Article pubs.acs.org/JPCB
Self-Organization of Glucose Oxidase−Polymer Surfactant Nanoconstructs in Solvent-Free Soft Solids and Liquids Kamendra P. Sharma,† Yixiong Zhang,† Michael R. Thomas,†,‡ Alex P. S. Brogan,† Adam W. Perriman,†,§ and Stephen Mann*,† †
Centre for Organized Matter Chemistry and Centre for Protolife Research, School of Chemistry, ‡Bristol Centre for Functional Nanomaterials, and §School of Cellular and Molecular Medicine, University of Bristol, Bristol BS8 1TS, United Kingdom S Supporting Information *
ABSTRACT: An anisotropic glucose oxidase−polymer surfactant nanoconjugate is synthesized and shown to exhibit complex temperature-dependent phase behavior in the solvent-free state. At close to room temperature, the nanoconjugate crystallizes as a mesolamellar soft solid with an expanded interlayer spacing of ca. 12 nm and interchain correlation lengths consistent with alkyl tail−tail and PEO−PEO ordering. The soft solid displays a birefringent spherulitic texture and melts at 40 °C to produce a solvent-free liquid protein without loss of enzyme secondary structure. The nanoconjugate melt exhibits a birefringent dendritic texture below the conformation transition temperature (Tc) of glucose oxidase (58 °C) and retains interchain PEO−PEO ordering. Our results indicate that the shape anisotropy of the protein− polymer surfactant globular building block plays a key role in directing mesolamellar formation in the solvent-free solid and suggests that the microstructure observed in the solvent-free liquid protein below Tc is associated with restrictions in the intramolecular motions of the protein core of the nanoconjugate.
1. INTRODUCTION We have recently reported the first known examples of solventfree liquid proteins.1−4 These viscous biofluids are obtained by thermally induced melting of samples of lyophilized protein− polymer surfactant nanoconjugates that are prepared in water by electrostatic complexation of anionic polymer surfactants to cationized globular proteins such as ferritin,1 myoglobin,2 hemoglobin,3 or lysozyme.4 In each case, re-engineering of the protein surface results in the formation of a densely packed polymer surfactant shell, which extends the range of the attractive intermolecular force field between the nanoconjugates and enables the liquid state to be accessed close to room temperature. In contrast, freeze-dried powders of the native proteins do not melt but degrade on heating. Significantly, the protein−polymer surfactant melts often comprise fewer than 15 water molecules per protein, which is approximately an order of magnitude lower than that required to produce a hydration shell around the biomolecules. Given the virtual absence of water molecules, it is surprising that the proteins in these viscous biofluids adopt structures and dynamics close to their room temperature native states and retain key biological functions such as dioxygen binding.2 Moreover, the solvent-free proteins display hyperthermophilic stability with half denaturation temperatures as high as 150 °C,5 exhibit slow unfolding pathways such that reactive intermediates can be isolated,4 show redox transitions in a highly viscous environment,6 and exhibit refolding pathways in the absence of water.5 As a consequence, this new class of nanostructured bioliquids could have important applications for example in barrier dressings for wound healing and artificial skin or diffusion-dependent biosensing platforms.7−9 More generally, studies of melting in © XXXX American Chemical Society
dehydrated protein−polymer surfactant materials should help to extend our understanding of other types of solvent-free colloidal liquids based for example on polymer-coated inorganic particles.10−12 The above investigations have focused on solvent-free liquid proteins produced by polymer surfactant complexation to globular biomolecules that are essentially isotropic in shape and hence exhibit no liquid crystalline behavior. However, evidence of potential liquid crystalline (LC) ordering across a narrow temperature range (32−37 °C) was reported for a solvent-free liquid of ferritin, even though this heteromeric 24-mer protein has a spherical architecture.1 This was attributed to chemical anisotropy in the isometric hybrid nanoconstruct that was associated with a heterogeneous distribution of the surfaceattached polymer surfactant molecules, which in turn originated from the nonuniform arrangement of different polypeptide subunit types in the quaternary structure of ferritin. Given these preliminary observations on solvent-free ferritin, we decided to investigate in more detail the possibility for exploiting anisotropy in protein structure as the basis for inducing higher order structuration in solvent-free biohybrid materials. For this, we use a relatively small anisotropic protein, glucose oxidase (GOx), to circumvent difficulties associated with the structural complexity of ferritin. GOx from Aspergillus niger is a homodimeric globular protein (subunit molecular mass = 80 kDa) with an elliptical molecular shape and dimer dimensions of 7.5 × 6.0 × 5.2 nm (aspect ratio of ca. Received: July 28, 2014 Revised: September 7, 2014
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dx.doi.org/10.1021/jp507566u | J. Phys. Chem. B XXXX, XXX, XXX−XXX
The Journal of Physical Chemistry B
