Article pubs.acs.org/Biomac
Electrostatic Stabilization of β‑lactoglobulin Fibrils at Increased pH with Cationic Polymers Jay Gilbert,† Osvaldo Campanella,‡ and Owen G. Jones*,† †
Purdue University, Department of Food Science, 745 Agriculture Mall Drive, West Lafayette, Indiana 47907, United States; Purdue University, Department of Agricultural and Biological Engineering, 225 South University Street, West Lafayette, Indiana 47907, United States
‡
S Supporting Information *
ABSTRACT: In order to improve the stability of βlactoglobulin fibrils formed in acidic conditions to increased pH values (pH 3−7), formation of electrostatic complexes between fibrils and cationic polymers chitosan (CH), amineterminated poly(ethylene glycol) (APEG), low molecular weight poly(ethylenimine) (LPEI), and high molecular weight poly(ethylenimine) (HPEI) was investigated by electrophoretic mobility, turbidimetry, and atomic force microscopy. Except for suspensions with APEG, addition of polycations increased ζ-potential values of the fibrils at pH 5, 6, and 7, verifying their interactions with fibrils. Maximal increase in ζpotential at pH 7, indicating optimal electrostatic interactivity, occurred at concentrations (w/w) of 0.05, 0.01, and 0.01% (corresponding to 6.9, 50, and 4 μmol·kg−1) for CH, LPEI, and HPEI, respectively. Turbidity of fibril solutions at pH 5, indicating isoelectric instability, was decreased significantly with increasing concentration of CH, LPEI, and BPEI, but not with added APEG. Turbidity was increased at pH 7 with added polycation, except for suspensions containing ≥0.02% HPEI. Fibril length and resistance to aggregation, as observed by atomic force microscopy, were increased at pH 5 with increasing concentration of CH and LPEI, yet only HPEI was capable of maintaining the morphology of fibrils at pH 7. Calculated persistence lengths of the fibrils, as compared to pure fibrils at pH 3 (∼4 μm), were only slightly reduced at pH 5 with CH and at pH 7 with HPEI, but increased at pH 5 with LPEI and HPEI. Improvement in the stability of β-lactoglobulin fibrils at higher pH conditions with the addition of polycations will contribute to their potential utilization in packaging, food, and pharmaceutical applications.
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INTRODUCTION A unique characteristic of proteins is their ability to assemble into amyloid fibril structures by noncovalent interactive forces between peptides of neighboring proteins, which is believed to be a common trait of most, if not all, proteins.1,2 These fibrils are comprised of β-sheets structured perpendicular to the fibril axis via hydrogen bonding, π−π stacking, and hydrophobic interactions.3 Much of the research surrounding amyloid fibrils has been targeted toward their association with neurodegenerative diseases such as Alzheimer’s, Huntington’s, and Parkinson’s diseases.2 In recent years these fibril structures assembled from natural proteins have been of interest to the food, medical, and pharmaceutical science fields due to their strength and rigidity.4,5 β-lactoglobulin, the major protein component of whey from milk processing, is a common food additive due to its unique emulsification and foaming properties.6,7 It is a globular protein containing 162 amino acids, a molecular weight of 18 400 g/ mol, an isoelectric point of 5.1, with an internal hydrophobic core surrounded by a hydrophilic exterior. In order for βlactoglobulin to form fibrils, the globular protein must first be refolded into protofilaments.8 This takes place under acidic © 2014 American Chemical Society
conditions where the protein possesses a dominant positive charge.9 Continued heating increases the contour length of these protofilaments to greater than 500 nm.4 These protofilaments then combine to form mature, multistranded, twisted fibrils.10 Rigidity of these biopolymers varies significantly depending on a number of factors, including protein type,11 ionic strength,12 or concentration.13 This rigidity, in combination with the significant contour length, makes these fibrils ideal for the production of percolated networks for a wide range of applications, such as scaffolds and drug delivery devices.14 There are a number of factors that account for the physical breakdown of amyloid fibrils, limiting their ability to be used in a wide range of applications. Some of these factors include increased temperature,15 denaturants,16 certain aromatic compounds,17,18 pH,19,20 surface-induced fragmentation,21 pressure,22 or interactions with charged polymers.23 In particular, previous research has shown that protein fibrils assembled at low pH, such as β-lactoglobulin fibrils, are prone Received: May 26, 2014 Revised: July 13, 2014 Published: July 14, 2014 3119
