Article pubs.acs.org/Biomac
Conformational Transition of Polyelectrolyte As Influenced by Electrostatic Complexation with Protein Yiping Cao,† Shugang Li,† Yapeng Fang,*,†,‡ Katsuyoshi Nishinari,†,‡ and Glyn O. Phillips† †
Glyn O. Phillips Hydrocolloid Research Centre, School of Food and Biological Engineering and ‡Hubei Collaborative Innovation Centre for Industrial Fermentation, Hubei University of Technology, Wuhan 430068, China S Supporting Information *
ABSTRACT: Conformation and conformational transitions are essential for the biological and technological functions of natural polyelectrolytes, for example, DNA. This study aims to clarify how the conformational transition of natural polyelectrolyte is affected and tuned by electrostatic complexation with protein as encountered in many biological processes. A model protein/polyelectrolyte system, β-lactoglobulin (β-lg) and κ-carrageenan (κ-car), was used for the investigation. The effect was found to be determined by the molecular state of β-lg/κ-car electrostatic complexation and the molecular weight of protein. β-lg/κ-car complexation in soluble state had a subtle effect on the coil-to-helix transition of κ-car, while that in insoluble state greatly suppressed it. On the basis of the McGheeHippel theory, a quantitative model was successfully developed to describe the effect of protein/polyelectrolyte electrostatic complexation on the conformational transition of polyelectrolyte. The model can also provide additional information on the change of tertiary structure of β-lg upon electrostatic complexation with κcar. Moreover, it was found that β-lg or its hydrolysates with a molecular weight larger than 2000 Da hindered the conformational transition of κ-car, while those with a molecular weight lower than 1000 Da promoted it. The observations offer a promising approach to control the conformational transition and related properties of polyelectrolytes for technological applications.
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INTRODUCTION
industry for applications such as thickening, gelling, and stabilization.7 Electrostatic complexation of polyelectrolyte with protein is also important in biological processes including DNA replication, transcription, and repair.8,9 Protein is considered first to bind nonspecifically to DNA via electrostatic interaction and move subsequently along the DNA strand until specific binding sites are identified.10 Protein/polyelectrolyte electrostatic complexation occurs naturally in many living organisms and triggers a variety of biological functions such as the adhesive property of sandcastle worms provided by the complexation of highly polar proteins11 and the mechanical properties of mammalian cartilages provided by the ternary complexes of aggrecan, hyaluronan, and cartilage link proteins.12 Protein/polyelectrolyte complexation has found numerous applications in the biotechnological and pharmaceutical industries including protein separation/purification,13 enzyme stabilization/immobilization,11 drug delivery, and gene therapy.14 In the food industry, protein/polyelectrolyte electrostatic complexation was used to design low-calorie and lowstarch foods,15 encapsulate flavors and probiotics,16,17 and stabilize emulsions and foams.18
Natural polyelectrolytes exhibit diverse conformations in solutions including random coil, single helix, multiple helix, aggregate, and sphere-like structure, etc.1−3 These conformations are involved in many biological processes and in human diseases. For instance, DNA with mutated conformation (e.g., hairpin, triplex, cruciform, and left-handed form) is associated with diseases such as myotonic dystrophy, Huntington’s chorea, fragile X syndrome, and Friedreich’s ataxia.1 In cell biology, conformational equilibria of ubiquitin can determine its specific binding with protein or DNA and thus regulate protein degradation, cell