soft interaction). The tendency observed for hΘini conveys that the hydrophilicity of the protein surface decreases, owing possibly to signicant intermolecular electronic attractive interaction of glucose-amino acid residues on the protein surface. Schneider and Trout reported that glucose is preferentially excluded from the vicinity of the surface of BSA in PBS buer (ionic medium) at pH of 6 and glucose concentrations between 0.5 to 2.0 M. 48,49 From their data, we estimated the non-intrinsic contribution to limiting partial molar volume of BSA at 1 M of glucose in PBS at constant molarity. The value obtained was 5614.05 cm3 /mol, which was close to our value established at very low concentration of glucose 6.94 × 10−2 M in pure water. In gure 4, the values obtained for ∆hΘiexp ni are shown, dened here as the value of hΘicarb ni in presence of carbohydrate minus the value hΘiw ni obtained in absence of carbohydrate on BSA at 1.52µm. For the case of BSA in glucose, all values of ∆hΘiexp ni are negative. In presence of sucrose we observe a dierent behavior, hΘini has a value very close to that observed in glucose at the same molar concentration 2.08 × 10−1 M, which is lower than the value obtained in water pure. Although, at a higher concentration of sucrose 2.78×10−1 M, hΘini and ∆hΘiexp ni increase, taking up positive values higher than those estimated in the rest of systems. These high positive magnitudes of ∆hΘiexp ni suggest that the contribution is due to an increase in proteincarbohydrate repulsive interactions which is the dominant factor upon attractive interactions. Qualitative study using SPT model. A distinct alternative to interpret the results obtained for the non-intrinsic contribution to the partial molar volume of solute at innite dilution V2∞ of BSA comes from the theoretical model based on the Scaled-Particle Theory (SPT) proposed by Lee and Graziano. 42,50 Accordingly, this model substantiates V2∞ as ∞ V2,m = V2spt + ∆V2r .


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(6)


The rest of intrinsic molar volume Vi to both sides of equation leads to ∞ − Vi = V2spt − Vi + ∆V2r . hΘiTni = V2,m

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The aforementioned considerations were taken into account before we carried out the theoretical evaluation of the change that occurs in hΘiTni , involved in the transfer of molecular cavity from pure water to a binary mixture (carbohydrate + water) as spt spt r r ∆hΘiTni = V2,m − V2,w + ∆V2,m − ∆V2,w

(7)

Here, we have dened hΘiTni as the theoretical non-intrinsic contribution to partial molar vol∞ is the molar volume at inume of protein, V2,m nite dilution of the solute cavity in a binary mixture of hard spheres and it is given by Vm spt 3 2 2 2 V2 = y + 3Ay + 3A y + A B , C (8) where A = (1 − ξ) / (1 + 2ξ), B = (1 − ξ) /ξ , C = (1 + 3A + 3A2 + A2 B). ξ is the volume packing density of the medium dened as the ratio of the physical volume of a mole of solvent molecules over the molar volume of the 3 solvent (i.e., ξ = πN σm /6Vm ) and y = σ2 /σm , where σ2 is the hard sphere diameter of the protein molecule (51.2 × 10−8 cm). 31 The parameter σm is the hard sphere diameter of the solvent molecules. For mixed solvent systems water+carbohydrate the calculations were made by assuming an ideal solution (pseudosolvent approximation), and thenσm = ΣXj σj , and Vm = ΣXj Vj , here Xj , Vj and σj is the molar fraction, the molar volume and molecular diameter of the species j , respectively. For water (w), glucose (g) and sucrose (s), we used the following parameters, σw = 2.8×10−8 cm, Vw = 18.07 cm3 ·mol−1 , ξw = 0.384, σg = 6.72 × 10−8 cm, Vg = 111.88 cm3 ·mol−1 , ξg = 0.856; and σs = 8.26 × 10−8 cm, Vs = 211.55 cm3 ·mol−1 , ξs = 0.842, respectively. The data for water, 19 glucose and sucrose 51 were taken from the data reported in literature. ∆V2r is a measure of the volumetric contribution from specic and non-specic van der Waals components, and the solvent reorganization around the protein. We have xed this quantity empirically as ∆V2r ∼ = 0.06V2SP T following the results reported by Graziano for small polar and no polar molecules in water. 50 Despite its simplicity, the SPT model studies the behavior of thermodynamic properties and their excess quantities in dierent molecular systems with great success. 6,19

= ∆V2spt + ∆V2r .