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to aggregation and reduction in length as the pH is increased.24 Jung and others, in individual papers, suggested electrostatic interactions with anionic surfactants25 or quick pH adjustment to neutral pH19 as means to retain some of the fibril integrity. Recent literature indicated that the latter approach significantly improved fibril properties at neutral pH,26 yet there were still unacceptable losses in fibril rigidity and length that would make the fibril products less valuable for structural applications. Protein fibrils, being composed of many of the same amino acids as the component protein,27 possess similar surface charge characteristics, such as an isoelectric point. Fibrils of βlactoglobulin were previously shown to have an isoelectric point close to pH 5.24 Several studies have investigated the electrostatic interaction of protein fibrils with charged molecules, such as sulfonic acid terminated poly(ethylene glycol),23 sodium-dodecyl sulfate,25 and κ-carrageenan.24 These electrostatic interactions imparted additional charge to the fibril surface, ultimately preventing aggregation and increasing stability.25 Another avenue to impart stability of polymers, aside from electrostatic interactions, is steric stabilization. For instance, the steric stabilization of DNA to improve its capacity for transfection has been reported through the addition of PEG−PEI brush structures to the DNA surface.28 Development of such electrostatic and steric barriers to protein−protein interaction can also be used to improve protein stability at and above the isoelectric point of proteins. Very limited research has been conducted between the interactions of cationic polymers with protein fibrils. Interactions with cationic polyfluorene polymers were shown to decrease the rate of fibrillation of α-synuclein, an amyloid protein association with Parkinson’s disease.29 To our knowledge, no publications have shown the influence of polycations on the behavior of already-formed fibrils in different pH conditions. In order to determine if addition of polymeric components to the exterior surface of fibrils would reduce aggregation and degradation of fibrils formed in acidic conditions during an increase of the pH, we investigated the interaction of chitosan (CH), poly(ethylenimine) of both low molecular weight (LPEI) and high molecular weight (HPEI), and amine-terminated poly(ethylene glycol) (APEG) with βlactoglobulin fibrils at different pH values. Chitosan is a polymer of 2-amino-glucose units extracted from crustaceans, insects, and mushrooms. LPEI and HPEI are typically branched polymers composed of one terminal amine and numerous secondary amines that are separated by two carbon units. APEG is a polymer structure containing an amino group at the terminus of a linear poly(ethylene glycol) chain. These polymers are frequently utilized in the food and pharmaceutical industries due to their commercial availability, antimicrobial activity,30,31 and film forming capacity.32 Degradation of β-lactoglobulin fibrils during pH neutralization appears to be due to a loss in surface charge causing hydrophobic aggregation of the fibrils and subsequent structural disintegration. This study observes the interaction between cationic polymers and protein fibrils over a wide pH range, with the ultimate goal of maintaining fibril length and integrity. It is believed that electrostatic interactions to the fibril surface will increase repulsions between fibrils at the isoelectric point, which would improve their value for material applications.
