Modification of β-Lactoglobulin by Oligofructose: Impact on Protein

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Modification of β-Lactoglobulin by Oligofructose: Impact on Protein Adsorption at the Air-Water Interface Daria Trofimova† and Harmen H. J. de Jongh*,†,‡ Wageningen Centre for Food Sciences, Diedenweg 20, 6700 AN Wageningen, The Netherlands, and TNO Nutrition and Food Research, Zeist, The Netherlands Received March 9, 2004. In Final Form: April 20, 2004 Maillard products of β-lactoglobulin (βLg) and fructose oligosaccharide (FOS) were obtained in different degrees of modification depending on incubation time and pH. By use of a variety of biochemical and spectroscopic tools, it was demonstrated that the modification at limited degrees does not significantly affect the secondary, tertiary, and quaternary structure of βLg. The consequence of the modification on the thermodynamics of the protein was studied using differential scanning calorimetry, circular dichroism, and by monitoring the fluorescence intensity of protein samples with different concentrations of guanidineHCl. The modification leads to lowering of the denaturation temperature by 5 °C and a reduction of the free energy of stabilization of about 30%. Ellipsometry and drop tensiometry demonstrated that upon adsorption to air-water interfaces in equilibrium modified βLg exerts a lower surface pressure than native βLg (16 versus 22 mN/m). Moreover, the surface elastic modulus increased with increasing surface pressure but reached significantly smaller values in the case of FOS-βLg. Compared to native βLg, modification of the protein with oligofructose moieties results in higher surface loads and thicker surface layers. The consequences of these altered surface rheological properties are discussed in view of the functional behavior in technological applications.

Introduction β-Lactoglobulin (βLg) is frequently used as functional and nutritional ingredient in food products. Focusing on the improvement of solubility, heat stability, foaming, and emulsifying capacity, numerous attempts have been undertaken to alter the functional properties of βLg through physical, chemical, or enzymatic treatment of the protein.1 βLg has been conjugated with methanol, ethanol, propanol, 1-butanol, or n-amyl alcohol,2 gluconic or melibionic acids,3 or phosphoric acid.4 Most of these methods utilize toxic chemical products and are not permitted for many industrial applications. A number of attempts were made to improve the functional properties of βLg by conjugation to the protein of sugars such as D-arabinose, D-galactose, D-rhamnose,5 D-glucose, D-fructose,6 or D-lactose7 or by coupling of polysaccharides such as carboxymethyl dextran.8 These modifications resulted generally in an increased foaming capacity, improved emulsifying properties, and a shift of the minimum in solubility toward more acidic pH. * Corresponding author: Wageningen University and Research Centre, Laboratory of Food Chemistry, PO Box 8129, Bomenweg 2, 6700 EV Wageningen, The Netherlands. Phone: +31 317 483208. Fax: +31 317 484893. E-mail: [email protected]. † Wageningen Centre for Food Sciences. ‡ TNO Nutrition and Food Research. (1) Haertle, T.; Chobert, J. M. J. Food Biochem. 1999, 23, 367-407. (2) Mattarella, N. L.; Richardson, T. J. Agric. Food Chem. 1983, 31, 972-978. (3) Kitabatake, N.; Cuq, J. L.; Cheftel, C. J. Agric. Food Chem. 1985, 33, 125-130. (4) Sitohy, M.; Chobert, J. M.; Haertle, T. J. Agric. Food Chem. 1995, 43, 59-62. (5) Chevalier, F.; Chobert, J.-M.; Popineau Y.; Nikolas, M. G.; Haertle, T. Int. Dairy J. 2001, 11, 145-152. (6) Broersen, K.; Voragen, A. G. J.; Hamer, R. J.; de Jongh, H. H. J. Biotechnol. Bioeng. 2004, 86, 78-87. (7) Gauthier, F.; Bouhallab, S.; Renault, A. Colloid Surf., B 2001, 21, 37-45. (8) Nagasawa, K.; Takahashi, K.; Hattori, K. Food Hydrocolloids 1996, 10, 63-67.

The first step in foam formation is the adsorption of proteins to an existing interface, resulting in a lowering of the surface tension (or increase of surface pressure). Although the kinetics of adsorption to air-water interfaces might be quite different between various proteins,9 the surface tension of these interfaces is typically lowered by about 20-25 mN/m upon adsorption of proteins. This lowering of the surface pressure is a result of proteinprotein interactions in the interface that occur generally above surface loads of 1 mg/m2.7 For a variety of technological applications it would be desirable to have biomolecules that do stabilize interfaces but with lower surface pressures. Such molecules could then for example be used to efficiently stabilize a freshly formed air-water interface but become replaced easily by other surfaceactive components that for example do not have this capacity but better network-forming properties. In this work it is aimed to produce proteins that do adsorb onto air-water interfaces but that have a hampered potential for intermolecular interactions at the interface. To achieve this, a limited number of bulky sugar moieties (oligofructoses) covalently linked to primary amine groups of solvent exposed lysine residues of βLg via the Maillard reaction. The conditions were chosen such that the globular fold of the protein remained unaffected to preserve other functional properties of the protein. The products are characterized both biochemically and thermodynamically and tested for their surface-active properties using ellipsometry and an automated drop tensiometer. Materials and Methods N,N-Dimethyl-2-mercaptoethylammonium chloride (DMA) and disodium tetraborate decahydrate (Borax) were purchased from Merck. Sodium dodecyl sulfate (SDS) was obtained from Serva, and o-phthaldialdehyde (OPA) was purchased from Sigma. Raftilose (oligofructose or fructose oligosaccharide (FOS); the (9) Martin, A. H.; Grolle, K.; Bos, M. A.; Cohen Stuart, M. A.; van Vliet, T. J. Colloid Interface Sci. 2002, 254, 175-183.

