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J. Phys. Chem. B 2009, 113, 13398–13404
Impact of Globule Unfolding on Dilational Viscoelasticity of β-Lactoglobulin Adsorption Layers B. A. Noskov,*,† D. O. Grigoriev,‡ A. V. Latnikova,† S.-Y. Lin,§ G. Loglio,| and R. Miller‡ St. Petersburg State UniVersity, Chemical Faculty, UniVersitetsky pr. 2, 198904 St. Petersburg, Russia, MPI fu¨r Kolloid- und Grenzfla¨chenforschung, Forschungcampus Golm, D14476 Golm, Germany, National Taiwan UniVersity of Science and Technology, Chemical Engineering Department, 43 Keelung Road, Section 4, Taipei, 106 Taiwan, and UniVersita degli Studi di Firenze, Dipartimento di Chimica Organica, Via della Lastruccia 13, 50019 Sesto Fiorentino, Firenze, Italy ReceiVed: June 9, 2009; ReVised Manuscript ReceiVed: August 17, 2009
The dynamic surface dilational elasticity, surface pressure, and adsorbed amount of the mixed solutions of β-lactoglobulin and guanidine hydrochloride were measured as a function of surface age and denaturant concentration. It was shown that the conformational transition from compact globules to disordered protein molecules in the surface layer leads to strong changes in the surface elasticity kinetic dependencies and thereby can be easily detected by measuring the surface dilational rheological properties. The corresponding changes of the kinetic dependencies of the surface pressure and adsorbed amount are not so pronounced but correlate with the results on surface dilational elasticity. Introduction Investigation of protein folding and unfolding is one of the main problems of modern protein physics.1 The protein unfolding at liquid-fluid interfaces is also important from the point of view of colloid and interface science. It is widely believed that the ability of globular proteins to unfold imparts high surface elasticity to the adsorption layer and thereby is responsible for the high stability of foams and emulsions containing proteins, which is of particular importance for the food industry.2-5 Although the structure of adsorbed protein layers at the liquid-gas interface has been intensively studied recently, the extent of protein unfolding is still to be determined even for relatively simple model systems.6-14 The first ideas on possible protein unfolding during adsorption probably arose from the observation that some enzymes lost their activity in the adsorbed state.15 Graham and Phillips assumed partial unfolding of ovalbumin and bovine serum albumin (BSA) in order to explain their data on protein adsorption3 and surface dilational viscoelasticity.4 The formation of highly cohesive surface protein networks with high surface viscoelastic properties was a strong argument in favor of protein denaturation.4,16 Another argument stems out from slow changes of the surface tension when the protein adsorbed amount is almost constant.17,18 The slow protein unfolding rearrangements which are accompanied by additional adsorption of hydrophobic amino acid residues give a probable explanation of the observed effect. Circular dichroism spectroscopy of protein adsorption layers also shows the increase of protein disordered structure and the high degree of unfolding of some proteins (BSA).12 On the other hand, the Fourier transform infrared reflectionabsorption (FTIR) spectroscopy indicates only slight changes in the conformation of most proteins (BSA, β-lactoglobulin, glycinin) upon adsorption at the air-solution interface.9-11 * Corresponding author. † St. Petersburg State University. ‡ MPI fu¨r Kolloid- und Grenzfla¨chenforschung. § National Taiwan University of Science and Technology. | Universita degli Studi di Firenze.
Neutron and X-ray reflectivity also shows that the thickness of protein adsorption layers is in good agreement with the dimensions of protein globules and thereby gives no indication of protein unfolding at least in the neutral range of solution pH.19-24 The conflicting conclusions on protein unfolding are probably a consequence of the limited number of experimental methods available for liquid-fluid interfaces, which can give only indirect information on protein conformation. Obviously, further progress in this field can be possible by applying new experimental techniques. The results on β-casein solutions show that the methods of dilational surface rheology have some perspectives in distinguishing between globular and unfolded protein conformations in the surface layer.25,26 β-casein has no tertiary structure in dilute aqueous solutions. As a result, the dilational surface properties of β-casein solutions differ significantly from the properties of globular protein solutions.2,4,27 Unlike for globular proteins, the dynamic surface elasticity of random coil proteins is relatively low ( 3 M is devoid of ordered secondary structure.28 The two remaining disulfide bonds do not influence noticeably the protein tertiary structure which approaches the random coil conformation. The same conformational transition probably occurs in the surface layer but at lower guanidine hydrochloride concentrations (cd g 0.25 M). Perriman et al. report indeed a noticeable increase of the adsorption layer thickness of β-lactoglobulin at cd ∼ 0.25 M and discuss possible causes of the observed effect.24 The nonmonotonous curves in Figure 3 show that the increase in layer thickness cannot be connected with a simple reorientation of the protein molecules in the surface layer and must be rather caused by significant changes of the protein tertiary structure. The adsorption of β-lactoglobulin dimers cannot also explain the observed peculiarities of the adsorption layer viscoelasticity because of the same reason. According to ref 24, a possible reason of the decrease of guanidine hydrochloride concentrations corresponding to the β-lactoglobulin unfolding in the surface layer as compared with the bulk phase consists of the difference in monomer concentrations. The β-lactoglobulin unfolding is a three-state process and occurs through a monomeric intermediate. The disintegration of dimers probably controls the unfolding rate in the bulk phase. However, the surface activity of monomers is much higher than that of dimers, leading to the predominance of the monomers in the surface layer and thereby to the easier protein unfolding at the liquid surface. The disordered unfolded protein molecules must behave like amphiphilic polymers and do not form long loops and tails in the adsorption layer at low surface pressures. If the surface pressure increases further in the course of adsorption, some parts of the molecules can be displaced into the subphase as loops and tails at a certain critical value of the surface pressure, leading to a local maximum of the dynamic surface elasticity (Figure 3). The noticeable dependence of the dynamic surface elasticity on the guanidine hydrochloride concentration in the region of the maximum at cd e 1.5 M is probably caused by the contribution of the adsorbed native protein monomers to the surface properties at low denaturant concentrations. The concentration of the native molecules decreases with the increase of guanidine hydrochloride concentration. At cd > 1.5 M, the adsorption layer consists mainly of unfolded molecules and the onset of the loop and tail formation corresponds approximately to the same surface elasticity of about 40 mN/m.
