Water Interface as Studied

Oct 20, 2010 - Pereira et al. studied the dilational surface rheological properties of BSA and β-casein solutions and discovered their high sensitivi...
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Bovine Serum Albumin Unfolding at the Air/Water Interface as Studied by Dilational Surface Rheology B. A. Noskov,*,† A. A. Mikhailovskaya,† S.-Y. Lin,‡ G. Loglio,§ and R. Miller †

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Department of Colloid Chemistry, St. Petersburg State University, Universitetsky pr. 26, 198504 St. Petersburg, Russia, ‡National Taiwan University of Science and Technology, Chemical Engineering Department, 43 Keelung Road, Section 4, Taipei, 106 Taiwan, §Dipartimento di Chimica Organica, Universita degli Studi di Firenze, Via della Lastruccia 13, 50019 Sesto Fiorentino, Firenze, Italy, and MPI f€ ur Kolloid- und Grenzfl€ achenforschung, Wissenschaftspark Golm, D-14424 Golm, Germany Received August 23, 2010 Measurements of the surface dilational elasticity close to equilibrium did not indicate significant distinctions in the surface conformation of different forms of bovine serum albumin (BSA) in a broad pH range. At the same time, the protein denaturation in the surface layer under the influence of guanidine hydrochloride led to strong changes in the kinetic dependencies of the dynamic surface elasticity if the denaturant concentration exceeded a critical value. It was shown that the BSA unfolding at the solution surface occurred at lower denaturant concentrations than in the bulk phase. In the former case, the unfolding resulted in the formation of loops and tails at surface pressures above 12 mN/m. The maximal values of the dynamic surface elasticity almost coincided with the corresponding data for the recently investigated solutions of β-lactoglobulin, thereby indicating a similar unfolding mechanism.

Introduction Protein conformational transitions at fluid-fluid interfaces are of fundamental interest in understanding colloid stability1-4 and the mechanism of physiological processes.5 At the same time, in spite of the fast progress in the investigation of protein folding and unfolding in bulk phases,6 any information on the influence of the interfaces on the protein secondary and tertiary structures is scarce and even sometimes controversial. The lost of the activity of some enzymes in the course of their adsorption at the liquid surface,7 the slow protein adsorption,8-10 and the formation of highly cohesive protein networks at liquid-fluid interfaces11,12 led some authors to the conclusion of globular protein unfolding at the transition from the bulk phase to the surface layer. Circular dichroism spectroscopy of bovine serum albumin (BSA) adsorption layers also indicated an increase in protein disordered structure and a high degree of unfolding of the protein.13 On the other hand, neutron and X-ray *Corresponding author. (1) Murray, B. S. Curr. Opin. Colloid Interface Sci. 2007, 12, 232. (2) Lucassen-Reynders, E. H.; Benjamins, J.; Fainerman, V. B. Curr. Opin. Colloid Interface Sci. 2010, 15, 264. (3) Martin, A. H.; Meinders, M. B. J.; Bos, M. A.; Cohen Stuart, M. A.; van Vliet, T. Langmuir 2003, 19, 2922. (4) Lad, M. D.; Birembaut, F.; Matthew, J. M.; Frazier, R. A.; Green, R. J. Phys. Chem. Chem. Phys. 2006, 8, 2179. (5) Cho, J.; Furie, B. C.; Coughlin, S. R.; Furie, B. J. Clin. Invest. 2008, 118, 1123. (6) Protein Folding Handbook; Buchner, J., Kiefhaber, T., Eds.; Wiley-VCH: Weinheim, Germany, 2005; Vols. 1-5. (7) James, L. K.; Augenste, Lg. Adv. Enzymol. Relat. Areas Mol. Biol. 1966, 28, 1. (8) Graham, D. E.; Phillips, M. C. J. Colloid Interface Sci. 1979, 70(403), 415–427. (9) Beverung, C. J.; Radke, C. J.; Blanch, H. W. Biophys. Chem. 1999, 81, 59. (10) Rao, C. S.; Damodaran, S. Langmuir 2000, 16, 9468. (11) Graham, D. E.; Phillips, M. C. J. Colloid Interface Sci. 1980, 76(227), 240. (12) van Aken, G. A. In Food Macromolecules and Colloids; Dickinson, E., Lorient, D., Eds.; The Royal Society of Chemistry: Cambridge, U.K., 1995; p 43. (13) Damodaran, S. Anal. Bioanal. Chem. 2003, 376, 182. (14) Lu, J. R.; Su, T. J.; Thomas, R. K.; Penfold, J.; Webster, J. Langmuir 1999, 15, 6975. (15) Lu, J. R.; Su, T. J.; Thomas, R. K. J. Colloid Interface Sci. 1999, 213, 426. (16) Lu, J. R.; Su, T. J.; Howlin, B. J. J. Phys. Chem. B 1999, 103, 5903. (17) Taylor, D. J. F.; Thomas, R. K.; Penfold, J. Adv. Colloid Interface Sci. 2007, 132, 69.

