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Langmuir 2001, 17, 3208-3215
Heat-Set Bovine Serum Albumin-Sodium Dodecyl Sulfate Gels Studied by Fluorescence Probe Methods, NMR, and Light Scattering Ank Valstar,*,† Marilena Vasilescu,‡ Ce´cile Vigouroux,§ Peter Stilbs,§ and Mats Almgren† Department of Physical Chemistry, Box 532, University of Uppsala, 751 21 Uppsala, Sweden, Romanian Academy, “I. G. Murgulescu” Institute of Physical Chemistry, Splaiul Independentei 202, 77208 Bucharest, Romania, and Physical Chemistry, Royal Institute of Technology, 100 44 Stockholm, Sweden Received November 23, 2000. In Final Form: February 5, 2001 In this work, concentrated protein-surfactant solutions and their corresponding heat-set gels were studied by fluorescence probe methods, NMR, and light scattering. Bovine serum albumin (BSA) was used as the protein, and sodium dodecyl sulfate (SDS) as the surfactant. Heating concentrated BSA solutions gives turbid gels. Heat-set BSA-SDS gels are transparent. From fluorescence measurements it was concluded that SDS forms micelle-like clusters on BSA, both in solution and in the corresponding heat-set gel. Aggregation numbers were found to be similar in solution and gel. Also, I1/I3 values in solution and gel were similar. 2H NMR relaxation measurements of specifically deuterated SDS at the R-carbon position next to the headgroup were performed, and the longitudinal relaxation rates R1 were found to be the same in solution and gel. High values for the transverse relaxation rate R2 (indicating slow motions of SDS bound to large aggregates) were obtained, and the largest R2 value was found for the gel. Dynamic light scattering on BSA-SDS gels was used to obtain the correlation length ξ, which defines a mean distance between two points of entanglements. The decrease of ξ with increasing [SDS]/[BSA] molar ratio was explained by the size of the BSA-SDS complex and the possibility that micelle-like structures might form cross-links between different BSA molecules. With static light scattering the extent of inhomogeneities in BSA and BSA-SDS gels was found to decrease with increasing SDS concentration. Also, the gel region in the ternary phase diagram BSA-SDS-3.1 mM NaN3 at room temperature and constant pressure (1 atm) was determined.
Introduction Gels can be made from concentrated aqueous solutions of globular proteins by heating above the denaturation temperature.1 These gels are important for the production of, e.g., foods and pharmaceuticals. The appearance of heat-set protein gels varies from turbid to transparent, depending on pH and ionic strength. In the food industry there is a certain interest in the production of transparent protein gels, mostly from an aesthetic point of view. The globular protein bovine serum albumin (BSA) is frequently used in studies concerning heat-induced gel formation. BSA has a molecular mass of 66 411 g mol-1 (calculated from the amino acid composition) and consists of 583 amino acids in a single polypeptide chain.2 The protein contains 17 disulfide bridges and one free SH group. The temperature of denaturation is ≈60 °C (pH 7, no added salt).3 The relative proportions of R-helix, β-structure, and random coil of native BSA are 67, 3, and 30%, respectively.4 The isoelectric point in 0.15 M NaCl * To whom correspondence should be addressed. † University of Uppsala. ‡ Romanian Academy, “I. G. Murgulescu” Institute of Physical Chemistry. § Royal Institute of Technology. (1) Clark, A. H.; Lee-Tuffnell, C. D. In Functional Properties of Food Macromolecules; Mitchell, J. R., Ledward, D. A., Eds.; Elsevier Applied Science Publishers Ltd.: London, 1985; pp 203-272. (2) Peters, T. J. All about Albumin Biochemistry, Genetics, and Medical Applications; Academic Press: San Diego, CA, 1996. (3) Yamasaki, M.; Yano, H.; Aoki, K. Int. J. Biol. Macromol. 1991, 13, 322-328. (4) Takeda, K.; Wada, A.; Yamamoto, K.; Moriyama, Y.; Aoki, K. J. Protein Chem. 1989, 8, 653-659.