signaling/trafficking, and DNA damage response.4 Conformation and conformational transitions of polyelectrolytes influence also their bioactivities. Triple helical schizophyllan shows strong inhibition against tumor cells, whereas its single coil counterpart has almost no bioactivity. This behavior is attributed to an increased interaction between the extended chain conformation and the immune system.5 Sulphation or phosphatization is known to greatly improve the inhibitivity of branched α-(1 → 4)-D-glucan against H-22 tumor cells. The effect is explained by a conformational transition from compact sphere to random coil upon the chemical modification, which confers the polyelectrolyte more flexibility to bind with relevant receptors on the tumor cell surface.6 Conformational transitions of polyelectrolytes are applied in © 2016 American Chemical Society
Received: September 7, 2016 Revised: October 25, 2016 Published: October 28, 2016 3949
DOI: 10.1021/acs.biomac.6b01335 Biomacromolecules 2016, 17, 3949−3956
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further separation. After dialysis against Millipore water at 4 °C for 96 h, the hydrolysates inside and outside the dialysis tube were collected by freeze-drying and designated as S1 and S2, respectively. A total hydrolysate of β-lg (S3) was obtained by heat treatment at 120 °C in 6.0 M HCl for 12 h, followed by removal of HCl using vacuum evaporation at 60 °C. Although S1, S2, and S3 were mixtures of peptide fragments of different length and chemical composition, as a whole, their average amino acid compositions were nearly the same as the mother protein β-lg without hydrolysis. This was confirmed by analysis using an automatic amino acid analyzer (Hitachi, L-8900). Weight-average molar mass measured by GPC-MALLS at 25 °C in 0.1 M phosphate buffer using a Sepax SRT SEC-150 column is β-lg (19100 Da) > S1 (2053 Da) > S2 (921 Da). The weight-average molar mass of S3 was calculated to be 127 Da according to the amino acid composition of β-lg.20,21 Solution Preparation. Stock solutions of 0.06−1.80 wt % β-lg and 0.90 wt % κ-car were prepared by dissolving appropriate amounts of the samples into 50 mM KCl. κ-car solutions were heated at 85 °C for 1 h under magnetic stirring. β-lg solutions were dissolved at ambient temperature overnight on a roller mixer. β-lg/κ-car mixtures at a fixed κ-car concentration (0.15%) and various β-lg/κ-car mixing ratios (w/ w, 0 < r < 10) were prepared by blending the stock solutions, followed by stirring at 60 °C for 10 min. The mixing temperature was chosen to avoid the denaturation of β-lg at high temperature and meanwhile ensure a sol state of κ-car. pH of the mixtures was adjusted to targeted values using 2 M NaOH or HCl. Differential Scanning Calorimetry (DSC). Conformational transition of κ-car in different β-lg/κ-car mixtures was examined by a high-sensitivity microcalorimeter DSC-III (Setaram, France). A 0.9 g sample was hermetically sealed into a stainless cell, and an equal amount of solvent was used as reference. The sample was heated from room temperature to 60 °C at a scan rate of 3 °C/min and was then held at 60 °C for 10 min. The subsequent cooling process from 60 to 0 °C at a scan rate of 0.5 °C/min was recorded. The onset temperature (T0) and enthalpy change (ΔH) of the conformational transition of κcar upon cooling were analyzed by the software Calisto Processing. Turbidity Measurements. Complexation of κ-car in the coil state with β-lg was investigated by measuring turbidity at 45 °C as a function of β-lg/κ-car mixing ratio. A TU-1900 UV−vis spectrophotometer (Persee, China) was used. β-lg/κ-car mixtures were placed into a copper cuvette fixed with optical quartz windows. After equilibration at 45 °C for 10 min, turbidity at 500 nm (τ) was recorded. The temperature was controlled by a Peltier device (Quantum Northwest, USA) with an accuracy of 0.01 °C. Isothermal