Noskov et al.
Figure 5. Refractive index of adsorption layer ns vs bulk guanidine hydrochloride concentration as evaluated from ellipsometric data.
Unlike for β-casein adsorption layers, the kinetic dependency of the surface elasticity of the adsorption layer of unfolded β-lactoglobulin has a single local maximum (Figure 3). This feature can be connected with its more homogeneous distribution of hydrophobic and hydrophilic amino acid residues along the protein chain.53 It has been shown recently that the kinetic dependence of the surface elasticity of copolymer solutions with randomly distributed segments of different hydrophobicity has really a single local maximum.54 The characteristic time of β-lactoglobulin unfolding in the guanidine hydrochloride solution bulk is a few seconds.28 The observed kinetic dependencies do not allow determination of the unfolding time in the surface layer, but the strong changes of surface pressure kinetics at cd ∼ 0.25 M indicate a significant contribution of unfolded molecules to the surface properties already during the initial adsorption step. Therefore, one can assume that the time scale of unfolding is much shorter than the characteristic adsorption time. Unfortunately, the application of ellipsometry to mixed β-lactoglobulin-guanidine hydrochloride solutions does not allow reliable determination of the adsorption layer thickness unlike for pure protein solutions55 or their mixtures with surfactants.56 Progressively increasing the concentration of guanidine hydrochloride leads to an increase of β-lactoglobulin monomer concentration and subsequently to the higher amount of unfolded protein molecules at the air-solution interface. The adsorption layer is composed of two species homogeneously distributed at the surface: relatively hard and thick globules and unfolded disordered molecules. Note that the ellipsometry deals only with the thickness and refractive index of the adsorption layer averaged over the illuminated spot at the interface. At low guanidine hydrochloride bulk concentrations, the fraction of globules in the adsorption layer is predominant and some of the properties are almost completely determined by globules. For example, the ellipsometry shows that the refractive index practically coincides with the value for pure β-lactoglobulin solution (Figure 5). Further increase of guanidine hydrochloride concentration in the bulk results in a quite sharp decrease of the average refractive index of the adsorption layer, indicating an increased surface fraction of disordered β-lactoglobulin. Since the disordered β-lactoglobulin molecules have a flat conformation in the adsorption layer (at low and moderate guanidine hydrochloride bulk concentration), the average ellipsometric adsorption layer thickness keeps decreasing and yields finally a thickness at which the ellipsometric method becomes unreli-
Dilational Viscoelasticity of β-Lactoglobulin
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Figure 7. Scheme of the changes of the surface layer structure in the course of β-lactoglobulin adsorption at cd < 0.25 M (a) and cd g 0.25 M (b). Figure 6. Kinetic dependencies of the adsorbed amount of β-lactoglobulin at different concentrations of guanidine hydrochloride of 0 (black squares), 0.012 (red squares), 0.1 (light-blue squares), 0.25 (pink squares), and 0.5 M (green squares).
able. That is why the refractive index of the adsorption layer in the β-lactoglobulin-gaunidine hydrochloride mixtures falls close to the water value already at a guanidine concentration of 0.1 M with a simultaneous apparent strong increase in the adsorption layer thickness (data not shown). A similar behavior was recently observed for protein-surfactant mixed solution in the range of surfactant concentrations where displacement of protein from the adsorption layer becomes significant and the ellipsometric method is no longer applicable.56 On the other hand, grave increase of bulk guanidine hydrochloride concentration leads to the appearance of the local maximum on the kinetic dependencies of the dynamic surface elasticity (Figure 3), which, as mentioned above, indicates the formation of the distal region in the adsorption layer containing tails and loops protruding deeper into the bulk phase. The formation of the loops and tails region should be therefore accompanied by the thickening of the adsorption layer, making again possible the application of ellipsometry to its characterization. Unfortunately, even in this range of guanidine concentrations, the quantitative application of ellipsometry is almost impossible. The reason for that lies in the increase of the bulk refractive index with increasing guanidine hydrochloride concentration. This results in a poor optical contrast between adsorption layer and bulk phase and, consequently, in high experimental errors in the calculated adsorption layer thickness. One can observe only the increase in the adsorption layer thickness with denaturant concentration, but the absolute values are not reliable enough. On the other hand, the determination of the adsorbed amount by ellipsometry usually leads to lower relative errors. This is typical for ellipsometric results because of the mutual compensation of errors for the refractive index and thickness in the calculation of the adsorbed amount.55 If the denaturant concentration is not high enough (cd < 0.25 M), one can observe a strong increase in the adsorbed amount at low surface ages (t < 10 min) and slight changes at longer times (Figure 6), in agreement with the adsorption mechanism discussed above. The increase of the guanidine hydrochloride concentration in the ranges from 0.01 to 0.1 M leads only to a slight increase in the adsorbed amount close to the error limits (