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fat, 1.0% ash, 4.7% moisture, and pH 6.8 (10% w/v solution), was graciously donated by Davisco Foods International, Inc. (Le Seur, MN). The β-lactoglobulin was purified of salts and aggregated protein using an established procedure.25 Briefly, β-lactoglobulin (10% w/w) solution was adjusted to pH 4.6 with 1.0 N HCl and centrifuged at 15000 rpm for 15 min at 20 °C (Sigma 3K3OH; Rotor 1215-H; 204000g) to remove precipitated material. Following adjustment of the supernatant to pH 2.0 with 1.0 N HCl, the protein solution was filtered through a 0.22 μm disc filter and dialyzed (MWCO = 6000− 8000 Da) against 0.01 N HCl for 5 days at 4 °C. Retentate (protein solution) pH was readjusted to pH 2.0 with 1.0 N HCl and lyophilized. Lyophilized protein powder was kept sealed and stored at −20 °C until use. Chitosan (CH; Lot # STBD1273 V; Mw ≈ 90 kDa), amine-terminated poly(ethylene glycol) (APEG; Lot # BCBD2802 V; Mw = 5 kDa), and polyethylenimine, both LPEI (Lot # MKBH2406; Mw = 1.8 kDa) and HPEI (Lot # MKBN3988 V; Mw = 25 kDa), were purchased from Sigma-Aldrich (St. Louis, MO) and utilized without further purification. Average molecular weight values of polymers were verified by static light scattering. Ultrapure water (σ ≥ 18 mΩ·cm) was obtained from a water filtration system (Barnstead E-pure, Thermo Scientific, Waltham, MA) and used for all solution preparations. Sodium hydroxide and hydrochloric acid were obtained from SigmaAldrich (St. Louis, MO) and used as received. Methods. Sample Preparation. Solutions of purified β-lactoglobulin (2% (w/w)) were fully solubilized in deionized water by stirring for 15 min at 300 rpm. Solutions were then acidified with continued stirring to pH 2.0 using 1.0 N HCl. Protein solution was heated in a preheated oil bath for 5 h at 90 °C, which has previously been shown to produce β-lactoglobulin at a conversion rate (from protein to fibril) of ∼75%.19 To ensure proper fibril formation, both the oil bath and the protein solutions were gently stirred with a magnetic stirring bar (60 rpm) throughout the heating process based on previously published findings linking low shear with increased fibril nucleation rates.33 After heat treatment, the fibrils were cooled in an ice bath and subsequently stored in the refrigerator until use. Stock solutions of cationic polymers CH, APEG, LPEI, and HPEI were prepared with 0.01 N HCl solution and solubilized overnight with continual stirring (300 rpm). The polymer stock solutions were diluted with 0.01 N HCl prior to mixing with dilute β-lactoglobulin solutions, and the resulting solution was readjusted to pH 2.0. Final concentrations of cationic polymer was varied experimentally and is summarized for both weight basis and approximate molar basis in Table S1, while β-lactoglobulin concentration was kept constant at 0.1% (w/w). In order to determine the influence of increased pH on the polymer/cationic polymer solutions, the pH was adjusted to pH 3, 4, and 5 (±0.08) under constant stirring (550 rpm) using 1.0 N NaOH and up to pH 6 and 7 (±0.08) using 0.1 N NaOH. Once the desired pH was reached, 1.8 mL of solution was removed and stored in labeled plastic vials and kept at room temperature for 3.5 h until measurements were performed. ζ-Potential. ζ-Potential of protein fibril suspensions was determined from laser Doppler electrophoresis measurements (Malvern Zetasizer Nano Series Nano ZS; Malvern Instruments, Worcestershire, U.K.) at 25 °C using the Smoluchowski approximation of the Henry equation with a dispersed phase refractive index of 1.45, a solution refractive index of 1.33, and a continuous phase viscosity of 0.8872 cP. Samples were analyzed in folded capillary cells (DTS1060, Malvern Instruments, Worcestershire, U.K.), which were previously rinsed with copious amounts of (sequentially) ultrapure water, methanol, and ultrapure water. Turbidity. Turbidity measurements of aqueous suspensions were performed on a PerkinElmer UV/vis Spectrometer Lambda 25 at a wavelength of 450 nm. Samples were analyzed in disposable polystyrene cuvettes with an interior volume of ∼1.4 mL. Turbidity was presented as 100 − %T, where T is the light transmitted through the sample and cuvette, in order to accentuate the increase in light scattering during colloidal interactions in the dilute solutions. Before initial measurement and at frequents points during sample measurements, T was set to 100% using ultrapure water as a blank.