Titration Calorimetry (ITC). An ITC 200 isothermal titration calorimeter (Malvern Instruments, U.K.) was used to quantify the complexation of κ-car with β-lg in the coil state (45 °C). β-lg and κ-car solutions were dissolved in phosphate buffer (5 mM) and then dialyzed against the buffer solvent to avoid pH and ionic strength mismatch. Two microliter aliquots of 2.5% β-lg solution were sequentially injected into a 200 μL reaction cell initially containing either buffer solvent (control) or 0.05% κ-car solution. There were a total of 20 injections for each measurement with an interval of 100 s between two successive injections. The stirring speed was set at 500 rpm for all the experiments. The binding isotherm obtained by integrating injection peaks was analyzed by a model of independent binding sites:30
Coupling of protein/polyelectrolyte electrostatic complexation with conformational transition was frequently encountered in biological processes and technological applications. Our previous studies have shown that protein/polyelectrolyte complexation is influenced by polyelectrolyte conformational transition via two interconnected mechanisms, namely, specific ionic binding and chain stiffening.19 It is necessary now to clarify how the conformational transition of polyelectrolyte is controlled by electrostatic complexation with protein. We demonstrate this behavior by reference to a model protein/ polyelectrolyte system, β-lactoglobulin (β-lg) and κ-carrageenan (κ-car). β-lg is the dominant globular protein found in whey protein.20 It has 162 amino acid residues and a molecular mass of 18.4 kDa.20,21 κ-car is a naturally anionic polysaccharide extracted from red seaweed species and is widely used in food, cosmetic, and pharmaceutical products as gelling agents, thickeners, stabilizers, and excipients.7 It is made up of alternating α-(1 → 3)-D-galactose-4-sulfate and β-(1 → 4)3,6-anhydro-D-galactose repeating units.7,22 The temperatureinduced conformational transition of κ-car has been a subject of debate for many years. It is traditionally accepted that κ-car, in the presence of ions (e.g., K+), undergoes a conformational transition from random coil to double helix and further to helical aggregates with lowering temperature.19,23 Recently, Schefer et al. elegantly showed that the κ-car chains first undergo a simple coil-to-helix transition, and then the single helices interwine or supercoil into supermolecular strands with a hierarchical chirality amplification using high-resolution atomic force microscopy.24−26 This represents a significant advancement of the debates on the conformational transition of carrageenans.
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EXPERIMENTAL SECTION
Materials. β-lg was kindly supplied by Davisco, which according to the supplier has a minimum purity of >95%. β-lg has a weight-average molar mass of Mw = 19.1 kDa, a polydispersity index of Mw/Mn = 1.1, and a radius of gyration of Rg = 6.5 nm, as determined by gel permeation chromatography-multiple angle laser light scattering (GPC-MALLS, Wyatt Technology Corporation, USA) at 25 °C in 0.1 M phosphate buffer (pH 7.0) using a Sepax SRT SEC-150 column (Sepax Technologies, USA). The isoelectric point (IEP) of the β-lg sample measured by Nano-ZS ZetaSizer (Malvern Instruments, UK) is 5.2 (Figure S1), which agrees with literature values.20,21,27 κ-car was a gift from FMC biopolymer (Gelcarin GP-911NF). It was converted into the sodium salt using ion-exchange resin (Amberlite IR-120, Sigma) and then lyophilized. The purified sample contains 6.32% Na, 0.067% K, 0.0027% Mg, and 0.0083% Ca as determined by atomic absorption spectrometry. The purification was to avoid the complicated effects of mixed counterions, which allowed a precise molecular characterization of κ-car. The molecular parameters measured by GPC-MALLS at 25 °C in 0.1 M NaI using a Shodex OHpak SB-805 separation