(9)

As mentioned, our results suggest that the concentration of both carbohydrates is dierent at the protein surface compared to the bulk. Therefore, it would be useful to determine the values of the non intrinsic contribution given by Eq. (9) for the complete molar fraction range and compare it to the experimental results here reported in both values and signs. The results obtained for the systems with glucose and sucrose of ∆hΘiTni are shown in gure 5 (see Table S5 for details). We note that for glucose ∆hΘiTni is positive between a molar fraction X3 of 0 to 0.6, then a decrease is observed and eventually this trend transitions to negative values. Interestingly, all the experimental values obtained of ∆hΘiexp ni for BSA in the presence of glucose fall in the range of the negative values of ∆hΘiTni corresponding to the theoretical region of high concentration of glucose (see gure 4), while the experimental concentration Xcarbohydrate of glucose in molar fraction used was lower than 0.01. A qualitative comparative analysis with ∆hΘiTni - suggests that the local concentration of glucose in the region close to the protein surface is higher than in the bulk. Additionally, the behavior indicates that there is possible high molecular crowding in the vicinity of the protein surface, due to the high volume density packing ξm for the mixture (glucose + water), in comparison to the value in pure water (ξm = 0.384). To estimate the role that the ionic forces play, we determined ∆hΘiexp ni using the data reported previously by Schneider and Trout 49 at a concentrations of 1 M (X3 ≈ 0.015) of glucose in PBS (pH 6). Our value calculated 3 −1 for ∆hΘiexp ni was 210 cm ·mol . We carried out the same comparative analysis using our data. From gure 5 we observe that to replicate the value of 210 cm3 ·mol−1 obtained by Schneider


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to glucose, the experimental values of ∆hΘiexp ni in presence of sucrose are positive and their magnitudes fall theoretically in the low concentration region of sucrose as expected, with the exception of negative value of ∆hΘiexp ni . Curiously, the experimental results of high concentration of sucrose exhibits a behavior similar to the theoretical prediction for low concentrations, suggesting that the repulsive forces are dominating. However, at an intermediate concentration, sucrose accumulates in the vicinity of the BSA surface, possibly owing to an interplay between repulsive sucrose-protein forces and sucrose-sucrose attractive forces. However, more studies are necessary in non ionic media to establish whether a relation between crowding, dipolarity-polarizability of water (polarization), and the local structure of water exists. Finally, the results reported here allows us to infer that in the initial step of non-enzymatic glycation reaction of BSA should involve the electronic polarization between the protein and glucose molecules, local dehydration at some sites on the protein surface, and changes in the dipolarity/polarizability of water in the vicinity of the protein.

Figure 5: Change in the non-intrinsic contribution ∆hΘiTni to limiting partial molar volume spt of cavity V2,m for the process of transference from water solution to carbohydrate solution at 298.15 K using SPT model. and Trout, a low molar fraction of glucose is to be used. This suggests that the local concentration of glucose at the protein surface is low in concordance with preferential exclusion expected in an ionic medium. Furthermore, our results advocate that high local concentration of glucose in non ionic medium is possible. Moreover, the shielding of charges of amino acid residues by the presence of ions induce the exclusion of glucose from the protein surface. It is likely to occur by the breaking of electronic intermolecular interactions and weak specic interactions: a glucose molecule contains ve highly electronegative hydroxyl groups, i.e. in absence of electrolytes, the glucose molecule may be very close to the protein surface. In the case of sucrose, the theoretical behavior of ∆hΘiTni is similar to the observed for glucose. However, in these conditions the values of ∆hΘiTni with sucrose are higher than in presence of glucose. A negative region appears after 0.8 in molar fraction X3 of this carbohydrate. Theoretically, this result is in concordance with a decrease in volume packing density in solutions with sucrose in comparison to glucose mixtures throughout the range of concentration (see Table S3). However, in contrast

Conclusions We have successfully demonstrated that the surface behavior of BSA in water/carbohydrate systems can be described using molecular polarizability measurements. We summarize the main conclusions as follows:

• The magnitude of static and dynamic electronic molar refraction of BSA are affected by the presence of carbohydrate, and their values are lower than the obtained in pure water. In particular, those magnitudes are less sensitive to the presence of sucrose which varies around an almost constant value. • The results from SFS show that BSA does not exhibit conformational change with the addition of either carbohydrates, and support the idea of inalterable native states.