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reflectivity evidenced only slight and insignificant changes of the tertiary structure of various proteins including BSA in the course of adsorption.14-18 According to the data of Fourier transform infrared reflection absorption (FTIR) spectroscopy, the secondary structure of BSA, β-lactoglobulin (BLG) and glycinin also changed only a little upon adsorption at the air-solution interface.4,19,20 Another argument in favor of the preservation of the globular protein structure in the course of adsorption at the liquid surface can be derived from the effect of chemical denaturants. It has been shown recently that the addition of urea or guanidine hydrochloride (G.HCl) to BLG solutions results in strong changes of the surface properties.18,21 If the denaturant concentration exceeds a critical value, the surface properties approach those of the solutions of random coil proteins. At the same time, one can also observe the influence of the interface on the protein conformation. The unfolding of BLG globules in the surface layer occurs at lower denaturant concentrations than in the bulk phase.18,21 A possible explanation of this distinction can be connected with strong BLG dimerization in the solution bulk. The disintegration of dimers probably controls the unfolding rate in the bulk phase. The surface activity of monomers is higher than that of dimers, leading to the predominance of monomers in the surface layer and thereby to an easier BLG unfolding at the surface. These conclusions can hardly be related to solutions of other globular proteins with different unfolding mechanisms where the peculiarities of the unfolding in the surface layer remain unclear. In this work the denaturation of another model globular protein BSA with a globular structure significantly different from that of BLG is studied in the surface layer with the main aim being to elucidate the influence of the interface on protein conformation. (18) Perriman, A. W.; Henderson, M. J.; Holt, S. A.; White, J. W. J. Phys. Chem. B 2007, 111, 13527. (19) Meinders, M. B. J.; de Jongh, H. H. J. Biopolymers 2002, 67, 319. (20) Martin, A. H.; Meinders, M. B. J.; Bos, M. A.; Cohen Stuart, M. A.; van Vliet, T. Langmuir 2003, 19, 2922. (21) Noskov, B. A.; Grigoriev, D. O.; Latnikova, A. V.; Lin, S.-Y.; Loglio, G.; Miller, R. J. Phys. Chem. B 2009, 113, 13398.

Published on Web 10/20/2010

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The properties of BSA and its human counterpart (HSA) in the bulk phase have been investigated probably to the most extent.2,23 At the same time, the surface properties of their aqueous solutions were studied less frequently than those of BLG.2,8,13-15,24,25 Unlike the simpler BLG tertiary structure, the globule of BSA of higher molecular weight consists of three distinct homologous domains and has a triangular or heart-like shape close to the isoelectric point (pH ≈ 5).22 Each of the three BSA or HSA main domains consists of two subdomains where some amino acid residues are held together by disulfide bonds. The secondary structure of albumins is predominantly helical. The change in pH results in at least four reversible conformational transitions, which are better studied in the range below the isoelectric point. At pH ≈ 4.3, the heart-like normal or N form transforms into a more elongated F form which passes into an even more extended E form at pH ≈ 2.7. These transitions are characterized by the decrease of the helical content up to about 35% for the E form and by the consecutive loosening of the physical bounds between the three main domains. There is an analogy between the structural transitions upon pH decrease and the first steps of the albumin chemical denaturation at neutral pH.26 The structure of B and A conformers at basic pH has not yet been determined to a sufficient extent.22,23 Pereira et al. studied the dilational surface rheological properties of BSA and β-casein solutions and discovered their high sensitivity to the intraprotein structural rigidity.24 The structure of macromolecules strongly influenced the dynamic surface elasticity leading to the different surface rheological properties of BSA solutions at different pH values. The solutions of F conformer at low pH had much lower surface elasticity than the solutions of N form close to the isoelectric point. Recently it has been shown that the measurements of nonmonotonous kinetic dependencies of the dynamic surface elasticity allows investigation of some details of the conformational transitions in protein adsorption layers.27,28 Small additions of cationic and anionic surfactants to β-casein solutions resulted in a different influence on the height and position of the local maxima in the surface elasticity thereby providing the possibility to estimate the parts of protein molecules interacting with the surfactants to the greatest extent and to corroborate the proposed mechanism of surface conformational transitions.28 On the other hand, measurements of the dynamic surface elasticity of BLG solutions containing G. HCl provided the possibility to trace the unfolding process in the surface layer and to determine the critical denaturant concentration corresponding to the formation of the distal region of the surface layer (the region of loops and tails) by the unfolded protein.21 In this work, this approach is applied to BSA solutions.