MATERIALS AND METHODS
Materials. Biopure β-lactoglobulin (lot JE 002−8−415; received 25−11−2009) containing 93.4% β-lactoglobulin (w/w protein), 0.1% 3120
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Atomic Force Microscopy (AFM). AFM topographical measurements of deposited protein fibrils were performed on an Asylum MFP3D instrument (Asylum Research, Santa Barbara, CA) in intermittent contact mode. Silicon cantilevers with a reflective aluminum coating (TAP150AL-G, Ted Pella, Inc., Redding, CA) were used that have a nominal resonance frequency of 150 kHz and a force constant of 5 N/m. A scan rate of approximately 0.7 Hz was used for all measurements. Prior to measurement, prepared fibril and polymer solutions were diluted 1:40 (v/v) in water that was previously adjusted to the appropriate pH; 12 μL of sample was deposited onto freshly cleaved mica and dried using filtered air. Samples were stored at 25 °C until analyzed. Persistence lengths of unaggregated deposited fibrils on mica were obtained from AFM topographical images of β-lactoglobulin fibrils using an established approach.34,35 Briefly, images were imported into an image processing software (ImageJ)36 and the contour and end-toend lengths were measured for each fibril within the image. Fibrils were only measured if both ends of the fibril were visible and no kinks or sharp bends were present to indicate damage during deposition. Fibrils longer than 15 μm were excluded due to the inability to accurately measure all fibrils that fall beyond those parameters due to the limited scan size available with the available AFM instrument. Fibrils smaller than 1 μm were also excluded from analysis, as they contribute less significantly to fitting parameters of the persistence length. Persistence length (P) was calculated from contour length (L) and end-to-end distance (R) using eq 1:
{
⟨R2⟩ = 4PL 1 −
2P (1 − e−L /2P) L
}
(1)
Use of eq 1 assumes that the measured fibrils are collapsed onto the two-dimensional plane of the mica slide and do not project into a poorly observable third dimension.34 Between 140 and 220 fibrils were measured for each sample variable in order to improve statistical significance of the persistence length value. Dynamic Light Scattering. Hydrodynamic radius of polycations was determined on an ALV-CGS3 light scattering goniometer (ALV, Langen, Germany). Scattered light from a HeNe laser (λ = 632.8 nm) was detected by ALV High Q.E. avalanche photodiode (APD) dual detectors in pseudo cross correlation mode. Hydrodynamic radius was determined for each polycation using the method of cumulants and extrapolation to zero angle and zero concentration.
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RESULTS AND DISCUSSION In order to utilize protein fibrils assembled from β-lactoglobulin for food or biomedical applications as rigid scaffolds or structural networks in gels or films, the integrity and rigidity of those fibrils must be maintained near neutral pH conditions. Stability of pure β-lactoglobulin fibrils, which were assembled by thermal treatment at pH 2, was determined by observing their structural morphology in topographical AFM images following neutralization of the pH to 3, 5, and 7 (Figure 1). At pH 3 (Figure 1a), fibrils were intact and there were no distinguishable differences in their morphology compared to fibrils at pH 2 (Figure S1). Median contour length of these fibrils at pH 3 was 4.8 μm, which is comparable with βlactoglobulin fibril contour lengths reported elsewhere.9,23,37−39 At pH 5, large agglomerates of shortened filaments were observed within AFM images, while no long and intact fibrils could be found, indicating significant aggregation and degradation of the fibrils (Figure 1b). Measurement of the ζpotential of the protein fibrils (Figure S2) showed that the isoelectric point was very similar to that of native βlactoglobulin (pH ∼ 5.2). The tendency of these fibril fragments to aggregate near the isoelectric point indicated that the loss of net-electrostatic repulsion contributed to the instability of the fibrils. Without electrostatic repulsions to
Figure 1. AFM height images of β-lactoglobulin fibrils at (a) pH 3, (b) 5, and (c) 7. Scale bars = 5 μm (Inset scale bars = 1 μm).