is about 4.7; bound chloride ions cause it to be lower than the isoionic point (about 5.2). The total charge at the isoionic point consists of about 100 each positive and negative charges.2 Heat denaturation changes only slightly the size and shape of the protein.5 Heat denatured BSA has substantial secondary structure left: the relative proportions of R-helix, β-structure, and random coil were determined to be 44, 13, and 43%, respectively.4 It has been suggested6 that during heat denaturation some of the internal hydrophobic groups flip to the outside and the denatured proteins then aggregate to reduce the exposure of the hydrophobic groups to the aqueous environment. During aggregation, intermolecular covalent disulfide bridges are formed.1 Depending on pH and ionic strength, aggregation could be, for example, random clumping or filamentous.6 Turbid gels with particulate gel structures7 are formed at pH close to the isoelectric point and at high ionic strength. At pH values far from the isoelectric point and at intermediate ionic strength, transparent gels are formed. The transparent BSA gels formed at pH values 4.0 and 6.5 (no added salt) were shown to consist of a network of linear polymers8 (or filaments9). (5) Matsumoto, T.; Inoue, H. J. Chem. Soc., Faraday Trans. 1991, 87, 3385-3388. (6) Tobitani, A.; Ross-Murphy, S. B. Macromolecules 1997, 30, 48454854. (7) Verheul, M.; Roefs, S. P. F. M.; Mellema, J.; Kruif, K. G. d. Langmuir 1998, 14, 2263-2268. (8) Murata, M.; Tani, F.; Higasa, T.; Kitabatake, N.; Doi, E. Biosci., Biotechnol., Biochem. 1993, 57, 43-46. (9) Clark, A. H.; Judge, F. J.; Richards, J. B.; Stubbs, J. M.; Suggett, A. Int. J. Peptide Protein Res. 1981, 17, 380-392.
10.1021/la0016221 CCC: $20.00 © 2001 American Chemical Society Published on Web 04/26/2001
Protein-Surfactant Heat-Set Gels
The interaction between BSA and the anionic surfactant sodium dodecyl sulfate (SDS) leads to the formation of a complex. The complex formation has been studied thoroughly at low protein and surfactant concentrations. Binding isotherms were determined showing the cooperative binding of SDS.10 Also, the hydrodynamic radii of the complexes were estimated.11,12 SDS forms micelle-like clusters on proteins10,13-15 and by using the fluorescence probe method it was shown that partially unfolded BSA wraps around the clusters.16,17 BSA saturated with SDS still contains secondary structure: the proportions of R-helix, β-structure, and random coil were determined to be 50, 10, and 40%, respectively.18 Our present results show that heating concentrated aqueous BSA solutions gives turbid gels. However, concentrated BSA solutions to which SDS has been added behave differently when heated, since the bound amphiphilic SDS molecules introduce additional negative charges and hydrophobic entities on the protein. In the present work, both the concentrated BSA-SDS solutions (before heating) and the corresponding heat-set BSASDS gels were studied. Structural information of the systems was obtained by using steady-state and dynamic fluorescence, NMR, and light scattering. Also, the gel region in the ternary phase diagram BSA-SDS-3.1 mM NaN3 at room temperature and constant pressure (1 atm) was determined. Experimental Section (A) Materials and Sample Preparation. (1) Fluorescence Probe Method and DLS. BSA (Albumin fraction V, lot no. K91048518, >97%) was supplied by Merck; SDS (especially pure) was obtained