Experimental Section BSA (Sigma-Aldrich, Germany) was used as received. The BSA solutions in phosphate buffer were prepared by dilution of a stock solution, which had been stored in a refrigerator at 2 °C not longer than one week. Most of the measurements in this work were carried out at a protein concentration of 3  10-8 M. The pH of (22) Carter, D. C.; Ho, J. X. Adv. Protein Chem. 1994, 45, 153. (23) Peters, T. All about Albumin Biochemistry, Genetics and Medical Applications; Academic Press: London, 1996. (24) Pereira, L. G. C.; Theodoly, O.; Blanch, H. W.; Radke, C. J. Langmuir 2003, 19, 2349. (25) Benjamins, J.; Lyklema, J.; Lucasen-Reynders, E. H. Langmuir 2006, 22, 6181. (26) Galantini, L.; Leggio, C.; Pavel, N. V. J. Phys. Chem. B 2008, 112, 15460. (27) Noskov, B. A.; Latnikova, A. V.; Lin, S.-Y.; Loglio, G.; Miller, R. J. Phys. Chem. C 2007, 111, 16895. (28) Latnikova, A. V.; Lin, S.-Y.; Loglio, G.; Miller, R.; Noskov, B. A. J. Phys. Chem. C 2008, 112, 6126.

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the solution was regulated by the addition of small amounts of HCl and NaOH, respectively. G.HCl (Sigma-Aldrich) was used as received. The substance was dissolved in a small quantity of buffer solution before addition to the protein solution. The volume of the solution was increased up to a required value after that. Sodium chloride from Vecton (St. Petersburg) was purified from organic impurities by heating in an oven up to 800 °C. All solutions in sodium phosphate buffer with an ionic strength of 0.02 M and pH = 6.7 were prepared by mixing Na2HPO4 and NaH2PO4 (SigmaAldrich) in appropriate amounts. Fresh triple-distilled water was used for the preparation of solutions. An all-Pyrex apparatus and alkaline permanganate were employed in the second and third stages of distillation. All measurements were carried out at 20 ( 1 °C. The surface tension was measured by the Wilhelmy plate method with a roughened glass plate attached to an electronic balance. The experimental setup for measurements of the complex dynamic surface elasticity by the oscillating barrier method together with the corresponding experimental procedure was described in detail elsewhere.27,29-31 We represent here only the basic principles of the employed experimental technique. The surface area of the solution in the Langmuir trough changed periodically with the given frequency by oscillations of the polytetrafluoroethylene (PTFE) barrier sliding along the polished brims of the trough. A mechanical generator transformed the rotation of an electromotor to the translational motion with reversion and provided the possibility to control frequency and amplitude. The moving part of the generator was connected to the PTFE barrier by a steel rod. In operation, the barrier glided back and forth along the Langmuir trough and produced oscillations of the liquid surface area δS with a relative amplitude of 3%. The corresponding surface tension oscillations δγ were measured by the Wilhelmy plate method. Both periodical functions of time γ(t) = γ(0) þ δγ(t) and A(t) = A(0) þ δA(t) could be represented in a complex form, and the complex surface elasticity ε at the angular frequency ω was determined as the ratio εðωÞ ¼