column (GE Healthcare Co., USA) are Mw = 467 kDa; Mw/Mn = 1.2; Rg = 85.0 nm, which represent the doubly associated helical conformation of κ-car without further aggregation.19,28 The zeta potential of κ-car, measured in the pH range of 3−8, is characteristic of strong polyelectrolytes with nearly a constant value of about −60 mV (Figure S1), which agrees with most literature reports.29 Preparation of β-lg Hydrolysates. Hydrolysates with different molecular weight were obtained by hydrolyzing β-lg to different extents. The 5.0% β-lg was heated at 85 °C for 5 min, followed by hydrolysis using the enzyme papain (3000 U, Biosharp) at the optimum conditions (45 °C, pH 4.5) for 4 h. The hydrolysate was separated using ultrafiltration tube (MWCO 3000, Biosharp), and the filtrate was loaded into dialysis tube (MWCO 1000, Biosharp) for
⎡ 1 + [M ]nK − Q = V ΔH ⎢[L] + ⎢ ⎣
⎤ (1 + [M ]nK − [L]K )2 + 4K[L] ⎥ ⎥ 2K ⎦
(1) where Q is accumulative heat, and V, [L], and [M] are the volume of cell, ligand concentration (i.e., β-lg), and macromolecule concentration (i.e., κ-car), respectively. Iterative curve fitting yielded thermodynamic parameters including the binding constant K, the binding enthalpy ΔH, and the stoichiometry n. Rheological Measurements. Rheological measurements were conducted to characterize the gelation and gel viscoelasticity of β-lg/κcar mixtures on a HAAKE RheoStress 6000 rheometer (Thermo 3950
DOI: 10.1021/acs.biomac.6b01335 Biomacromolecules 2016, 17, 3949−3956
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Figure 1. DSC curves of κ-car during cooling at different β-lg/κ-car mixing ratio r at (a) pH 5.6 and (d) pH 4.7; enthalpy change (ΔH) of the conformational transition of κ-car as a function of r at (b) pH 5.6 and (e) pH 4.7; turbidity (τ) of β-lg/κ-car mixtures at 45 °C as a function of r at (c) pH 5.6 and (f) pH 4.7. β-lg/κ-car mixing ratio was indicated beside the DSC curves in panels a and d. The broken line in panels e and f marks the turning points in ΔH-r and τ-r curves. The concentration of κ-car was fixed at 0.15%. Fisher Scientific, USA). A serrated parallel-plate geometry (diameter 35 mm; gap 1.0 mm) was used to prevent slippage. κ-car mixtures with β-lg or its hydrolysates were loaded onto the preheated geometry at 60 °C and were covered with a thin layer of silicon oil to prevent evaporation. Temperature dependence of storage (G′) and loss (G″) moduli of the mixtures upon cooling was measured under dynamic oscillation mode at a frequency of 1 Hz and a stress of 0.5 Pa. The temperature was controlled by an external circulating water bath from 60 to 10 °C at a cooling rate of 0.5 °C/min. The stress was checked to be within the linear viscoelastic region. After the completion of temperature dependence measurement, the samples were subjected to dynamic frequency sweep measurements at 10 °C to evaluate the frequency dependence of G′ and G″ at a stress of 0.5 Pa and within a frequency range of 0.01−10 Hz.
electrostatic complexation on conformational transition was investigated using β-lg/κ-car mixtures at different pHs and mixing ratios. Two typical examples are illustrated here for pHs above and below the IEP of β-lg (pH 5.2). Figure 1a shows DSC peaks of κ-car during cooling in mixtures with β-lg at pH 5.6. The DSC curve of pure κ-car (r = 0) contains a single asymmetric exothermic peak, which is assigned to the coil-tohelix transition of κ-car.19 With increasing β-lg/κ-car mixing ratio (r), the exothermic peak is hardly changed with a constant onset temperature of T0 = 40.6 °C and a constant enthalpy change of ΔH = 59.6 mJ/g (Figure 1b). The results indicate that mixing β-lg and κ-car at pH 5.6 had a negligible effect on the conformational transition of κ-car upon cooling. This is ready to understand since no electrostatic complexation occurs between κ-car and β-lg at pH > IEP.31 It is because both κ-car and β-lg are overall negatively charged at this pH, with a zeta potential of −60 mV and −25 mV, respectively (Figure S1). The absence of electrostatic complexation at pH 5.6 is