from BHD. The samples were prepared by mixing appropriate amounts of neat components, i.e., BSA, SDS, and 3.1 mM NaN3 solution (or water). The 3.1 mM NaN3 solution was used in the light scattering experiments, water was used in the fluorescence experiments. All samples were mixed by stirring. The solutions were kept at 85 °C in an oven for several hours (3-5 h). Since BSA resists thermal denaturation when SDS is added at low molar ratio [SDS]/[BSA],19 the rather high temperature of 85 °C was used. The samples that were used in the light scattering experiments were filtered into the light scattering cells through Sartorius Minisart N filters with pore size 0.2 µm to remove dust. (2) NMR. BSA (98% monomer, Sigma A-1900, lot no. 75H9305) and specifically deuterated SDS at the R-carbon position next to the surfactant headgroup (R-CD2)20 were mixed in deuteriumdepleted water (Isotec Inc.) containing 3.1 mM NaN3 to prevent bacterial growth. The solutions were stirred for 24 h, and the NMR tubes (5 mm) were flame-sealed. The samples contained 14 wt % BSA and/or 5 wt % SDS. Gels were obtained by heating at 85 °C for 3 h then allowing the sample to cool overnight. (3) Phase Diagram. The samples were prepared by mixing appropriate amounts of neat components, i.e., BSA (Albumin (10) Oakes, J. J. Chem. Soc., Faraday Trans. 1 1974, 70, 2200-2209. (11) Takeda, K.; Sasaoka, H.; Sasa, K.; Hirai, H.; Hachiya, K.; Moriyama, Y. J. Colloid Interface Sci. 1992, 154, 385-392. (12) Valstar, A.; Almgren, M.; Brown, W.; Vasilescu, M. Langmuir 2000, 16, 922-927. (13) Shirahama, K.; Tsujii, K.; Takagi, T. J. Biochem. 1974, 75, 309319. (14) Guo, X. H.; Zhao, N. M.; Chen, S. H.; Teixeira, J. Biopolymers 1990, 29, 335-346. (15) Ibel, K.; May, R. P.; Kirschner, K.; Szadkowski, H.; Mascher, E.; Lundahl, P. Eur. J. Biochem. 1990, 190, 311-318. (16) Turro, N. J.; Lei, X.-G.; Ananthapadmanabhan, K. P.; Aronson, M. Langmuir 1995, 11, 2525-2533. (17) Vasilescu, M.; Angelescu, D.; Almgren, M.; Valstar, A. Langmuir 1999, 15, 2635-2643. (18) Takeda, K.; Shigeta, M.; Aoki, K. J. Colloid Interface Sci. 1987, 117, 120-126. (19) Yamasaki, M.; Yano, H.; Aoki, K. Int. J. Biol. Macromol. 1992, 14, 305-312. (20) Ginley, M.; Henriksson, U.; Li, P. J. Phys. Chem. 1990, 94, 4644.
Langmuir, Vol. 17, No. 11, 2001 3209 fraction V), SDS, and 3.1 mM NaN3 solution, or SDS was dissolved in a BSA-NaN3 solution. NaN3 was added to prevent bacterial growth. All samples were mixed by slow stirring (several hours) and left unperturbed to equilibrate at room temperature. (B) Experimental Setup and Methods. (1) Fluorescence Probe Method. The pyrene concentration (10-5 to 5 × 10-5 M) and excitation wavelength (i.e., λex ) 340 nm in steady-state fluorescence measurements and λex ) 325 nm in time-resolved fluorescence measurements) have been so selected to obtain intense and well-resolved emission spectra and to avoid intrinsic protein fluorescence and light scattering, important for these high protein concentrations (10-14 wt %). The absence of pyrene excimer emission was checked in all experiments. All measurements were carried out for both the BSA-SDS solution and the corresponding heat-set gel. (2) Steady-State Fluorescence Measurements. The fluorescence emission spectra of pyrene solubilized in the investigated systems were obtained using a SPEX Fluorolog 16 spectrofluorometer, combined with SPEX DM3000 software. The slits were 1.5 mm (excitation) and 0.3 mm (emission). The frontal face arrangement was preferred for the recording of emission spectra in these scattering samples. The ratio of the first over the third vibronic peak of pyrene fluorescence, I1/I3, was used to monitor the polarity of the probe microenvironment. The aggregation numbers were determined using the static fluorescence quenching method (SSFQ) based on Turro and Yekta’s21 theory, with dodecylpyridinium chloride (DPCl) as the quencher. (3) Dynamic Fluorescence Measurements. The pyrene fluorescence lifetime, τ0, was determined by the time-correlated single photon counting technique using the same instrumentation and conditions as described in an earlier study.17 Its value is higher in micelle-like clusters than in free micelles, partly due to the better protection from water and partly owing to the higher microviscosity of the clusters compared to that of the SDS micelles.22 The aggregation numbers were also determined by timeresolved fluorescence quenching measurements (TRFQ) using the Infelta23,24-Tachiya model.25-27 As in the SSFQ measurements, DPCl was used as the quencher. A detailed description of the TRFQ method and equipment is given elsewhere.28,29 The pyrene fluorescence decay curves with quencher were fitted to the equation
ln[F(t)/F(0)] ) -k0t + n[exp(-kqt) - 1]
(1)
where F(t) describes the time evolution of the fluorescence intensity, F(0) is the fluorescence intensity at time zero, τ0 ()1/ k0) is the natural lifetime of pyrene obtained from separate experiments without quencher, n is the average number of quenchers in a micelle, and kq is the first-order quenching rate constant. Using the n value determined by fitting the experimental data to eq 1, the micelle aggregation number, Nagg, can be calculated
Nagg ) n[Sm]/[Qm]
(2)
where [Sm] and [Qm] are, respectively, the concentration of the surfactant and quencher in the micelles. Because the free concentrations of surfactant and quencher are negligible, [Sm] and [Qm] may be replaced by the total concentrations. (4) NMR Theoretical Background. 2H spin relaxation rates are detailed and quantitative sources of information on the (21) Turro, N. J.; Yekta, A. J. Am. Chem. Soc. 1978, 100, 5951-5952. (22) Vasilescu, M.; Almgren, M.; Angelescu, D. J. Fluoresc. 2000, 10, 339-346. (23) Infelta, P. P.; Gratzel, M.; Thomas, J. K. J. Phys. Chem. 1974, 78, 190-195. (24) Infelta, P. P. Chem. Phys. Lett. 1979, 61, 88-91. (25) Tachiya, M. Chem. Phys. Lett. 1975, 33, 289-292. (26) Tachiya, M. J. Chem. Phys. 1982, 76, 340-348. (27) Tachiya, M. J. Chem. Phys. 1983, 78, 5282-5283. (28) Almgren, M. Adv. Colloid Interface Sci. 1992, 41, 9-32. (29) Almgren, M.; Hansson, P.; Mukhtar, E.; Stam, J. v. Langmuir 1992, 8, 2405-2412.
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Valstar et al.
dynamics of deuterated surfactant molecules.30,31 For a nucleus with spin I ) 1, the longitudinal (R1) and the transverse (R2) relaxation rates (R1,2 ) 1/T1,2) are32
3π2 2 χ (2J(ω0) + 8J(2ω0)) 40
(3)
3π2 2 χ (3J(0) + 5J(ω0) + 2J(2ω0)) 40
(4)
R1 ) R2 )
with the following spectral densities, expressing the dynamics of the molecular reorientation in the case of a single correlation time τ
J(0) ) 2τ J(ω0) )
J(2ω0) )
2τ 1 + ω02τ2
(5)
gE2(t) - 1 ) β|gE1(t)|2
2τ 1 + 4ω02τ2