dγ ¼ εr þ iεi d ln S

The elasticity modulus was determined from the amplitude ratio of the oscillations of surface tension and surface area, while the phase shift between the oscillations of the two measured parameters (surface tension and surface area) determined the phase angle of the dynamic surface elasticity. At frequencies less than about 0.2 Hz, the length of surface longitudinal waves far exceeded the length of the Langmuir trough, and they did not influence the surface tension oscillations in the trough.25,29 Nevertheless, the surface tension can change slightly from point to point in the trough probably because of the influence of the trough walls on the liquid flow. If the Wilhelmy plate was not fixed at a given position relative to the brims, this effect could lead to an insufficient accuracy of the surface elasticity measurements (about 10%). Therefore, all the results in this work correspond to a fixed Wilhelmy plate position in the center of the Langmuir trough. This configuration allowed a significant reduction of the relative experimental error. The surface shear viscosity of BSA layers is negligible at concentrations under investigation and cannot influence measurements of surface dilational properties.11 Most of the results in this work were obtained at a frequency of 0.1 Hz. The changes of the frequency in the range from 0.02 to 0.15 led only to small variations of the surface elasticity within (10%, close to the error limits. (29) Noskov, B. A.; Akentiev, A. V.; Bilibin, A. Yu.; Zorin, I. M.; Miller, R. Adv. Colloid Interface Sci. 2003, 104, 245. (30) Noskov, B. A.; Akentiev, A. V.; Bilibin, A. Yu.; Grigoriev, D. O.; Loglio, G; Zorin, I. M.; Miller, R. Langmuir 2004, 20, 9669. (31) Noskov, B. A.; Grigoriev, D. O.; Lin, S.-Y.; Loglio, G.; Miller, R. Langmuir 2007, 23, 9641.

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Figure 1. Kinetic dependencies of surface pressure of BSA solutions (0.00003 mM) of ionic strength 0.02 M at pH 2.7 (yellow squares), 4.1 (red squares), 5.1 (black squares), 8 (blue squares), and 10.7 (green squares).

Figure 2. Kinetic dependencies of the real part of the dynamic surface elasticity of BSA solutions (0.00003 mM) of ionic strength 0.02 M at pH 2.7 (yellow squares), 4.1 (red squares), 5.1 (black squares), 8 (blue squares), and 10.7 (green squares) and at a frequency of 0.1 Hz.

Results The surface properties of BSA solutions without a denaturant at different pH were measured in this work at a protein concentration of 3  10-8 M and ionic strength of 0.02 M. The constancy of the ionic strength was provided by the addition of an appropriate amount of sodium chloride. The dynamic surface properties close to the isoelectric point changed at these concentrations from zero to an almost steady-state value within the time of measurement (∼5 h). Figure 1 shows the kinetic dependencies of the surface pressure at five pH values corresponding to BSA forms N, F, E, B, and A in solution. The solutions of conformers F and B exhibit slightly slower surface pressure changes in comparison with the N form as observed earlier at higher concentrations.24 In the case of E and F conformers, the surface pressure increased much slower and reached only 3 and 5 mN/m in five hours. The rate of increase of the dynamic surface elasticity changed in a similar way at the increase of pH (Figure 2). The high surface elasticity values for solutions of N, F, and B conformers close to equilibrium (75-82 mN/m) are characteristic Langmuir 2010, 26(22), 17225–17231

Figure 3. Kinetic dependencies of the real part of the dynamic surface elasticity of BSA solutions (0.00003 mM) in 0.003 (red squares), 0.02 (blue squares), 0.04 (green squares), 0.1 (magenta squares), and 2 M NaCl (yellow squares) at pH = 2.7, and at a frequency of 0.1 Hz.