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RESULTS AND DISCUSSION Conformational Transition of κ-car upon Electrostatic Complexation with β-lg. At pH values below or slightly above its IEP, protein was reported to form electrostatic complexes with anionic polyelectrolyte.31 The effect of 3951
DOI: 10.1021/acs.biomac.6b01335 Biomacromolecules 2016, 17, 3949−3956
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transition of κ-car is influenced to a limited extent by the electrostatic complexation with β-lg. As electrostatic complexation further proceeds (Figure 2b), more and more β-lg are bound to κ-car, and cross-linking between κ-car occurs. This causes the formation of insoluble β-lg/κ-car complexes. κ-car molecules are sterically blocked by β-lg and have limited freedom to restructure to form helices. The conformational transition of κ-car in this instance is greatly suppressed by electrostatic complexation with β-lg. It should be pointed out that although the exact conformations of κ-car are to some extent controversial (i.e., single helix, double helix, or supermolecular helical strand),24−26 the proposed mechanism, as schematically shown in Figure 2, applies to different situations. Theoretical Modeling. As already discussed, the conformational transition of κ-car is greatly suppressed by electrostatic complexation with β-lg, and the origin of this effect is κ-car being physically occupied with β-lg. Assuming that the relative extent of conformational transition of κ-car is proportional to the number of repeating units that are unoccupied with β-lg, a quantitative model can be developed to describe the effect of protein/polyelectrolyte electrostatic complexation on the conformational transition of polyelectrolyte (see Supporting Information) based on the DNA−protein binding theory proposed by McGhee- Hippel:33
supported by the turbidity measurements in Figure 1c where τ remains close to zero at all mixing ratios at 45 °C. In contrast, at pH 4.7 < IEP, the exothermic peak associated with the coil-to-helix transition of κ-car was significantly reduced with increasing β-lg/κ-car mixing ratio (Figure 1d). The exothermic peak tends to disappear completely at r > 8.00. This indicates that the conformational transition of κ-car is greatly suppressed when mixed with β-lg. This can be attributed to the electrostatic complexation between β-lg and κ-car, which impairs the helix formation of κ-car. Figures 1e plots the enthalpy change (ΔH) of the conformational transition of κ-car as a function of r at pH 4.7. ΔH shows a two-step decrease with increasing r, and a turning point is located at r = 0.67. Beyond the turning point (r > 0.67), the decrease in ΔH is apparently accelerated. It means that the conformational transition of κ-car is more seriously impaired beyond the turning point. Interestingly, the turning point in ΔH-r curve coincides with that obtained in τ-r curve at 45 °C (Figure 1f). Turbidity is relatively low at r < 0.67, whereas it increases abruptly at r > 0.67. Similar coincidence was found at other pHs, and the turning point is at r = 0.33, 1.45, 2.70, and 4.00, respectively, for pH = 4.4, 4.9, 5.0, and 5.1 (see Figure S2). Turbidity was seen as a sensitive measure of electrostatic complexation of protein/ polyelectrolyte.11,31,32 The abrupt increase in τ at the turning points is attributed to the transition from soluble to insoluble electrostatic complexes.31,32 When β-lg molecules cross-link different κ-car chains, large aggregates that are insoluble are formed, and thus the turbidity increases.31,32 Note that further decrease in pH below 4.4 caused extensive precipitation, which prevented the measurement of the conformational transition of κ-car. The evolution of electrostatic complexes as a function of pH and mixing ratio has been studied in detail in our previous works.29,30 The molecular state of β-lg/κ-car complexation seems to control the conformational transition of κ-car (Figure 2). During the initial stage of electrostatic complexation (Figure 2a), a small number of β-lg are bound to κ-car, and the complexes remain soluble. β-lg molecules pose limited steric effect, and κ-car molecules have sufficient freedom to form helices through pairing. Therefore, the conformational