where ω0 represents the Larmor frequency of the deuterium nuclei for a given field strength, χ, the deuterium quadrupolar coupling constant, assumed to be 167 kHz.33 For an aqueous surfactant system above the critical micelle concentration (cmc), the “two-step” model34 gives a good link between spin relaxation and dynamics of the surfactant molecules. This model is based on the separation of two independent time scales, one representing the local reorientation of the surfactant in the micelle (fast and slightly anisotropic motion) and the other one the rotation of the micelle and the diffusion of the surfactant along the surface of the micelle (slow isotropic motion). In a protein-surfactant system, a multisite model has to be considered, since the surfactant molecules may exist as monomers, micelles, and also in the protein-surfactant complex that may then aggregate as well. Since the surfactant molecules can be found in i different sites, with fast exchange the observed NMR parameter is a population-weighted average according to eq 6
R1,2 )
∑ pR i
i 1,2
(6)
i
R1,2 represents the experimental parameter, pi the fraction of surfactant in site i, and R1,2i the corresponding relaxation rate in site i. Changes in the experimental values of relaxation rates may arise from variations in the populations of the surfactants at the different sites as well as from alterations in the intrinsic relaxation rates. The latter ones can originate from changes in the aggregate size (mainly affecting J(0)) or changes in the properties of the surfactant bound to the aggregates, modifying the local dynamics of the surfactant and the fraction of the quadrupolar interaction relaxed by the slower motions, i.e., the amplitude of the motion the surfactant undergoes. (5) NMR Relaxation Measurements. The experiments35 were made on a Bruker DMX 500 MHz spectrometer, operating at 76.8 MHz for 2H nucleus at 25 °C. The R1 measurements were performed with the inversion recovery method using 32 different interpulse delay times. Signal line widths ∆ν1/2 were also measured in order to obtain R2* ()1/T2*) with (7)
T2* ) (π∆ν1/2)-1
Since a strong increase in the NMR signal line widths in the BSA-SDS systems was at hand, R2* was considered to be equal to R2. (6) Dynamic and Static Light Scattering Measurements. The light scattering measurements were performed using a frequency-stabilized Coherent Innova Ar ion laser emitting vertically polarized light at 488 nm. The signal analyzer was an ALV-5000 digital multiple-τ correlator (Langen GmbH) with 288 exponentially spaced channels. The measurement temperature was 25.0 ( 0.1 °C. (7) Dynamic Light Scattering. In a DLS experiment the time-averaged intensity autocorrelation function, gT2(t), is measured. In the vast majority of cases the time-averaged correlation function gT2(t) is equal to its ensemble average (however not in gels, see below). The Siegert relation relates the normalized ensemble-averaged intensity autocorrelation function, gE2(t), to the normalized ensemble-averaged autocorrelation function of the electric field, gE1(t)
(7)
(30) So¨derman, O.; Walderhaug, H.; Henriksson, U.; Stilbs, P. J. Phys. Chem. 1985, 89, 3693. (31) So¨derman, O.; Stilbs, P. Prog. NMR Spectrosc. 1994, 26, 445. (32) Abragam, A.; The Principles of Nuclear Magnetism; Clarendon Press: Oxford, U.K., 1961. (33) More´n, A. K.; Nyde´n, M.; So¨derman, O.; Khan, A. Langmuir 1999, 15, 5480-5488. (34) Halle, B.; Wennerstro¨m, H. J. Chem. Phys. 1981, 75, 1928. (35) Canet, D. Nuclear Magnetic Resonance, Concepts and Methods; John Wiley & Sons: New York, 1991.