10.1021/la049390j CCC: $27.50 © 2004 American Chemical Society Published on Web 05/25/2004

Modification of β-Lactoglobulin latter abbreviation will be used throughout this work) was produced by the partial enzymatic hydrolysis of chicory inulin and was provided by Orafti (Wijchen, The Netherlands). The molecular mass of FOS varied between 180 and 1260 Da (monoto heptamers) with the following distribution as determined by mass spectrometry: 5% 180 Da, 5% 360 Da, 27% 540 Da, 27% 720 Da, 18% 900 Da, 13% 1080 Da, and 5% 1260 Da. So, the ensemble-averaged degree of oligomerization is 4.1 units. βLg was isolated and purified (>98% purity) from fresh bovine milk (type A-B ratio 60:40) using the protocol as described by de Jongh and co-workers,10 where solely mild isolation conditions were applied. All other chemicals used were of analytical grade. 1. Modification of β-Lactoglobulin. The details of the modification procedure have been described before.6 Freeze-dried βLg was solubilized in demineralized water and mixed with a FOS solution in demineralized water at the weight ratio 1:1 to give a final molar ratio of FOS relative to the number of primary amino groups on the protein of about 2. The pH of the mixture was then adjusted to 7.0 or 8.0 using NaOH. The solution was subsequently frozen at -20 °C and lyophilized at 50 mbar at -50 °C for 48 h. The dry powder mixture was then incubated at 60 °C at a relative humidity of 65% (exposed to a saturated NaNO2 solution). The incubation was carried out for 12-78 h. Next, demineralized water was added until all material was dissolved and the samples were subsequently extensively dialyzed (membrane cutoff 12-14 kDa) against demineralized water at 4 °C. Finally, the material was freeze-dried and stored at -20 °C until further use. Prior to each experiment the material was checked for the degree of modification to ensure the stability of the product. We choose to prepare Maillard products in a dry form over the aqueous dissolved method since it was reported that dry-way modification has a less damaging effect on the structural integrity compared to heat treatment in solution.6,11 Moreover, in our hands the reproducibility of such modification procedure appeared much higher in the dry form. Separation of Conjugates by Anion-Exchange Chromatography. To fractionate the proteins conjugated with FOS, the material was applied in 10 mM sodium phosphate buffer (pH 6.5) to a Source-Q column equilibrated with the same buffer. Elution was carried out using a linear gradient from 0 to 1 M sodium chloride in phosphate buffer. The gradient’s length was 20 times the column volume. The elution of the proteins from the column was monitored by detection of the absorbance at 280 nm and each eluted peak was collected, extensively dialyzed against demineralized water at 4 °C during 48 h, lyophilized, and stored at -20 °C. 2. Analytical Methods for Product Characterization. 2.1. Protein Concentration. The protein concentrations were determined spectrophotometrically by measuring the absorbance at 280 nm, using an extinction coefficient of 0.959 mL‚mg-1‚cm-1 for both native and modified βLg. In all experiments the number of native and modified protein molecules was kept comparable by calibration of the samples on their absorption at 280 nm. Since the different modified proteins varied quite significantly in hygroscopicity, referencing to the absorption at 280 nm appeared much more reliable than by weighing. 2.2. Degree of Modification. OPA Analysis. The degree of modification (DM) is defined as the number of moles FOS attached per mole monomer of protein. The DM was determined indirectly by a chromogenic assay described in principle by Church and co-workers12 based on the specific reaction between o-phthaldialdehyde (OPA) and free primary amino groups in proteins. In the presence of DMA, these amino groups react to alkylisoindole derivatives that show absorbency at 340 nm. In short, the OPA reagent was prepared by dissolving 40 mg of OPA in 1 mL of methanol, followed by the addition of 25 mL of 0.1 M Borax buffer, 200 mg of DMA, and 5 mL of 10% SDS. Finally, the volume was adjusted to 50 mL with demineralized water. A quartz cuvette was filled with 3 mL of this reagent, and the absorbance at 340 (10) de Jongh, H. H. J.; Groneveld, T.; de Groot, J. J. Dairy Sci. 2001, 84, 562-571. (11) Morgan, F.; Molle, D.; Henry, G.; Venien, A.; Leonil, J.; Peltre, G.; Levieux, D.; Maubois, J.; Bouhallab, S. Int. J. Food Sci. Technol. 1999, 34, 429-435. (12) Church, F. C.; Swaisgood, H. E.; Porter, D. H.; Catignani, G. L. J. Dairy Sci. 1983, 66, 1219-1227.