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1.44).13−15 The enzyme is glycosylated with acetylglucosamine and mannose residues (sugar content ∼18 wt %)16−18 and together with cofactor FAD catalyzes the oxidation of β-Dglucose to D-glucono-1,5-lactone, which subsequently undergoes nonenzymatic conversion to D-gluconic acid accompanied by reduction of GOx-FAD to GOx-FADH2. The enzyme is then regenerated often in association with the reduction of dioxygen to hydrogen peroxide. Interestingly, differential scanning calorimetry (DSC) experiments performed on dry powders of GOx have shown a first-order endothermic transition at temperatures between 50 and 60 °C, which was described as a glass transition temperature (Tg) arising from a change in the intramolecular dynamics of the polypeptide chain.19−22 Strictly speaking, Tg for small molecules or synthetic polymers describes a second-order transition associated with a change in the heat capacity of a material as an amorphous glass turns into a rubbery solid, and accordingly, this transition will be described herein as a conformation transition temperature (Tc). In this paper, we synthesize and characterize an anisotropic GOx−polymer surfactant nanoconjugate and employ a range of physical methods to determine the structure and phase behavior of this hybrid nanoscale object in both the solventfree liquid and solid state. Our results indicate that the shape anisotropy of the GOx−polymer surfactant globular building block plays an important role in directing mesolamellar formation in the solvent-free solid. We also show that birefringent microstructures are observed in the solvent-free GOx−polymer surfactant liquid below a critical transition temperature related to changes in the intramolecular dynamics of the dimeric protein. Overall, our observations suggest that anisotropy in protein structure can provide a rational basis for inducing higher order structuration in solvent-free biomaterials comprising discrete protein−polymer surfactant nanoscale objects.
pH 6.5 for 6 h with controlled additions of 0.2 M HCl. The resultant mixture was centrifuged, filtered (Millex Millipore, 0.22 μm), and dialyzed against sodium phosphate buffer (10 mM, pH 6.9) for 48 h to produce a stable solution of the DMPA-cationized enzyme (cGOx). The cationization efficiency was monitored using MALDI-TOF, which suggested that ca. 81% of the surface accessible glutamic and aspartic acid residues combined with DMPA (Supporting Information, Figure S1). Cationized GOx/polymer surfactant conjugates (S1-cGOx) were prepared by mixing 10 mL of aqueous cGOx (1.5 mg mL−1) with 42 mg of poly(ethylene oxide)-4-nonylphenyl 3sulfopropyl ether (S1). The solution was stirred for 24 h, any resulting precipitate removed by centrifugation, and the supernatant dialyzed (Visking dialysis tubing 12−14 000 Da MWCO) against sodium phosphate buffer (10 mM) for 24 h to remove unbound polymer surfactant molecules. The resulting aqueous S1-cGOx conjugate solution was then lyophilized for 48 h to produce a light yellow low-density powder, which melted to produce a yellow solvent-free viscous liquid after annealing at 60 °C and which thickened to form a soft solid at room temperature. Characterization. Dynamic light scattering (DLS) measurements of the aqueous protein dispersions were performed on a Malvern Zetasizer Nano-ZS at a protein concentration of ≈1 mg cm−3. Small-angle X-ray scattering (SAXS) experiments were performed using a Bede Microsource laboratory SAXS system operating at 40 kV and 1 mA (Cu Kα X-rays (λ = 1.54 Å)) with a wire array detector (detector distance, 0.90 m). The solvent-free S1-cGOx soft solid was placed between a 0.5 mm Teflon spacer that was mounted between two transparent poly(4,4′-oxidiphenylene pyromellitimide) (Kapton) films. This provided an accessible q-region of 0.03−0.6 Å−1. Collection times were typically in the region of 2 h for each sample, and data calibration was performed using a silver behenate standard.23 Wide-angle X-ray scattering (WAXS) experiments were performed using a commercial Philips X’Pert PRO diffractometer with a Cu Kα source (λ = 1.54 Å) operating at 40 kV and 30 mA. Measurements were made in the 2θ range of 2°−60°. Temperature-controlled measurements were undertaken using an Anton Paar environment with liquid nitrogen cooling. Temperature-dependent rheology measurements on the S1cGOx solvent-free soft solid or liquid were performed using a Bohlin stress-controlled rheometer (model C-VOR 200; Malvern Instruments Ltd., UK) with a torque range of 0.0005−100 mN m and frequency range of 10 μHz−100 Hz. The sample was spread between a stationary bottom plate and a 20 mm diameter cone with a tapering angle of 1°. The gap between the cone and plate was kept at 0.03 mm, and the temperature of the plates was controlled using an internal water circulation attachment connected to a Peltier water bath cooling system. Oscillatory frequency sweep experiments were conducted at 0.1% strain, after finding the linear region using a strain sweep test. DSC experiments were performed on the polymer surfactant S1, native GOx (lyophilized powder), and S1-GOx solvent-free soft solid/liquid using a DSC Q100 machine (TA Instruments, hermetic pan/lid). Samples were cycled through a temperature range of −70 to 70 °C. Thermogravimetric analyses (TGA; TA Q500 instrument) of native GOx (lyophilized powder) and S1cGOx solvent-free soft solids were undertaken at temperatures between 25 and 800 °C using a platinum plate.