(8)
where β is a factor that accounts for deviations from ideality (β e 1). gE1(t) is characterized by a distribution of relaxation times A(τ)
gE1(t) )
∫
∞
0
A(τ) exp(-t/τ) dτ
(9)
(8) DLS on Gels. This study describes DLS measurements on heat-set protein-surfactant gels. The transparency of these gels indicates of a network of fine-stranded structures6,7 formed by the colloidal particles. The colloidal particles are restricted by cross-links to particular regions of the sample and are only able to execute limited Brownian motion about a fixed average configuration.36 Different parts of a gel (i.e., different scattering volumes) are characterized by different average configurations. Thus the time-averaged gT2(t) obtained from a single DLS measurement (a single scattering volume) on a gel is not equal to its ensemble average and a gel is to be considered as a nonergodic medium. The electric field of the scattered light by a gel can be written as the sum of two components,37 i.e., a fluctuating component and a constant component. The fluctuating component is associated with the restricted Brownian motions of the colloidal particles and the constant field arises from the “frozen-in” density fluctuations or large-scale inhomogeneities.37 Different methods have been used to obtain the ensembleaveraged gE1(t) of a gel. Joosten et al.37 performed a large number of gT2(t) measurements (≈100). All measurements were done at different scattering volumes and accumulated without clearing the memory of the correlator between the measurements. To obtain gE2(t), the total correlation function data were normalized by the total number of photon counts and the number of summations. gE1(t) was obtained using the Siegert relation (eq 8). Another method is the nonergodic approach, developed by Pusey et al.,36,37 where gT2(t) for a single scattering volume is related to gE1(t). In this work the light scattering cell was continuously rotated during the DLS measurement (1 rpm) to obtain the ensembleaveraged gE2(t). Rotating the sample places an upper limit on the observable relaxation times. This is shown in Figure 1. Here two DLS measurements on the same sample are compared: (A) the sample is stationary (no rotation) and (B) the sample is rotated. Correlation function A shows a fast decay followed by a long tail. The fast decay is related to the restricted Brownian motion of the colloidal particles. The reduced initial amplitude of gT2(t) < 1, implies the presence of “frozen-in” fluctuations or inhomogeneities.37 The ensemble-averaged correlation function B is also characterized by the fast decay and an additional second decay is seen. This second decay (τ ≈ 10-3 s), however, is not a feature of the system, but an artifact caused by rotation. This (36) Pusey, P. N.; Megen, W. v. Physica A 1989, 157, 705-741. (37) Joosten, J. G. H.; McCarthy, J. L.; Pusey, P. N. Macromolecules 1991, 24, 6690-6699.
Protein-Surfactant Heat-Set Gels
Langmuir, Vol. 17, No. 11, 2001 3211 (II). However, since poor fits were obtained, the subtraction technique39 was applied. This technique involves the elimination of a relaxation mode (i.e., peak II) from the relaxation-time distribution spectrum, followed by reconversion to gE2(t) (Figure 2c). Thereafter, the corrected correlation spectrum is reanalyzed with REPES (Figure 2d). (9) SLS on Gels. The scattered intensity I(t)q)cst, at a particular scattering angle, was measured while the light scattering cell was continuously rotated (1 rpm). The mean value equals the ensemble-averaged intensity 〈I(q)〉E. The parameter q is the scattering vector: q ) (4πns/λ0) sin(θ/2), where ns is the refractive index of the sample, λ0 is the wavelength of the radiation in a vacuum, and θ is the scattering angle. Toluene was used as a reference, and normalized intensities are calculated from 〈I(q)〉E/ Itol.
Results and Discussion
Figure 1. Intensity autocorrelation functions obtained at scattering angle θ ) 90° on the same gel sample (14 wt % BSA with [SDS]/[BSA]) 10): (A) the sample was stationary; (B) the sample was rotated during the measurement (1 rpm). Correlation function (C) was obtained when a glass rod was rotated during the measurement (1 rpm).
Figure 2. (a) Intensity autocorrelation function at scattering angle θ ) 90° for a gel consisting of 14 wt % BSA and [SDS]/ [BSA] ) 10, the sample was rotated during the measurement. (b) Inverse Laplace transform (ILT) analysis using REPES of the correlation curve shown in (a). (c) Correlation function from (a) after subtraction of the mode related to rotation (mode II). (d) ILT analysis of the correlation function in (c). is verified by a DLS measurement on a glass-rod (a rigid, nonfluctuating medium), resulting in correlation function C (Figure 1). To extract the distribution of relaxation times A(τ) from gE1(t) (eq 9), an inverse Laplace transformation has to be performed. Here, REPES38 is applied, which operates directly on gE2(t). The results of such an analysis are shown in Figure 2b. The distribution function of relaxation times shows two modes: a peak characteristic of the gel system (I) and one related to rotation (38) Jakes, J. Czech. J. Phys. B 1988, 38, 1305-1316.
Heat-Set Gels. Heat-set gels were studied in the concentration ranges of 9-14 wt % BSA and 0.4-18 wt % SDS; i.e., [SDS]/[BSA] ) 10-425. This range of concentrations was used for the following reasons. Solutions containing