Langmuir, Vol. 20, No. 13, 2004 5545 nm was determined. Subsequently, 15 µL of a βLg solution was added, and after an incubation time of 30 min at room temperature, the absorbance at 340 nm was determined again. A calibration curve of L-leucine was obtained by preparing samples with a final concentration range from 6.6 to 95.0 µM L-leucine. All measurements were performed in triplicate. Phenol-Sulfuric Acid Reaction Assay. Total fructose content of the conjugates was determined using the phenol-sulfuric acid reaction as described in detail by Dubois and co-workers.13 Fructose was used as standard. The reproducibility of the determination was within ∼5%. 2.3. SDS-Page. The molecular weight of native and modified βLg was estimated using the Phast System (Pharmacia). Ready to use homogeneous 12.5% PhastGels were used, stained with Coomassie brilliant blue G-250, and destained in 30% methanol and 10% acetic acid solution. A low molecular weight calibration sample with proteins ranging from 14.4 to 94 kDa (Pharmacia) was applied to each gel. 2.4. Isoelectric Focusing. The apparent isoelectric points (IEPs) of native and modified βLg were determined using the Phast System (Pharmacia). Ready to use IEF 4-6.5 PhastGels were used, stained with Coomassie brilliant blue G-250, and destained in 20% trichloroacetic acid, 30% methanol, and 10% acetic acid solution. A calibration sample with proteins ranging in IEP from 2.5 to 6.5 (Pharmacia) was applied to each gel. 2.5. Gel Permeation Chromatography. Quaternary structure of native and modified βLg in a 10 mM phosphate buffer (pH 7.0) containing 50 mM NaCl at 20 °C was determined by using a 24 mL Superdex 75 column HR 10/30 (Pharmacia Biotech). Two hundred microliters of a 5 mg/mL sample was applied to the column, and the flow rate was set to 0.4 mL/min. Detection of eluting proteins was performed at 280 nm. 2.6. Intrinsic Viscosity. A concentration range of 0-10 mg/ mL protein was prepared by dilution of a stock solution in 50 mM phosphate buffer (pH 7.0). The intrinsic viscosity was determined by measuring kinematic viscosity for a concentration range of the βLg by means of Ubbelohde tube viscosimeter at 25 °C ((0.1 °C). The Ubbelohde viscosimeter was calibrated with demineralized water and rinsed twice with the sample prior to the actual measurement. All experiments were performed at least in duplicate. The intrinsic viscosity [η] was estimated using the Huggins equation for extrapolation to infinite low protein concentration, which reads as follows: ηsp/c ) [η] + κH[η]2c, where ηsp represents the specific viscosity ()(η - η0)/η0, with η being the recorded viscosity of a protein sample and η0 the viscosity of the protein-free sample), κH is the Huggins constant, and c is the protein concentration. 2.7. Conformational Analysis. Tryptophan Fluorescence. Fluorescence measurements were carried out on 0.03 mg/mL of native and modified βLg in 20 mM phosphate buffer (pH 7.0) or in solutions containing 0-6 M of guanidinium using a luminescence spectrometer, LS-50B (Perkin-Elmer). Spectra of the samples were recorded from 300 to 450 nm upon sample excitation at both 295 and 274 nm with a resolution of 1 nm. Typically, four spectra were averaged. Slit widths were set at 5 nm, and a scan speed of 100 nm/min was used. Circular Dichroism Spectroscopy. Far-UV circular dichroism (CD) spectra of 0.1 mg/mL of native and modified βLg in 20 mM phosphate buffer (pH 7.0) were recorded at 20 °C in the range from 190 to 260 nm with a spectral resolution of 0.2 nm on a Jasco J-715 spectropolarimeter (Jasco Corporation Japan). Spectra were recorded as averages of eight spectra. The scanning speed was 100 nm/min, and the response time was 0.125 s with a bandwidth of 1 nm. Quartz cells with an optical path of 0.1 cm were used. The spectra were corrected for the corresponding protein-free sample. Noise reduction was applied to the recorded spectra based on inverse Fourier transformation methodology using supplier’s software. The spectra were far-UV CD spectra analyzed for the secondary structure content of the proteins using a nonlinear least-squares fitting procedure using reference spectra as described previously.14 (13) Dubois, M.; Gilles, K. A.; Hamilton, J. K.; Rebers, P. A.; Smith, F. Anal. Chem. 1956, 28, 350-354. (14) de Jongh, H. H. J.; Goormaghtigh, E.; Killian, J. A. Biochemistry 1994, 33, 14521-14528.