for globular proteins.2,25 The kinetic dependencies for F and B forms are much closer to that for the N form than the corresponding results in ref 24. This distinction is probably caused by the higher protein concentration and especially the lower salt concentration in ref 24. Pereira et al. explained the low surface elasticity of the solutions of F and B forms as being due to the less rigid molecular structure compared to that of the N form.24 Although the strong difference between the surface elasticities of the solutions of rigid globules and random coil macromolecules is doubtless,2,21,25 the differences in the molecular structure of the conformers are probably not the main cause of the observed effect. The faster increase of the surface elasticity at the increase of the solution ionic strength (Figure 2 as compared with Figure 7 of ref 24.) indicates an influence of the adsorption kinetics on the surface elasticity measured within the limited time of experiment. It is well-known that the addition of inorganic salts strongly accelerates polyelectrolyte adsorption at the expense of shielding electrostatic repulsion between the adsorbing macromolecules and the liquid surface charged by the previously adsorbed polyelectrolyte molecules.32-34 Note that A and E forms correspond to stronger deviations from the isoelectric point and, consequently, have higher charges than the F and B forms. This results in a higher adsorption kinetic barrier and slower surface elasticity changes even at an ionic strength of 0.02 M (Figure 2). It is reasonable to assume that if the time of measurement was not limited, one could obtain at the approach to equilibrium close values of the dynamic surface elasticity in the whole pH range from 2 to 11. In order to verify this assumption, the kinetic dependencies of the surface elasticity of the E conformer solutions were determined at different sodium chloride concentrations (Figure 3). The dynamic surface elasticity was almost zero for 5 h at a sodium chloride concentration of 0.003 M and reached about 70 mN/m at the approach to equilibrium for concentrations of 0.1 M or higher. In the latter case, the deviation from the value for solutions of N conformers close to equilibrium was only about 12%. The obtained (32) Noskov, B. A. Curr. Opin. Colloid Interface Sci. 2010, 15, 229. (33) Cohen Stuart, M. A.; Hoogendam, C. W.; de Keizer, A. J. Phys.: Condens. Matter 1997, 9, 7767. (34) Noskov, B. A.; Nuzhnov, S. N.; Loglio, G.; Miller, R. Macromolecules 2004, 37, 2519.

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Figure 4. Kinetic dependencies of the surface pressure of BSA solutions (0.00003 mM) in phosphate buffer on the additions of G. HCl (squares) and NaCl (circles) at the concentrations 0 (black squares), 0.002 (yellow circles), 0.01 (red squares), 0.1 (pink circles), and 0.2 M (blue squares).

Figure 5. Kinetic dependencies of the real part of dynamic surface elasticity of BSA solutions (0.00003 mM) in phosphate buffer on the additions of G.HCl (squares) and NaCl (circles) at the concentrations 0 (black squares), 0.002 (yellow circles), 0.01 (red squares), 0.1 (pink circles), and 0.2 M (blue squares), and a frequency of 0.1 Hz.

results confirm the assumption made above and show that the significant weakening of the physical bounds between domains I, II, and III of the BSA molecule and the decrease of the helicity by more than 20% at the transition from N to E conformers22 is not accompanied by strong changes in the dynamic surface elasticity close to equilibrium. Small additions of G.HCl at concentrations of about 0.2 M and less to BSA solutions (3  10-8 M) in phosphate buffer led to a noticeable acceleration in the change of the surface properties (cf. Figures 4 and 5). All the kinetic dependencies were monotonous in this concentration range, but the equilibrium values were not reached within 5 h. Almost the same effect can be observed for small additions of sodium chloride (Figures 4 and 5). These results indicate a similar influence of these two substances on the protein adsorption kinetics. Both NaCl and G.HCl form free ions in aqueous solution, and the increase of their concentration leads to 17228 DOI: 10.1021/la103360h

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Figure 6. Kinetic dependencies of surface pressure of BSA solutions (0.00003 mM) in phosphate buffer on the additions of G.HCl and NaCl (squares) at the concentrations 0.65 (yellow filled squares and black filled circles), 1 (blue diamonds), 1.5 (brown open squares and green stars) and 4 M (violet circles).

Figure 7. Kinetic dependencies of the real part of dynamic surface elasticity of BSA solutions (0.00003 mM) in phosphate buffer on the additions of G.HCl and NaCl (squares) at the concentrations 0.65 (yellow filled squares and black filled circles), 0.8 (red triangles), 1 (blue diamonds), 1.5 (brown open squares and green stars), 2 (pink crosses), and 4 M (violet circles), and at a frequency of 0.1 Hz.

the shielding of electrostatic interactions between macromolecules. As a result, the electrostatic adsorption barrier decreases, leading to the acceleration of the adsorption rate, and the adsorbed segments take a more compact configuration in the surface layer. The additions of sodium chloride at concentrations higher than 0.2 M resulted in a further increase of the rates of surface property changes without any noticeable changes in the shape of the kinetic curves (Figures 6 and 7). At the same time, the addition of a denaturant of the same concentration leads to a higher increase of the adsorption rate and thereby to a stronger surface pressure rise to about 19 mN/m (Figure 6). Note that at high G.HCl concentrations (>1 M), the accuracy of the surface tension measurements by the Wilhelmy plate method decreases fast within about an hour after creation of the surface due to the formation of a solid G.HCl film on the plate in the region close to the three-phase contact line. Langmuir 2010, 26(22), 17225–17231