2. EXPERIMENTAL SECTION Materials. Glucose oxidase (Aspergillus niger (G7141); type X-S, activity 100 000−250 000 units/g solid (without added oxygen)), N-(3-(dimethylamino)propyl)-N-ethylcarbodiimide hydrochloride (EDC; E7750), poly(ethylene oxide)-4-nonylphenyl 3-sulfopropyl ether potassium salt (S1; 473197), peroxidase from horseradish (HRP; P8250, type II, activity 150−250 units g−1, solid), and 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS; 11557) were purchased from Sigma-Aldrich. N,N′-Dimethyl-1,3-propanediamine (97%) (DMPA; A16901) and D-glucose (101174Y) were purchased from VWR International Ltd. Preparation of Solvent-Free Liquid Glucose Oxidase. Solvent-free liquids of GOx were prepared using a two-step sequential process involving cationization of the enzyme followed by electrostatic conjugation with an anionic polymer surfactant.1,2 Cationization of native GOx was undertaken via carbodiimide-mediated coupling of the surface accessible glutamic and aspartic acid residues to N,N′-dimethyl-1,3propanediamine (DMPA). In a typical procedure, 2 mL of a DMPA solution (2.45 M) solution was added dropwise with stirring to 5 mL of purified glucose oxidase (4 mg mL−1; sodium phosphate buffer (10 mM, pH 6.9); enzyme purification by centrifugation and dialysis (12−14 000 Da MWCO)), and the coupling reaction was initiated by the addition of 200 mg of 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDC). The reaction mixture was maintained at B
dx.doi.org/10.1021/jp507566u | J. Phys. Chem. B XXXX, XXX, XXX−XXX
The Journal of Physical Chemistry B
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Figure 1. Representative photographs of solvent-free S1−cGOx nanohybrid. (a) Low-density fluffy light yellow powder obtained after freeze-drying of solution of S1−cGOx. (b) After melting the freeze-dried powder in an oven at 60 °C for 30 min, showing viscous yellow solvent-free liquid. (c, d) Cooling of the solvent-free liquid below 40 °C produces a yellow soft solid that can be cast into self-supporting waxy monoliths and films. Samples shown are at room temperature.
Figure 2. (a) DSC profiles for polymer surfactant S1 (blue) and solvent-free hybrid S1−cGOx (red) showing different phase transitions. S1 exhibits an exothermic crystallization at −16 °C and an endothermic melting transition at 33 °C, whereas S1−cGOx shows a crystallization transition at 16 °C and a large melting transition at 40 °C followed by a small endothermic transition centered at 58 °C. (b) ATR-FTIR spectra of solvent-free S1− cGOx left for 60 h at 25 °C (blue), 44 °C (red), or 60 °C (green), showing absorptions of surfactant S1 at 1512 cm−1, amide II at 1544 cm−1, and amide I at 1655 cm−1.
of horseradish peroxidase (HRP). Stock solutions of sodium phosphate buffer (10 mM), enzyme (3.1 × 10−8 M), D-glucose (5−100 mM), ABTS (50 mM), and HRP (25 units mL−1) were prepared in deionized water. Concentrations of different GOx solutions were determined using the absorbance at 450 nm (ε = 18 600 M−1 cm−1). In a typical assay procedure, 200 μL of Dglucose and 100 μL of ABTS/HRP were mixed with 1.6 mL of sodium phosphate buffer solution followed by addition of 100 μL of enzyme (for a total volume of 2 mL) to initiate the reaction. The initial rate of reaction was obtained by measuring the increase in the absorbance of the product (ABTS•+) at 414 nm (ε = 36 000 M−1 cm−1) using UV−vis spectroscopy over 2 min. Each assay was performed three times, and the average initial rate values were used to determine the catalytic turnover number (kcat) and Michaelis constant (Km) using the Michaelis−Menten equation.