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2.8. Thermostability. Circular Dichroism Spectroscopy. Near-UV CD spectra of native and modified βLg (1 mg/mL) in 20 mM phosphate buffer at pH 7.0 were recorded from 250 to 350 nm at temperatures ranging from 25 to 95 °C with 5 °C intervals. The spectra were accumulated as described for the far-UV spectra, with the exceptions that the response time was set to 0.25 s and a cell with a 1 cm path length was used. Temperature scans were performed on comparable samples from 25 to 95 °C, while monitoring the ellipticity at 293 nm with a resolution of 0.2 °C. The heating rate was 30 deg/h. Differential Scanning Calorimetry. Differential scanning calorimetry (DSC) was performed on a MicroCalorimeter VP-DSC, MicroCal. Samples with protein concentrations between 1 and 5 mg/mL of native and modified βLg in 20 mM phosphate buffer at pH 7.0 were heated from 20 to 95 °C with a heating rate of 30 °C/h. The denaturation temperature (TD) and the onset temperature of denaturation (Ton) were determined using standard software provided by the supplier. 2.9. Interfacial Properties at the Air-Water Interface. Automated Drop Tensiometer. Automated drop tensiometer (ADT, ICT (France)) was used to measure the interfacial tension between liquid and gas phase. The interfacial tension was determined by means of drop shape analysis of a bubble of air formed within a cuvette containing 0.035 mg/mL of native or modified βLg solution in 20 mM phosphate buffer at pH 7.0. The bubble was illuminated by a light source, and its profile was imaged and digitized by a CCD camera and a computer. The profile was used to calculate the interfacial tension using Laplace’s equation. The modulus was obtained by dynamic oscillation of the bubble area. During the time of the experiment (10.000 s) elasticity measurements were alternated with periods where the surface area was kept constant. Starting with a mean area of 17 mm2, five compression-expansion cycles with a period of 10 s and amplitude of 0.75 mm2 were followed by five cycles where the area was kept constant at 17 mm2. Temperature was kept constant at 22 ((1 °C). All samples were measured in duplicate on two separate occasions. Ellipsometry. A multiskop ellipsometer (Optrell, Germany) was used to determine the adsorbed amount of proteins and the layer thickness of the surface layer between water and air. The angle of incidence used was 50°, and the laser wavelength was 632.8 nm. Before measurements were started, the surface was cleaned by vacuum suction of the air/water interface. As reference a solution of buffer only was used. For the ellipsometry measurements 0.01 mg/mL protein solutions were used in 20 mM phosphate buffer (pH 7.0). The temperature of the chamber was controlled and set at 22 ((0.5 °C). From the change in ellipsometric angles ∆ and Ψ, the layer thickness and the refractive index were calculated using the software supplied by the manufacturer. The refractive index increment, dn/dc, of the various protein solutions was determined using a refractometer using 100 mg/mL protein solutions in 20 mM phosphate buffer (pH 7.0).

Results Protein Modification and Product Characterization. Coupling of oligofructose (FOS) moieties to βLg through the Maillard reaction between the -amino groups in the protein and the reducing end of the oligosaccharide is achieved by incubation of these mixed materials at 60 °C and 65% relative humidity. The degree of modification, as determined from the number of nonreacted -amino groups in the protein using the OPA analysis as a function of incubation time, is shown in Figure 1 for incubation at pH 7.0 and 8.0. Both the higher pH and the longer incubation time promote the degree of covalent coupling. During the first stages of the modification reaction, the number of modified groups increases steadily. However, upon prolonged incubation the availability of reactive groups decreases, and thereby the reaction rate. The same relation has been reported previously6 for fructosylation of βLg. Since the pKa of the lysine -amino group is about

Trofimova and de Jongh

Figure 1. Effect of incubation time at pH 7.0 (open circles) and 8.0 (closed squares) on the degree of modification of βLg with FOS. The degree of modification is expressed as the number of reacted lysine residues per protein, as reversibly assayed by the OPA analysis.