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The shape of the kinetic curves of the dynamic surface elasticity changed abruptly at G.HCl concentrations higher than 0.2 M. The surface elasticity increased in the course of the first adsorption step, went through a local maximum at a surface age between 20 and 40 min, and decreased abruptly thereafter (Figure 7). The behavior was similar to that of recently studied BLG/G.HCl solutions. The maximal value of the real part of the surface elasticity of BSA/G.HCl solutions decreased from ∼65 to ∼40 mN/m upon a denaturant concentration increase from 0.65 to 4 M. The dynamic surface elasticity reached a limit value corresponding to small perturbations of the equilibrium solution within 1-3 h after the surface formation. This value also decreased from ∼50 to ∼15 mN/m upon the denaturant concentration increase to 4 M. Note that the real part of the surface elasticity significantly exceeded its imaginary part for all G.HCl concentrations, and the adsorption film was almost purely elastic. The increase of BSA concentration did not noticeably influence the G.HCl concentration range of abrupt changes in the kinetic surface elasticity dependencies. A local maximum of the kinetic dependency of the dynamic surface elasticity indicates a change of the adsorption mechanism and a conformational transition in the surface layer.11,21,27-30,32,34 The maximum of the surface elasticity of solutions of nonionic polymers29,30,32 and nonglobular proteins11,27,28 appears at the transition from a thin almost two-dimensional adsorption layer to a three-dimensional structure of the adsorption layer with long loops and tails protruding into the bulk phase (the formation of a distal region of the surface layer). In this case, the relaxation of surface stresses can proceed at the expense of segment exchange between the distal and proximal regions of the surface layer, and the surface elasticity drops abruptly as a result.29,32 Therefore one can conclude that the local maximum of the kinetic dependency of the dynamic surface elasticity of BSA solutions (Figure 7) shows a destruction of the protein tertiary structure and at least partial destruction of the secondary structure, resulting in an increased flexibility of the macromolecules and thereby the formation of loops and tails in the surface layer. The loss of BSA globular structure at denaturant concentrations above 0.2 M also leads to a more compact packing of the hydrophobic amino acid residues and their higher local concentration in the proximal region of the surface layer, which determines the surface tension value. Therefore the surface tension of the solutions containing G.HCl drops faster with the surface age than for solutions containing the same molar NaCl concentrations and reaches lower values in the former case (Figure 6). At denaturant concentrations higher than 1.5 M, the kinetic dependencies of the surface elasticity coincide in the error limit, indicating that the conformation of BSA molecules in the surface layer does not change any more. One can assume that the tertiary structure is entirely destroyed in this case, the secondary structure can remain to an insignificant extent, and the BSA conformation is not too far from a random coil.

Discussion The experimental results on the influence of pH and ionic strength on the dynamic surface dilational elasticity of BSA solutions (Figures 1-3) show that the protein globular structure, although modified, is preserved in the surface layer for different conformers, and even the dynamic surface properties of E conformer solutions close to equilibrium differ significantly from those of random coil proteins. The high dynamic surface elasticity of BSA solutions can be attributed to the formation of a rigid network in the surface layer at the expense of strong interactions (35) Wijmans, C. M.; Dickinson, E. Langmuir 1998, 14, 7278.