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References

• The non-intrinsic contribution to partial molar volume at innite dilution of BSA determined in presence of carbohydrate conrmed that hΘini is always positive ∞ representing around of 14% of V2m .

(1) Hall, D.; Minton, A. P. Macromolecular crowding: qualitative and semiquantitative successes, quantitative challenges. Biochim. Biophys. Acta 2003, 1649, 127 139.

• Electronic polarization evinces changes in the size of solvation shell of BSA by dehydration induced by the presence of electronic interactions and absence of specic interactions between glucose and protein surface.

(2) Ferreira, L. A.; Breydo, L.; Reichardt, C.; Uversky, V. N.; Zaslavsky, B. Y. Eects of osmolytes on solvent features of water in aqueous solutions. J. Biomol. Struct. Dyn. 2016, 35, 114.

• A comparative analysis between our results with the obtained using the SPT model proposed by Lee-Graziano rationally advocates the occurrence of accumulation of glucose in the vicinity of protein surface. The complex behavior of sucrose supports the idea of exclusionaccumulation-exclusion close to the protein surface. Also, a possible relation between crowding and local dipolarity/polarizability of water in the vicinity of the protein surface by structural reorganization of solvent is plausible.

(3) Kuznetsova, Breydo, L.; sky, V. N. ume eects: the crowded 13771409.

I. M.; Zaslavsky, B. Y.; Turoverov, K. K.; UverBeyond the excluded volmechanistic complexity of Milieu. Molecules 2015, 20,

(4) Hilaire, M. R.; Abaskharon, R. M.; Gai, F. Biomolecular crowding arising from small molecules, molecular constraints, surface packing, and nano-connement. J. Phys. Chem. Lett. 2015, 6, 25462553. (5) Rosen, J.; Kim, Y. C.; Mittal, J. Modest protein-crowder attractive interactions can counteract enhancement of protein association by intermolecular excluded volume interactions. J. Phys. Chem. B 2011, 115, 26832689.

Supporting Information Refractive index and density values for BSA in aqueous solution of glucose and sucrose, static and dynamic average electronic molecular polarizability and electronic molar refraction of BSA in presence and absence of carbohydrate, synchronous uorescence without inner lter correction spectra at ∆λ=15 nm of the BSA/water system in the presence of carbohydrate, geometric parameters and volumetric properties used for SPT calculations.

(6) Graziano, G. A view on the dogma of hydrophobic imperialism in protein folding. J. Biomol. Struct. Dyn. 2013, 31, 1016 1019.

Acknowledgement The authors thank the nancial support to Fondo Nacional de Ciencia, Tecnologia e Innovacion (FONACIT, Mision Ciencia (grant 2007000881)), the convenio inter-institucional IVIC-LUZ-INZIT. Thanks are given to Yiseli Gonzalez by her useful help in the preparation of the manuscript.

(8) Damoradan, S. Electrodynamic pressure modulation of protein stability in cosolvents. Biochem. 2013, 52, 83638373.

(7) Benton, L. A.; Smith, A. E.; Young, G. B.; Pielak, G. J. Unexpected eects of macromolecular crowding on protein stability. Biochem. 2012, 51, 97739775.

(9) Sharp, K. A. Analysis of the size dependence of macromolecular crowding shows that smaller is better. Proc. Natl. Acad. Sci. 2015, 112, 79907995.


10

Page 11 of 14 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


(10) Sharp, K. A. Unpacking the origins of incell crowding. Proc. Natl. Acad. Sci. 2016, 113, 16841685.

(19) Graziano, G. On the molecular origin of cold denaturation of globular proteins. Phys. Chem. Chem. Phys 2010, 12, 1424514252.

(11) Poddar, N. K.; Ansari, Z. A.; Brojen Singh, R. K.; Moosavi Movahedi, A. A.; Ahmad, F. Eect of oligosaccharides and their monosaccharide mixtures on the stability of proteins: a scaled particle study. J. Biomol. Struct. Dyn. 2010, 28, 331341.