minimize inter- and intrafibril interactions, the protein fibrils aggregated and broke into shorter fragments. As the pH was increased to 7, fewer aggregates were present, but the fibrils were also observed as shortened fragments when compared to their original morphology at low pH (Figure 1c). Accordingly, the median contour length of fibrils at pH 7 was less than 1 μm. Results showed that at pH values above the isoelectric point of the protein, the fibrils possessed a net-negative charge (Figure S2) that facilitated the dispersal of the aggregates formed at pH 5, yet the fibrils were already shortened by the 3121
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contribute a thick steric layer to prevent fibril−fibril interactions due to the highly hydrated polyethyelene-glycol chains. Failure of APEG to reduce fibril−fibril interactions implied that steric stabilization alone was insufficient to preserve fibril stability at pH 5. Interestingly, complex formation between a sulfate-terminated hydrocarbon (sodium-dodecyl sulfate) and β-lactoglobulin fibrils was found to reduce precipitation of the fibrils at pH 5, although no evidence was given that the structure of the fibril was maintained.25 Further experiments investigated the interaction between βlactoglobulin fibrils and polycations, which would contribute a greater repulsion between fibrils compared to the attached APEG due to the increased charge density. The charge of βlactoglobulin fibrils with increasing concentrations of CH, LPEI, and HPEI was increased at pH values above 4, indicating that interactions between fibrils and polycations were taking place (Figure 2). ζ-Potential values of fibril suspensions
neutralization process. Similar aggregation and degradation of β-lactoglobulin fibrils has been found in our laboratory for fibril solutions at low pH possessing >100 mM of added salts (not shown). This supports the concept that fibril stability in solution was maintained by a strong effective surface charge. Within confined aggregates that were formed at pH 5, the protein fibrils experienced crowding effects that would have altered the hydration and hydrophobic forces exerted on the individual fibrils.40 Without knowledge of the ordering of water molecules along the fibril surface, it could only be concluded that these forces were unfavorable to the fibril structure and contributed to degradation of the fibrils to shorter strands. Degradation of the fibrils at increased pH value agrees with previous observations of β-lactoglobulin fibrils using electron microscopy and AFM.24 A recent study indicated that much of the structure of β-lactoglobulin fibrils was preserved at pH values above the isoelectric point, but this conclusion was based largely on the lack of individual peptide fragments resulting from full dissolution of the fibrils.26 As shown in Figure 1c, some of the fibril structure was preserved at neutral pH in the form of the small fragments that did not degrade to the constituent peptides but were still significantly shortened compared to the original fibrils at low pH. These shortened fibril fragments could still contribute to improved gelation properties compared to the native protein, such as shown for neutral-pH gels of β-lactoglobulin fibrils cross-linked by calcium ions, yet the percolation concentration of potential networks is greatly diminished if the fibril length and rigidity are reduced. Jung and others suggested increasing the speed at which fibril solutions are neutralized in order to limit aggregation at the isoelectric point and preserve much of the fibrils’ original persistence length.19 However, initial observations of βlactoglobulin fibril morphology at pH 5 and 7 with faster pH adjustment and less incubation time prior to measurements indicated that fibrils still undergo a significant degree of aggregation and degradation by passing through the isoelectric point (not shown). Since the aim of this study was to preserve the utmost fibril integrity during pH neutralization, fibril contour lengths of 50% of the amine groups would have been charged in the studied pH range. 3122
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Figure 3. Turbidity of β-lactoglobulin fibrils (0.1% w/w) as a function of solution pH (a) alone or with increasing concentrations of added (b) CH, (c) LPEI, and (d) HPEI.
Figure 4. AFM height images of β-lactoglobulin fibrils with increasing concentrations (listed above each image) of cationic polymers at pH 5. Scale bars = 5 μm (Inset scale bars = 1 μm).