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between rigid compact molecules.24,35,36 Numerical simulations indicate that strong intermolecular interactions and molecular rigidity are the main conditions required to explain the mechanical properties of adsorption layers of globular proteins.35 Figures 5 and 7 show that the addition of G.HCl at concentrations below 0.2 M and sodium chloride also do not lead to significant alterations of the surface layer structure. On the other hand, strong changes of the dynamic surface elasticity at higher G.HCl concentrations (>0.65 M) indicate the destruction of the BSA globular structure at the surface (Figure 7). The transition from the monotonous kinetic dependencies of the dynamic surface elasticity to dependencies with a local maximum occurs in a narrow concentration range close to the G. HCl concentration of 0.65 M. At first glance, comparison with the results on the bulk phase denaturation of BSA and homologous HSA leads to the conclusion that the globule unfolding begins at the interface and in the bulk phase at similar concentrations. According to the precise data on small-angle X-ray scattering, the first step of HSA denaturation takes place in the G.HCl concentration range of 0.3-1 M.26 However, the first step consists only in the loosening of the connection between domain III and the whole globule and between the loops of this domain.26 In this case, the secondary structures of HSA and BSA and the hydrodynamic radii of their globules remain almost constant.26,37,38 Obviously this step, which is similar to the transition between N and E conformers, cannot be accompanied by noticeable changes of the macromolecular flexibility, which are required for the formation of loops and tails in the surface layer.29,32,39 More significant changes occur in the bulk phase in the G.HCl concentration range 1.5-2.3 M.26 Here the increase of the G.HCl concentration results in a loosening of the bounds between all domains and subdomains of the protein molecule and a decrease of the fraction of R-helices. This leads to rather abrupt changes of the protein hydrodynamic radius.26,36 The almost complete loss of secondary structure occurs at even higher denaturant concentrations (g4 M).35 It is hardly possible now to estimate the extent of the macromolecular flexibility required for the formation of loops and tails in the surface layer. The results of refs 26 and 35 show that the HSA molecular conformations in the bulk solution approach those of random coils, and the molecular flexibility can increase strongly only at G.HCl concentrations higher than 2 M. At the same time, it follows from Figure 7 that BSA molecules can meet these conditions in the surface layer already at a denaturant concentration of about 0.65 M, i.e., the destruction of the globular structure in the surface layer occurs at concentrations less than those in the bulk phase. The same conclusion has been drawn recently from investigations of the dilational surface rheology21 and X-ray reflectivity18 of BLG solutions containing G.HCl. The denaturation of BLG in the surface layer and in the bulk solution, respectively, takes place at G.HCl concentrations differing by more than 1 order of magnitude. The difference is smaller for the system under investigation here probably because of the distinctions in structures of the two proteins. The probability of dimer formation in aqueous solutions is much lower for BSA than for BLG. The unfolding process in the latter case proceeds through two steps at least, and the first of them is disaggregation.18 The adsorption layers of BLG solutions contain mainly monomers having a higher surface activity than dimers. The monomer unfolding proceeds through a (36) Jones, D. B.; Middelberg, A. P. J. Langmuir 2002, 18, 5585. (37) Santra, M. K.; Banerjee, A.; Krishnakumar, S. S.; Rahaman, O.; Panda, D. Eur. J. Biochem. 2004, 271, 1789. (38) Adel, A.; Nadia, M.; Mohamed, O.; Abdelhafidh, G. Mater. Sci. Eng. 2008, 28, 594. (39) Noskov, B. A. Colloid Polym. Sci. 1995, 273, 263.

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single step, and therefore this process in the surface layer requires much lower G.HCl concentration than in the bulk phase. The insignificant aggregation of native BSA in aqueous solutions as compared to BLG23 results in a smaller difference between the G. HCl threshold concentrations required for the protein denaturation in the bulk phase and in the surface layer, correspondingly. At the same time, the observed distinction of these concentrations for BSA solutions indicate some perturbations of the protein tertiary structure at the transition of the protein molecules from the bulk phase into the surface layer, which facilitates the subsequent denaturation process. The kinetic dependencies of the dynamic surface elasticity of solutions of both BLG and BSA have the only local maximum at high G.HCl concentrations up to 4 M when there is no tertiary structure and the secondary one is also mainly destroyed. On the other hand, the solutions of other proteins without a tertiary structure and with a weak secondary structure (for example β-casein) are characterized by two local maxima in the dynamic surface elasticity.27 This distinction is probably connected with a more homogeneous distribution of hydrophilic and hydrophobic amino acid residues in the macromolecules BSA and BLG. The β-casein molecule can be separated into two parts with mainly hydrophobic and hydrophilic properties and represents a kind of

Figure 8. Real part of dynamic surface elasticity of BSA solutions (0.00003 mM) in phosphate buffer with the additions of G.HCl at the concentrations 0.65 (black circles), 0.8 (red triangles), 1 (blue diamonds), 1.5 (green stars), and 4 M (violet circles) as a function of the surface pressure.