(20) Xi, E.; Remsing, R. C.; Patel, A. J. Sparse sampling of water density uctuations in interfacial environments. J. Chem. Theory Comput. 2016, 12, 706713. (21) Timashe, S. N. Control of protein stability and reactions by weakly interacting cosolvents: the simplicity of the complicated. Adv. Protein Chem. 1998, 51, 355 432.

(12) O'Connor, T. F.; Debenedetti, P. G.; Carbeck, J. D. Stability of proteins in the presence of carbohydrates: experiments and modeling using scaled particle theory. Biophys. Chem. 2007, 127, 5163.

(22) Lee, J. C.; Timashe, S. N. The stabilization of proteins by sucrose. J. Biol. Chem. 1981, 256, 71937201.

(13) Singh, L. R.; Poddar, N. K.; Dar, T. A.; Rahman, S.; Kumar, R.; Ahmad, F. Forty years of research on osmolyte-induced protein folding and stability. J. Iran. Chem. Soc. 2011, 8, 123.

(23) Timashe, S. N. Protein-solvent preferential interactions, protein hydration, and the modulation of biochemical reactions by solvent components. Proc. Natl. Acad. Sci. 2002, 99, 97219726.

(14) Saunders, A. J.; Davis-Searles, P. R.; Allen, D. L.; Pielak, G. J.; Erie, D. A. Osmolyte-induced changes in protein conformational equilibria. Biopolymers 2000, 53, 293307.

(24) Chalikian, T. V. Excluded volume contribution to cosolvent-mediated modulation of macromolecular folding and binding reactions. Biophys. Chem. 2016, 209, 18.

(15) Auton, M.; Rosgen, J.; Sinev, M.; Holthauzen, L. M. F.; Bolen, D. W. Osmolyte eects on protein stability and solubility: A balancing act between backbone and side-chains. Biophys. Chem. 2011, 159, 9099.

(25) Guzey, D.; McClements, D. J.; Weiss, J. Adsorption kinetics of BSA at air-sugar solution interfaces as aected by sugar type and concentration. Food Res. Int. 2003, 36, 649660. (26) Auton, M.; Bolen, D. W.; Rösgen, J. Structural thermodynamics of protein preferential solvation: Osmolyte solvation of proteins, aminoacids, and peptides. Proteins 2008, 73, 802813.

(16) Graziano, G. How does sucrose stabilize the native state of globular proteins? Int. J. Biol. Macromol. 2012, 50, 230235. (17) Chen, L.; Shukla, N.; Cho, I.; Cohn, E.; Taylor, E. A.; Othon, C. M. Sucralose destabilization of protein structure. J. Phys. Chem. Lett. 2015, 6, 14411446.

(27) Damoradan, S. On the molecular mechanism of stabilization of proteins by cosolvents: role of Lifshitz electrodynamic forces. Langmuir 2012, 28, 94759486.

(18) Palaniappan, L.; Velusamy, V. Impact of cosolvent (glucose) on the stabilization of ovalbumin. Food Hydrocolloid 2013, 30, 217223.

(28) Marenich, A. V.; Cramer, C. J.; Truhlar, D. G. Reduced and quenched polarizabilities of interior atoms in molecules. Chem. Sci. 2013, 4, 23492356.


11


(29) Ghosh, K. S.; Sen, S.; Sahoo, B. K.; Dasgupta, S. A spectroscopic investigation into the interactions of 3-O-Carboxy esters of thymidine with bovine serum albumin. Biopolymers 2009, 91, 737744.

Page 12 of 14

aqueous media in macromolecular crowding eects. J. Biomol. Struct. Dyn. 2016, 34, 92103. (36) Chalikian, T. V.; Breslauer, K. J. On volume changes accompanying conformational transitions of biopolymers. Biopolymers 1996, 39, 619626.

(30) Taboada, P.; Barbosa, S.; Castro, E.; Gutiérrez-Pichel, M.; Mosquera, V. Effect of solvation on the structure conformation of human serum albumin in aqueous-alcohol mixed solvents. Chem. Phys. 2007, 340, 5968.

(37) Bru¹dziak, P.; Adamczak, B.; Kaczkowska, E.; Czub, J.; Stangret, J. Are stabilizing osmolytes preferentially excluded from the protein surface? FTIR and MD studies. Phys. Chem. Chem. Phys 2015, 17, 2315523164.