m−1 ⎡ 1 − ϕ(r ) ⎤ (1 − ϕ(r ))⎢ϕ(r ) + ⎥ ⎣ ⎦ m P ⎡ CM ⎤ N (1 − ϕ(r )) ⎥ p w − Kmϕ(r )m ⎢r − =0 C P ⎢⎣ M wPr ⎥⎦ m
(2)
where r is the protein/polyelectrolyte weight ratio; ϕ(r) is the relative extent of conformational transition of polyelectrolyte; m represents the number of repeating units consecutively covered by one protein molecule and is the reciprocal of the binding stoichiometry n; K is the binding constant; Cp is the molar concentration of polyelectrolyte; MPw and MPr w are the molecular weight of polyelectrolyte and protein, respectively; and N is the total number of repeating units of polyelectrolyte. Thus, ϕ(r) could be experimentally measured as the ratio of the enthalpy change of conformational transition of protein/ polyelectrolyte mixture (ΔH(r)) to that of pure polyelectrolyte (ΔH(r = 0)). Figure 3 plots ϕ as a function of β-lg/κ-car mixing ratio at different pHs. Except at pH 5.6, which is above the IEP of β-lg, ϕ at pH = 5.1, 5.0, 4.9, 4.7, and 4.4 all exhibit a two-step decrease with r. The decrease is more pronounced at lower pH. This is due to an increased electrostatic complexation of β-lg/κ-car at lower pH, which exerts a stronger effect on suppressing the conformational transition of κ-car. Intriguingly, the turning points of the two-step decrease at different pHs occur at the same value of ϕ = 0.93 (Figure 3b). This probably indicates a common critical binding threshold beyond which insoluble β-lg/κ-car complexes start to be formed and the conformational transition of κ-car is seriously inhibited. This threshold corresponds to 7% repeating units of κ-car being bound/occupied with β-lg. By taking N = 572, Cp= 6.42 × 10−6 mol/L, MPr w = 19 100 Da, and MPw = 233 500 Da for the present system, ϕ measured by DSC can be well fitted to eq 2 (solid lines in Figure 3a). The fittings yielded the thermodynamic binding parameters m and K at different pHs, as listed in Table 1. The parameters are in good agreement with those obtained from ITC measurements
Figure 2. Schematic representation of the effect of β-lg/κ-car electrostatic complexation on the conformational transition of κ-car upon cooling: (a) β-lg/κ-car complexation in soluble state; (b) β-lg/κcar complexation in insoluble state. κ-car and β-lg molecules are illustrated in black and red, respectively. During the conformational transition, coiled κ-car chains transform into ordered helical conformations (bold lines), which can be single helix, double helix, or even supermolecular helical strand. 3952
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Figure 3. (a) Relative extent of the conformational transition of κ-car (ϕ) as a function of β-lg/κ-car mixing ratio r at different pHs: pH = 5.6 (black square); pH = 5.1 (red circle); pH = 5.0 (blue diamond); pH = 4.9 (green triangle); pH = 4.7 (pink ex); pH = 4.4 (orange plus); (b) enlarged part of panel a showing two-step decrease in ϕ. The solid lines represent the curve fittings to eq 2. The dot-dash lines at ϕ = 0.93 mark the turning points of ϕ-r curves.
Table 1. Thermodynamic Parameters of the Binding of β-lg to κ-car Obtained from DSC and ITC method
parametera
pH = 4.4
pH = 4.7
pH = 4.9
pH = 5.0
pH = 5.1
DSC
m K m K
25.2 1.7 × 108 25.5 1.9 × 108
17.1 2.3 × 106 16.6 2.1 × 106
13.9 3.1 × 103 14.2 3.0 × 103
13.2 3.5 × 102 12.8 3.9 × 102
12.2 5.1 × 101 12.3 6.2 × 101
ITC a
m = 1/n; n is the binding stoichiometry of β-lg to κ-car.