10.515 and lysine residues must be deprotonated to act as satisfactory nucleophiles, the reaction rate of the Maillard reaction increases at higher pH. The modification leads to the formation of conjugates with an average number of oligofructose moieties per βLg monomer from two after 13 h at pH 7.0 up to nine moieties upon incubation for 68 h at pH 7.0 or 8.0. SDS-PAGE analysis (not shown) confirmed qualitatively the increase in molecular weight of the conjugates. From the latter analysis it was evident that, since FOS ranged from monomers up to heptamers and the DM displays a Gaussian distribution,6 the heterogeneity in the reaction mixtures is significant. This complexity hinders a detailed characterization of the material by, for example, mass spectrometry. The coupling of FOS to the protein was confirmed quantitatively by determination of the number of fructose residues of the conjugates, using the phenol-sulfuric acid analysis (see method section). Typically, the number of fructose groups covalently linked to the protein was always about four times higher than the number of reacted groups (from OPA analysis). This number is similar to that of the ensemble average length of the FOS moieties, suggesting that the reactivity of all oligosaccharides present in the FOS mixture is comparable. The formation of the conjugates occurred at relatively low temperature. Generally, no browning of the material was observed, except that at longer incubation times (>72 h) the product appeared slightly yellowish. Also no odor formation could be detected, suggesting that the Maillard products were typically at the initial stage of the complex reaction.6 The conjugates formed appeared relatively stable, since prolonged storage (weeks to months) of the materials in dry form at -20 °C did not result in lower DM. Also no change in the DM could be detected for protein solutions that were kept for 1-2 days at 20 °C in an aqueous solution at neutral pH (results not shown). Impact on Structural Integrity. Secondary Structure. While modifying the primary amino groups of βLg, it was aimed to preserve the globular fold of the protein by applying relatively mild conditions during the modification, for example, well below the denaturation temperature of the protein. A (partial) unfolding of the protein might interfere with deriving conclusive results on the role of particle bulkiness on adsorption properties. The effect of the modification on the secondary structure of βLg was studied using far-UV circular dichroism (CD). The CD (15) Lee, C. M.; Sherr, B.; Koh, Y.-N. J. Agric. Food Chem. 1984, 32, 379-382.

Modification of β-Lactoglobulin

Figure 2. Testing the structural integrity of native and modified βLg at a secondary (panel A) and tertiary (B) folding level: (A) far-UV circular dichroism and (B) fluorescence (excitation wavelength at 274 nm) spectra of native (black lines) and FOS-βLg (gray lines; DM ∼ 9).

spectrum (Figure 2A) of native βLg displays a positive extreme around 195 nm, a zero crossing at 204 nm, and a broad negative band centered around 218 nm. Such a spectrum is typical for a highly secondary structured protein containing a high content of β-strands. Estimates of the secondary structure content can be obtained by spectral analysis providing values of 17% R-helix, 44% β-sheet, 27% random coil, and 12% β-turn for the native βLg, in close agreement with the reported X-ray structure.16 Spectra recorded of material with only a moderate degree of modification were comparable to that of the native protein (not shown). The spectrum of the modified βLg with the highest DM prepared (DM ∼ 9) is also shown in Figure 2A. The shape of the spectrum of this most extreme sample does display a shift of the zero crossing to lower wavelength with approximately 4 nm, suggesting the loss of secondary structure. Spectral analysis demonstrated that in this modified material approximately 8% of the secondary structure has been unfolded compared to the structure of native βLg. The CD spectrum of the protein subjected to the same modification conditions, but in the absence of polysaccharides, did not show a significantly different spectrum, illustrating that the processing conditions as such are not destructive for the secondary structure (result not shown). Tertiary Structure. To obtain information at the tertiary folding level, near-UV CD spectra and tryptophan fluorescence spectra of native and modified βLg were recorded. All CD spectra (not shown) are characterized by a single broad band with a positive extreme between 280 and 290 nm, reflecting the combined effect of tyrosine and tryptophan signals. No significant differences in the spectra of native and modified βLg could be observed in the band shape or the intensity, not even for the highest degree of (16) Brownlow, S.; Morais-Cabral, J. H.; Cooper, R.; Flower, D. R.; Yewdall, S. J.; Polikarpov, I.; North, A. C. T.; Sawyer, L. Structure 1997, 5, 481-495.

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modification, suggesting that the tertiary packing of these proteins is comparable. Also from fluorescence spectra of native and modified βLg upon excitation at 274 nm (tyrosine) no significant differences could be observed in the band shape and position (Figure 2B). This again suggests that the global packing, as far as sensed by the local environment of the tyrosine and tryptophan residues, is not affected by modification. Moreover, when the fluorescence spectra upon excitation at 274 and 295 are compared (not shown), it is can be observed that the FOS-βLg conjugate shows a comparable intensity as observed for the native protein. This illustrates that the energy transfer from tyrosine to tryptophan residues was not affected by the oligofructosylation, indicating that the intramolecular distances between tyrosine and tryptophan and their relative orientation were not significantly affected. Quaternary Structure. The quaternary structure of native and modified βLg was studied using gel permeation chromatography (GPC). From the chromatograms (not shown) it was concluded that native βLg appeared to be mainly dimeric under the conditions used here (low ionic strength, neutral pH) with about 10% in a monomeric form. FOS-βLg conjugates with a relatively moderate degree of modification (DM ∼ 2-6) elute predominantly as dimer. With increase in the degree of modification, the monomer contribution increases. Only extensive modification (DM > 9) leads to formation of significant amounts of monomer and high molecular weight material and decreased amount of the dimeric form (only ∼60% left). Prolonged incubation at elevated temperature also resulted in the formation of (covalent) dimers (up to 20%), as observed by SDS-PAGE analysis (not shown), irrespective of the presence of FOS. These results were attributed to the specific molecular species formed at elevated temperatures, which includes monomers and unfolded covalent homodimers of βLg molecules with a high tendency to self-association via noncovalent interaction.17 Apparent IEP. Since the attachment of an uncharged moiety to the βLg molecule eliminates a positive charge (of the lysine residue), variation in the DM results in a variation of isoelectric point of the material.18 Native βLg showed upon isoelectric focusing analysis two bands at pH 5.2 and 5.3, corresponding to βLg genotypes A and B. With increasing DM broad electrophoretic bands appeared on the gel at lower isoelectric points ranging up to pH 4.5 for a DM of 9 (results not shown). The apparent IEP decreases with increasing DM, but the dependence appears not linear. Selection of a Specific FOS-βLg Conjugate. A mixture of FOS-βLg conjugates with different DM was prepared in a larger quantity and subsequently subjected to anion-exchange chromatography. The elution pattern of a batch of FOS-βLg conjugates is shown in Figure 3. OPA analysis of the eluting peaks illustrated that, in contrast to what one would expect on basis of the theoretical net charge (and demonstrated by isoelectric focusing analysis), the proteins with of highest degree of modification eluted first. This phenomenon might be related to an increase in the hydrodynamic volume of the modified proteins due to the bulky compounds fixed on them, which could obstruct the contact between the protein (17) McSwiney, M.; Singh, H.; Campanella, O.; Creamer, L. K. J. Dairy Res. 1994, 61, 221-232. (18) Kosters, H.; Broersen, K.; de Groot, J.; Simons, J.-W. F. A.; Wierenga, P.; de Jongh, H. H. J. Biotechnol. Bioeng. 2003, 84, 61-70.