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these protein materials and the added polycations were not observed, indicating that there was a strong tendency for the polycations to interact with the fibrils. Fibrils with added APEG were not stable at pH 5 at any tested polymer concentration (Figure S3), which indicated that addition of the hydrated yet uncharged polymer to the surface of the fibril was incapable of stabilizing the fibril structure. Imparting charge to the fibril surface by addition of polycations was then a more successful strategy for improving fibril integrity at and above its isoelectric point. At pH 7, HPEI was the only added polycation that maintained much of the original fibril length, as fibrils with added CH and LPEI were observed as small aggregates of short fragments (Figure S4), similar to the pure fibrils at pH 7 (Figure 1c). Fibril morphology with increasing concentrations of HPEI at pH 7 by AFM measurements is shown in Figure 5. Low concentrations (800 nm (Table S2). At pH 3, fibrils with added polycations possessed median contour lengths between 3.9 and 7.9 μm, which was comparable to observed contour length of the pure fibrils (median = 4.8 μm). Since pure fibrils aggregated and degraded to short fragments at pH 5 and 7, no contour length values are reported at these higher pH values. Increasing concentrations of added CH, LPEI, and HPEI at pH 5 led to a corresponding increase in median contour length, demonstrating the capacity of added polycation to improve fibril stability. Median contour lengths at pH 5 with these added polycations were between 2.9 and 5.9 μm, which is comparable to contour lengths of fibrils at pH 3. At pH 7, only HPEI was able to stabilize fibrils and maintain fibril lengths greater than 1 μm, with the median contour length increasing from 3.2 to 4.8 μm with increasing HPEI concentration. Calculated persistence length values indicated that the fibrils at low pH, as well as the fibrils with added polycations at increased pH, were semiflexible with values between 1.9 and 6.7 μm (Table S2). Persistence length of pure fibrils at pH 3 was 4.1 μm, which was not significantly altered in the presence of the polycations. These persistence lengths values are large relative to some of the reported values in the literature20,25,44 yet were consistent with persistence length values reported for similarly produced fibrils of β-lactoglobulin.33 Persistence length of protein fibrils has been previously shown to depend on the formation conditions, such as pH and ionic
Turbidity measurements were performed in order to determine the influence of added polycations on the aggregation and phase stability of β-lactoglobulin fibrils at increased pH (Figure 3). Pure suspensions of β-lactoglobulin fibrils became quite turbid near pH 5, while pure polycations did not significantly contribute to turbidity at any of the pH values tested (except for CH, which became slightly turbid at pH 7 due to reduced solubility; Figure 3a). Turbidity of the pure fibrils demonstrated the aggregation and phase separation of fibrils at pH values near the isoelectric point. Addition of CH, LPEI, and HPEI at and above concentrations of 0.0125, 0.0016, and 0.0024% (w/w), respectively, significantly reduced the turbidity of the fibrils at pH 5 (Figure 3b−d). This indicated that aggregation of the fibrils was suppressed with the addition of increasing concentrations of the polycations. At pH 6 and 7, fibril suspensions with CH and LPEI became increasingly turbid, which is similar to the observed turbidity increases during the electrostatic interaction between polyelectrolytes and proteins to form complex coacervates.43 Increased turbidity of fibril-CH suspensions above pH 5 were also likely influenced by the decreasing charge, and associated decrease in solubility, of CH as it surpassed its isoelectric point of 6.3. Fibril suspensions with higher concentrations of added HPEI (>0.01% w/w), however, were not turbid above pH 5 and indicated a complete suppression of large-scale aggregation among the protein fibril−polycation system (Figure 3d). Considering that the crucial difference between LPEI and HPEI is the molecular weight, and therefore the total number of charges per added polymer, the enhanced capability of the higher molecular weight HPEI to stabilize protein fibrils at pH 7 was attributed to a greater attachment of cationic charges per attachment site on the fibril. This agrees with the relatively greater positive charge of fibrils at pH 7 with HPEI compared to fibrils with LPEI at polycation concentrations greater than 0.01% (w/w), as observed by electrophoretic mobility measurements (Figure 2b,c). Charge of fibril−polycation complexes at pH 7, in terms of ζ-potential, reached a maximum value of ∼20 mV with increasing addition of LPEI, while a maximum value of ∼30 mV was reached with increasing addition of HPEI. Experimentally observed ζ-potential values of