troscopy (ATR-FTIR) shows that the β-sheet and α-helix content of β-lg are both decreased upon complexation with κcar at lower pHs (see Figure S5). It was also found that protein structures were altered upon complexation with polyelectrolyte in α-gliadin/gum arabic or β-lg/acacia gum systems.32,36 The structural changes revealed by DSC and ATR-FTIR may be responsible for the geometric elongation of β-lg, as predicted by the theoretic model in eq 2. The situation is similar to the binding of proteins onto negatively charged surfaces (e.g., mica, silica) at pHs < IEP or hydrophobic surfaces. The strong electrostatic interaction or hydrophobic interaction upon adsorption onto the surfaces caused somewhat denaturation and spreading/elongation of the proteins along the surfaces.37−39 On the other hand, β-lg was reported to exhibit a number of pH-induced, local and global structural transitions between pH 1.0 and pH 13 including the dimer-to-monomer transition (pH 2.5−4.0), the Q-to-N transition (pH 4.5−6.0), and the Tanford transition (>pH 6.5), etc.35 In the pH range investigated (pH 4.4−5.6), β-lg exists predominately as dimers and undergoes a conformational change from the compact native form (N) at pH 6.0 to the more extended acidic form (Q) at pH 4.5. The hydrodynamic diameter of β-lg molecule was reported to increase from 5.16 to 5.44 nm, accordingly.40 Such a pHinduced size expansion (only by 5%) could contribute to but was not fully responsible for the observed elongation of β-lg upon electrostatic complexation with κ-car (Table 1). Moreover, Taulier and the co-worker observed a slight change in β-lg tertiary structure (i.e., increase in volume and compressibility) during the Q-to-N transition, which however was not accompanied by any significant alteration in secondary structure.35 This slight change in tertiary structure seems not to have conspicuous influence on the electrostatic complexation of β-lg with κ-car, as the complexation behaviors at different pHs can be described by a common theoretical model.
at 45 °C (see Figure S3). The ITC results represent the binding of β-lg to κ-car in coil state and just prior to the coil-to-helix transition and therefore to large extent can approximate their dynamic binding during the subsequent conformational transition. The agreement validates the theoretical model as developed in eq 2. With lowering pH from 5.1 to 4.4, K increases from 5.1 × 101 to 1.7 × 108 M−1. The increase in K is apparently attributed to an increase in electrostatic attraction. Note that the binding constants with magnitude as high as 108 at lower pHs might represent strong out-of-equilibrium bindings, where precipitation of β-lg/κ-car complexes occurred. m is also increased by lowering pH, but its change around IEP (i.e., pH 5.1 and 5.0) is insignificant. Considering a repeating unit length of 1.03 nm for κ-car,34 m = 12.2 at pH 5.1 corresponds to a consecutive binding length of 12.6 nm, which is very close to the diameter of β-lg (2Rg = 13 nm) as measured by GPC-MALLS. It implies that the weak binding between β-lg/κ-car close to IEP is determined by the size of β-lg (most likely in dimeric form).35 In contrast, m = 25.2 at pH 4.4, which corresponds to a consecutive binding length of 30.0 nm. This length is more than double the diameter of β-lg. It could be due to an increase in protein surface charge, which increases its effective Debye length. Another possibility is that the strong binding between βlg/κ-car at lower pH induces a geometric elongation of β-lg. DSC measurements (see Figure S4) show that at pH values (i.e., pH 5.1 and 5.6) close to IEP, there is almost no difference in protein denaturation temperature between pure β-lg and βlg/κ-car mixture. However, at lower pHs (e.g., pH 4.4), the denaturation temperature of pure β-lg is considerably higher than that in the β-lg/κ-car mixture. The reduction in denaturation temperature indicates a less stable conformation or tertiary structure of β-lg upon electrostatic complexation with κ-car. Furthermore, structural component analysis by attenuated total reflectance Fourier transform infrared spec3953
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Figure 4. (a) Onset temperature (T0) and (b) relative extent (ϕ) of the conformational transition of κ-car, as affected by electrostatic complexation with β-lg or its hydrolysates. κ-car 0.15%; pH = 4.7.
Figure 5. (a) Gelation profiles of storage modulus G′ versus temperature during cooling for κ-car at different β-lg/κ-car mixing ratios r; (b) frequency sweep of storage (G′, closed symbol) and loss moduli (G″, opened symbol) of β-lg/κ-car mixtures at 10 °C. κ-car 0.15%; pH = 4.7.