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Trofimova and de Jongh

Figure 3. Anion exchange chromatography elution profile of a typical sample of a FOS-βLg conjugate. The DM values, as determined by OPA analysis, of the elution peaks are indicated in the figure. The peaks with a DM of 6 are pooled and further used in this study. The dashed line represents the ionic strength gradient applied.

and the chromatography support.19 For every degree of modification two eluted peaks are apparent. Most probably these reflect the A and B genotype of βLg in the sample. The proteins that contained on average six FOS moieties were pooled and used for all further experiments described in this work. In this material 21 ((1) mol of fructose groups was present per monomer of βLg, indicating that the average length of the oligofructose moieties is 3-4 fructose units. This material does not display any detectable changes at a secondary or tertiary folding level and is still predominantly dimeric (>95%) under the conditions used here (results not shown). Molecular Properties Relevant for Interface Adsorption. Brownian Diffusion. For adsorption to interfaces the proteins first have to encounter the interface to which either they can stick or to “bounce” back into the solution. The existence of such a kinetic barrier was demonstrated previously.10 From gel permeation chromatography (not shown) it was illustrated that the selected FOS-βLG conjugate eluted like the native dimer, suggesting that the radius of gyration was not significantly altered. An increase in molecular mass of about 20% (21 fructose units per monomer protein) will affect theoretically the Brownian diffusion rate by 6%. To compare native with modified protein in terms of having the same number of “hits” with the interface the particle concentration of the modified sample was in all cases 6% higher than for native protein. Structural Stability. Adsorption of βLg onto air-water interface is known to result in (partial) unfolding.21,22 Since such unfolding might be essential for the surface rheological properties, the structural stability of native and FOS-βLG is compared. The denaturation temperatures and the corresponding enthalpic contribution to these transitions as determined by differential scanning calorimetry are presented in Table 1. From the thermograms (19) Gaerthner, H. F.; Puigserver, A. J. Enzyme Microb. Technol. 1992, 14, 150-155. (20) Wierenga P. A.; Meinders M. B. J.; Egmond M. R.; Voragen A. G. J.; de Jongh, H. H. J. Langmuir 2003, 19, 8964-8970. (21) Meinders, M. B. J.; van den Bosch, G. G. M.; de Jongh, H. H. J. Eur. Biophys. J. 2001, 30, 256-267. (22) Meinders, M. B. J.; de Jongh, H. H. J. Biopolymers 2002, 67, 319-322.

Table 1. Molecular Characteristics of Native and FOS-βLg As Determined by Various Methodologiesa

native βLg FOS-βLg

∆Hb (kJ/mol)

∆GH2O c (kJ/mol)

340 290

27.7 25.2

denaturation temp (°C) DSCd CDe 78.0 72.1

74.5 69.0

viscosityf [η] (dL/g) KH 6.0 3.3

60 37

a The change in enthalpy (∆H) upon denaturation is determined by DSC, the change in free energy of unfolding (∆G) is quantified by guanidinium titration studies monitored by the fluorescence at 320 nm, the denaturation temperatures are determined both by DSC and far-UV CD, and the intrinsic viscosity and Huggins constant are determined using the Ubbelohde viscometer. b Obtained from integrating the heat flow; reproducibility is typically (30 kJ/mol. c Obtained by extrapolation from guanidine titration study according to ∆G[Gu] ) ∆GH2O - m[Gu]. d Temperature at maximum heat flow. e Temperature at midpoint of conformational change, obtained by taking the first derivative of the ellipticity signal over the temperature. f The intrinsic viscosity is determined as the intercept from a plot of the ηsp/c versus c, where the slope of the line gives the Huggins constant KH.