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block copolymer.27 The two parts of the molecule form loops and tails at different surface pressures leading to two local maxima of the dynamic surface elasticity. The dynamic surface elasticity at all G.HCl concentrations reaches maximum values at approximately the same surface pressure (∼ 12 mN/m) when some parts of the protein molecules begin to be squeezed out from the surface as loops and tails (Figure 8). At the same time, the maximum surface elasticities decrease with increasing denaturant concentration indicating different degrees of unfolding. The unfolding of globular proteins is a fast process as compared to the adsorption and takes usually a few seconds.40 Therefore one can consider that in the G.HCl concentration range between 0.65 and 2 M, the further unfolding of partially unfolded and native protein molecules happens almost immediately after adsorption (Figure 9). The degree of unfolding in the bulk phase and in the surface layer is probably the same at G.HCl concentrations higher than 2 M. The unfolding process in the surface layer is accompanied by the spreading of macromolecules taking a larger area of the surface. When the surface pressure exceeds 12 mN/m after adsorption, the spreading results in the formation of loops and tails (Figure 9), and the surface elasticity starts to decrease. It is noteworthy that the maximum dynamic surface elasticity in the range of the G.HCl threshold concentrations corresponding to the beginning of the denaturation in the surface layer is almost the same for BSA and BLG solutions and amounts to ∼65 mN/m. This value decreases to about 40 mN/m at higher denaturant concentrations. One can assume that these elasticity values are characteristic for most of the globular proteins. The latter one corresponds to the beginning of the formation of tails and loops in the surface layer at the adsorption of nonstructured chains of amino acid residues connected by peptide covalent bounds. The former value is higher because it corresponds to the chains with the maximum contribution of the secondary structure compatible with the formation of loops and tails. The dynamic surface elasticity begins to decrease after the maximal value. The rate of the decrease is higher, the corresponding peak is sharper, and the limit value at high G.HCl concentrations is lower (∼ 15 mN/m) in the case of BSA solutions as compared to BLG solutions where the limit value is about 30 mN/m. These distinctions are probably connected with the differences in the protein molecular weights. Although the disulfide bridges provide approximately the same restrictions on the formation of loops and tails for both proteins, the formation of the distal region of the surface layer becomes more probable for the larger BSA molecules. This leads to an easier segment exchange between the

Figure 9. Scheme of the changes of BSA conformations during the process of adsorption. 17230 DOI: 10.1021/la103360h

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Noskov et al.

Article

two regions of the surface layer at the relaxation of mechanical surface stresses and consequently to the lower dynamic surface elasticity. However, the charged groups and disulfide bounds restrict the formation of tails and loops even for BSA solutions leading to the higher real part of the dynamic surface elasticity than in the case of nonionic polymers where the corresponding characteristic values are on the order of 2-5 mN/m.29,30,32

Conclusions Measurements of the kinetic dependencies of dynamic surface properties of BSA solutions show that the main distinctions for different BSA conformers are caused by the different charges of the protein globules leading to strong differences in the adsorption rate. The surface elasticities at the approach to equilibrium are rather close for solutions of different BSA conformers. The addition of G.HCl at low concentrations (e0.2 M) results in the increase of the rate to approach the equilibrium as observed also for additions of NaCl due to the decrease of the electrostatic (40) Viseu, M. I.; Melo, E. P.; Carvalho, T. I.; Correia, R. F.; Costa, S. M. B. Biophys. J. 2007, 93, 3601.

Langmuir 2010, 26(22), 17225–17231

adsorption barrier. On the other hand, the further increase of G. HCl concentrations leads to strong changes of the shape of the dynamic surface elasticity curves. This quantity as a function of surface age displays a local maximum indicating the formation of loops and tails in the surface layer according to the theory of the dilational surface viscoelasticity.29,39 The maximum surface elasticity values are close to the corresponding data for other globular proteins such as BLG.21 The appearance of the maximum indicates the destruction of the protein tertiary and secondary structures in the surface layer. The BSA unfolding in the surface layer occurs at G.HCl concentrations lower than in the bulk phase as observed also for BLG solutions. This effect is probably a consequence of the interfacial effect on the protein conformation. The obtained results show that the methods of dilational surface rheology is very suitable for studies of the details of protein unfolding in the surface layer. Acknowledgment. This work was financially supported by the Russian Foundation of Basic Research (RFFI No. 08-03-00207_a) and the National Science Counsel of Taiwan (joint project RFFI No. 09-03-92002-HHC_a).

DOI: 10.1021/la103360h

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