As discussed above, the suppressing effect of β-lg or its hydrolysates with a molecular weight larger than 2000 Da is due to their steric effect, which physically hinders the conformational transition of κ-car upon electrostatic complexation. K and m for S1 binding to κ-car at pH 4.7 were calculated to be 4.1 × 102 M−1 and 6.8, respectively, based on the curve-fitting using eq 2. The values are significantly smaller than those of 2.3 × 106 M−1 and 17.1 for β-lg binding to κ-car at pH 4.7, which indicate a decreased binding affinity with decreasing hydrolysate molecular weight. m = 6.8 corresponds to a consecutive binding length of 6.8 × 1.03 nm = 7.0 nm, which agrees well with the size of S1 of 7.2 nm (containing 19 amino acids, each being approximately 0.38 nm in length).41 The steric effect disappears completely when the molecular weight of β-lg hydrolysates is decreased to around 1000 Da. With further decreasing molecular weight below 1000 Da, the ionic strength effect of the short hydrolysates becomes pronounced due to the increasing number of amino and carboxyl groups. This facilitates the coil-to-helix transition of κcar and stabilizes the formed helices during cooling. The promoting effect of β-lg hydrolysates with a molecular weight lower than 1000 Da is similar to that of adding electrolytes such as NaCl.22 The addition of NaCl was reported to increase also the onset temperature of the conformational ordering of κ-car, while it did not change noticeably the transition enthalpy at high ionic concentration. The promoting effect was attributed to the screening of intra- or intermolecular electrostatic repulsion and the alteration of hydration properties of κ-car chains, which favors the formation of ordered helices.42
Effect of Protein Molecular Weight. Since protein/ polyelectrolyte electrostatic complexation suppresses the conformational transition of polyelectrolyte via the physical hindrance of protein, it is desirable to investigate in detail the effect of protein molecular weight or size. For this purpose, β-lg was hydrolyzed into different extents by enzymatic or acidic treatment, and three hydrolysates of different molecular weights were fractionated using ultrafiltration and dialysis: S1 (2053 Da) > S2 (921 Da) > S3 (127 Da). The three fractions have almost the same average amino acid compositions as the mother protein β-lg. Figure 4 shows how differently the β-lg hydrolysates affect the conformational transition of κ-car upon electrostatic complexation at pH 4.7. The effect of lysine was also compared, as it is one of the most typical and abundant amino acids in βlg. When the hydrolysate molecular weight is >2000 Da (e.g., βlg and S1), the onset temperature (T0) and relative extent (ϕ) of conformational transition of κ-car are markedly reduced with increasing r, which suggests a suppressing effect of the hydrolysates. When the hydrolysate molecular weight is around 1000 Da (e.g., S2), T0 and ϕ are nearly constant with increasing r. It indicates that the electrostatic complexation in this range of hydrolysate molecular weight has no effect on the conformational transition of κ-car. Significantly, when the hydrolysate molecular weight is 2000 Da) had an inhibitory effect on the conformational transition of κ-car, whereas shorter hydrolysates ( G″, indicative of a typical elastic gel.44 With increasing r, G′ and G″ become more and more frequency-dependent and cross over at high frequencies. This is an indication of weak viscoelastic gels or viscous solutions.44 The results demonstrate that by mixing with protein at different ratios, electrostatic complexation can be used to tune the gelling behavior of polyelectrolytes to give viscoelastic properties ranging from elastic gels to viscous solutions. The effect is in line with that on the conformational transition of κ-car, as shown in Figure 3a. Figure 6 demonstrates tuning the gelation and gel viscoelasticity of κ-car by electrostatic complexation with β-lg hydrolysates of different molecular weights. At r = 10.0 and pH = 4.7, the presence of β-lg or S1 (>2000 Da) significantly reduces the gelling ability and viscoelasticity of κ-car (Figure 6a). The corresponding frequency sweep exhibits weak gel type or liquid behavior (Figure 6b). The presence of S2 (∼1000 Da) has almost no effect on the gelation and gel viscoelasticity of κcar. The gelation profile and frequency sweep of S2/κ-car overlap with those of pure κ-car. In contrast, the low molecular weight hydrolysates, S3 and lysine (