(not shown) it can be deduced that native βLg has a denaturation temperature (Td) of 77.4 ((0.5) °C with a symmetric band shape. Integration of this band yields an enthalpy change of 340 ((30) kJ/mol upon thermal denaturation. FOS-βLg displayed a slightly broader band, shifted 6 °C to lower temperatures compared to that of the native protein, with a corresponding enthalpy change of 290 ((30) kJ/mol. As an alternative to determine denaturation temperatures, temperature scans of the native and modified proteins were taken from 25 to 95 °C while monitoring the CD ellipticity at 293 nm. This wavelength was chosen since the strongest near-UV CD intensities (reflecting tertiary packing of the protein) were observed at this frequency. By taking the first derivative of these temperature scans, an accurate evaluation of the denaturation temperatures could be established. The denaturation temperature of native βLg was 75.4 ( 0.5 °C and for modified βLg was 69.0 ( 0.5 °C (Table 1). Again a 6 °C lower denaturation temperature for the modified material was found, but both temperatures are about 2 °C lower compared to the denaturation temperatures as determined by DSC. Possibly the difference in protein concentration used might be responsible for that.

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Figure 4. Determination of the free energy of unfolding by titration of native (closed squares) and FOS-βLg (open circles, DM ) 6) with guanidinium as monitored by the intrinsic tryptophan fluorescence. The inset shows the analysis of the data using a two-state unfolding process, where the free energy of unfolding in the absence of urea can be deduced by extrapolation of the data to 0 M denaturant.

To obtain insight in the free energy change associated to the loss of structural stability of native and modified protein, guanidine titration studies are performed at 20 °C where the tryptophan fluorescence of the proteins in equilibrium between the folded and unfolded conformers is monitored. Upon titration with guanidinium the intrinsic fluorescence emission of βLg shifts from 337 nm to longer wavelengths due to the increased exposure of the two tryptophan residues to a more polar (aqueous) environment. The quantum yield increases when βLg is denatured in guanidine chloride solution. This could possibly be related to a decrease in quenching as a result of the increased distance of tryptophan-61 from the cysteines 66-160 disulfide bond as reported previosuly,23 but also a less efficient energy transfer from tyrosines to tryptophans (upon excitation at 274 nm) or a reduced tryptophan-tryptophan quenching could be responsible for this. The guanidinium titrations of the two βLg forms are shown in Figure 4. The curves display a rather symmetric sigmoidal shape, supporting the assumption that these curves can be analyzed using a two-state transition (∆G ) -RT ln((θN - θ)/(θ - θD)), with θN and θD representing the fluorescence intensity of the native and fully denatured protein, respectively, θ the intensity recorded at a given denaturant concentration, R the gas constant, and T the absolute temperature (i.e., 293 K). Extrapolation of the obtained linear dependence of ∆G as a function of the denaturant concentration allows the extrapolation to ∆G in the absence of denaturant. For the native protein a ∆GH2O of 27.6 kJ/mol was found and for modified βLg 25.1 kJ/mol (summarized in Table 1) was found with midpoints for unfolding at 2.55 and 2.51 M guanidinium, respectively. Intermolecular Interactions. To evaluate the intermolecular interactions the intrinsic viscosity of the two proteins was determined using an Ubbelohde tube viscometer. The intrinsic viscosity ([η]) of native and FOSβLg was obtained by determination of the viscosity for a series of protein concentrations and by subsequent (23) Manderson, G. A.; Hardman, M. J.; Creamer, L. K. J. Agric. Food Chem. 1999, 46, 3617-3627.

extrapolation to infinitely low protein concentration (results not shown). The intrinsic viscosity of the FOSβLg conjugate appeared 1.8 times lower compared to the native protein (3.3 and 6.0 dL/g, respectively). The Huggins constant, as calculated from the slope (dηsp/dc)/[η]2, is about 1.6 times lower for the conjugate compared to that found for the native protein. Both the intrinsic viscosity and the Huggins constant (summarized in Table 1) demonstrate that FOS-βLg behaves like a more deformable particle compared to the native protein. Interfacial Properties. To study the impact of coupling oligofructose moieties to βLg on the adsorption to air-water interfaces, both the adsorption process itself and the rheological properties of the formed layer might be affected. These two properties are described below. The Adsorption Process. The development of a surface pressure in time upon creation of an essentially proteinfree air-water interface has been studied using both the automated drop tensiometer and a Langmuir trough. With the first technique the surface tension is determined by analysis of the shape of an air bubble, with controlled inner pressure, in an aqueous protein solution, while with the latter technique the surface tension is measured using a Wilhelmy-plate. Both methods gave identical results. Figure 5 shows the evolution of the surface pressure (72 mN/m minus the detected surface tension) in time as monitored by the automated drop tensiometer. The absence of the so-called lag phase for both the native and FOS-βLg indicates that on a second time scale the adsorbed amounts reach levels above 1 mg/m2, a typical value for proteins required to exert a significant surface pressure. After about 1500 s the change in surface pressure levels off but, in both cases, still increases slowly in time. It is evident, however, that the surface pressure in “equilibrium” of the FOS-βLg (16 mN/m) is significantly lower than that for the native protein (22 mN/m). For comparison, βLg was also modified with monofructose instead of FOS, with the same degree of modification (results not shown). The equilibrium surface pressure of this monofructosylated material was comparable to that of native βLg, about 22 mN/m (Figure 5).