(31) Alvarado, Y. J.; Rodríguez-Lugo, P.; VeraVillalobos, J.; Ferrer-Amado, G.; Ferrebuz, A.; Restrepo, J.; Romero, F. Nonintrinsic contribution to the limiting partial molar volume of globular proteins in water: a study comparative between a new refractometric strategy and densitometric classical approach. Biointerface Res. Appl. Chem. 2015, 5, 916925.

(38) Mohamadi-Nejad, A.; MoosaviMovahedi, A. A.; Hakimelahi, G. H.; Sheibani, N. Thermodynamic analysis of human serum albumin interactions with glucose: insights into the diabetic range of glucose concentration. Int. J. Biochem. Cell B. 2002, 34, 11151124.

(32) Alvarado, Y. J.; Ballestas-Barrientos, A.; Restrepo, J.; Vera-Villalobos, J.; FerrerAmado, G.; Rodríguez-Lugo, P.; Ferrebuz, A.; Infante, M.; Cubillán, N. Volume-related properties of thiophene and furan-2-carboxaldehyde phenylhydrazone derivatives in DMSO: A discussion about non-intrinsic contribution. J. Chem. Thermodyn. 2015, 85, 210215.

(39) Pravdin, A. B.; Kochubey, V. I.; Mel'nikov, A. G. Fluorescent eosin probe in investigations of structural changes in glycated proteins. Opt. Spectrosc. 2010, 109, 193196. (40) Awang, T.; Wiriyatanakorn, N.; Saparpakorn, P.; Japrung, D.; Pongprayoon, P. Understanding the eects of two bound glucose in Sudlow site I on structure and function of human serum albumin: theoretical studies. J. Biomol. Struct. Dyn. 2016, 35, 110.

(33) Illien, B.; Ying, R. Molar mass, radius of gyration and second virial coecient from new static light scattering equations for dilute solutions: application to 21 (macro)molecules. ChemPhysChem 2009, 10, 10971105.

(41) Samanta, N.; Luong, T. Q.; Das Mahanta, D.; Mitra, R. K.; Havenith, M. Effect of short chain poly(ethylene glycol)s on the hydration structure and dynamics around human serum albumin. Langmuir 2016, 32, 831837.

(34) Hérail, M.; Le Guennec, M.; Le Go, D.; Proutiére, A. Molecular weight determination from light scattering and refraction in solutions. A new and coherent theoretical equation. J. Mol. Struct. 1996, 380, 171 193.

(42) Graziano, G. Non-intrinsic contribution to the partial molar volume of cavities in water. Chem. Phys. Lett. 2006, 429, 420 424.

(35) Ferreira, L. A.; Madeira, P. P.; Breydo, L.; Reichardt, C.; Uversky, V. N.; Zaslavsky, B. Y. Role of solvent properties of


12

Page 13 of 14 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


(43) Chalikian, T. V. Volumetric properties of proteins. Annu. Rev. Bioph. Biom. 2003, 32, 207235. (44) Te, J. A.; Tan, M.; Ichiye, T. Solvation of glucose, trehalose, and sucrose by the soft sticky dipole-quadrupole-octupole water model. Chem. Phys. Lett. 2010, 491, 218 223. (45) Docoslis, A.; Giese, R.; van Oss, C. Inuence of the water-air interface on the apparent surface tension of aqueous solutions of hydrophilic solutes. Coll. Surf. B: Biointerf. 2000, 19, 147 162. (46) Van Oss, C. J. Interfacial forces ous media ; CRC press, 2006.

in aque-

(47) Fucaloro, A. F. Reporting Molar Refractions. J. Solution Chem. 2002, 31, 601 605. (48) Arakawa, T.; Timashe, S. N. Stabilization of protein structure by sugars. Biochem. 1982, 21, 65366544. (49) Schneider, C. P.; Trout, B. L. Investigation of cosolute-protein preferential interaction coecients: new insight into the mechanism by which arginine inhibits aggregation. J. Phys. Chem. B 2009, 113, 20502058. (50) Graziano, G. Partial molar volume of nalcohols at innite dilution in water calculated by means of scaled particle theory. J. Chem. Phys. 2006, 124, 134507. (51) Fucaloro, A.; Pu, Y.; Cha, K.; Williams, A.; Conrad, K. Partial molar volumes and refractions of aqueous solutions of fructose, glucose, mannose, and sucrose at 15.00, 20.00, and 25.00 C. J. Solution Chem. 2007, 36, 6180.


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