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Figure 5. The surface pressure versus time for native (black line), FOS-βLg (gray solid line, DM ) 6), and monofructosylated βLg (gray dashed line, mono-F-βLg (DM ) 6) as monitored using the automated drop tensiometer.

Figure 6. (A) The adsorbed amount of native (black symbols) and FOS-βLg (gray symbols, DM ) 6) in time as monitored by ellipsometry. Panel B shows the apparent surface layer thickness in time for the two proteins. For the analysis for both proteins a refractive index of 0.181 mL/g was used, as determined using a refractometer on protein solutions.

Using ellipsometry the adsorbed amount can be monitored in time (Figure 6A). Again it can be seen that the rate of accumulation of material at the interface is comparable between native and modified protein, but for the modified material the surface load (in mg/m2) is always significantly higher than that for the native protein. The maximum amount of adsorbed native βLg reached after 1.5 h was found to be about 1.4 mg/m2 while for FOS-βLg the amount was 1.9 mg/m2. Analysis of the data using manufacturer supplied software illustrated that for the

modified material the surface layer thickness was at any time point about two times larger than that found for the native protein. To allow such analysis the refractive index of both that native and FOS-βLg were determined using a refractometer on a 100 mg/mL protein solution and appeared not significantly different (results not shown). Surface Rheology. Insight in the viscoelastic properties of the surface layers formed can be obtained using the automated drop tensiometer. By regulation of the bubble volume by the automated syringe loop, the surface area of the bubble can be increased or reduced in an oscillating mode. Typical deformations of 4% in area are applied, and the resulting changes in surface pressure can be monitored on-line. Such dynamic measurements yield the modulus of the surface layer (defined as dπ/d(ln(area))) and provide direct insight in the intermolecular interactions in this layer. From Figure 7 it can be seen that at low surface pressures the modulus is comparable for native and FOS-βLg and gradually increases with increasing surface pressure. At higher surface pressures above 13 mN/m, the modulus of the native protein increases sharply, while that of the modified protein increases more steadily. At the maximal surface pressure exerted by these two proteins, the modulus of the native is almost twice that of the FOS-βLg. The presence of noncovalently coupled FOS in the aqueous solution had no effect on the surface pressure or on the rheological properties of the native protein (results not shown). Discussion Most of the globular proteins behave rather comparable in their ability to stabilize air-water interfaces. They typically form surface layers with local concentrations of 100-300 mg/mL and may range from 8 to 70 nm thick in equilibrium.21,22,24 Only the kinetics of adsorption to interfaces may differ significantly, depending for example on the exposed hydrophobicity20 or net charge (unpublished results) of the protein. It is the aim of this study to determine the impact of bulky groups covalently (24) Kudryashova, E. V.; Meinders, M. B. J.; Visser, A. J. W. G.; van Hoek, A.; de Jongh, H. H. J. Eur. Biophys. J. 2003, 32, 553-562.

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Figure 7. The viscoelastic modulus versus time for native (black symbols), FOS-βLg (gray symbols) and monofructosylated βLg (gray dashed line, mono-F-βLg, DM ) 6) as determined using the automated drop tensiometer. The oscillating area was 4% and the frequency of area modulation was 0.2 Hz.

attached on the protein surface (here oligofructose and βLg, respectively) on the behavior at air-water interface. Since, as mentioned previously,25 protein denaturation will affect exposed hydrophobicity and thereby alter the kinetics of protein adsorption and their reorganization at the air-water interface, it was important that during the modification of the protein with oligofructose the native structure of the protein was retained. This work demonstrates that the coupling of oligosugars to proteins through the Maillard type reaction using the dry method is a useful option to obtain such conjugates. Conjugates with a desirable degree of modification were obtained in a reproducible and controlled manner by Maillard reaction between oligofructose and βLg. Wild type βLg contains 16 potential sites (15 lysine residues and 1 N-terminal group) for coupling of oligofructose molecules. FOS-βLg conjugates with two to none attached groups per monomer of protein were obtained (see Figure 1). Only at a degree of modification (DM) of 9 or higher are small changes in the secondary structure observed, but not at a tertiary folding level (Figure 2). The ability to form dimers at neutral pH conditions and ambient temperature was also shown not to be affected at lower DM (