Optical properties of native GOx (lyophilized powder) and S1-cGOx solvent-free soft solids/liquids were observed under crossed and parallel polarizers (OLYMPUS BX50 optical microscope, 10× and 20× objective lenses). The samples were sandwiched between a glass slide and coverslip and sealed in a Linkam THMS 600 heating stage for temperaturedependent optical experiments undertaken between 20 and 65 °C (Linkam TP92/LNP 2 heating/cooling system). Liquid nitrogen was used to ensure a constant cooling rate. Images were taken using an OLYMPUS C-5060 camera. ATR-FTIR spectroscopic measurements were performed on a PerkinElmer Spectrum One FTIR spectrometer fitted with a universal attenuated total reflectance (ATR) accessory. Measurements were performed on S1-cGOx solvent-free soft solids/liquids prepared after annealing a sample for 60 h at 25, 44, or 60 °C. In each case, measurements were taken within a period of 30 s after removal from the oven. Synchrotron radiation circular dichroism (SRCD) spectroscopy was performed at Diamond Light Source (beamline B23) on a solvent-free soft solid that was cast as a thin film between two quartz plates. Enzyme kinetic assays were performed on aqueous solutions of native GOx, cGOx, and S1-cGOx conjugates by catalytic oxidation of D-glucose at 25 °C. Formation of hydrogen peroxide was monitored by 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) in the presence
3. RESULTS AND DISCUSSION Enzyme/polymer surfactant nanoconjugates were prepared in phosphate buffer by cationization of glucose oxidase (GOx; Supporting Information, Figure S2a) followed by electrostatic conjugation with poly(ethylene oxide)-4-nonylphenyl 3-sulfopropyl ether (S1). Conjugation was confirmed by zeta potential and dynamic light scattering (DLS) measurements on aqueous solutions of native GOx, cationized GOx (cGOx), and the S1− cGOx conjugate, which showed respectively surface potentials C
dx.doi.org/10.1021/jp507566u | J. Phys. Chem. B XXXX, XXX, XXX−XXX
The Journal of Physical Chemistry B
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Figure 3. SAXS profiles for solvent-free S1−cGOx at different temperatures ranging from 30 to 60 °C. (a) Effect of heating the sample annealed at 30 °C for 2 h. The arrows indicate the presence of Bragg peaks corresponding to a mesolamellar soft solid which persists until 35 °C but becomes disrupted at 40 °C. Further heating leads to formation of an isotropic liquid at 60 °C. (b) Cooling curves at different temperatures showing similar trend as seen in (a) with the appearance of an ordered mesostructure at 35 °C. Formation of the lamellar microstructure is indicated by the arrows. Inset shows changes in low q forward scattering as the sample temperature is reduced from 60 to 43 °C and then annealed at 43 °C for 24 h. Data in (a) and (b) have been vertically shifted for clarity of presentation. The low q scattering shown in the inset of (b) is from unscaled data.
of −10.7, +33.2, and +15 mV and mean hydrodynamic diameters of 7.6 ± 0.8, 8.1 ± 1.1, and 8.9 ± 1.6 nm (Supporting Information, Figure S2b). The observed increase in size was consistent with the coupling of a polymer− surfactant shell around the cationized protein core. Comparison of the ratios of the protein amide II (∼1540 cm−1) and polymer surfactant C−C phenyl ring stretching absorbances (∼1512 cm−1) in FTIR spectra of solvent-free solid S1−cGOx with calibration curves generated from formulations of cGOx and S1 with known compositions gave a S1:GOx dimer ratio of 1.2:1 (Supporting Information, Figure S3). Enzyme kinetic spectrophotometric assays performed on aqueous solutions of native GOx, cGOx, and S1−cGOx conjugates gave similar turnover numbers (kcat) (Supporting Information, Figure S4), indicating that there was minimal effect on the catalytic activity of the enzyme on cationization and subsequent conjugation with the polymer surfactant. However, the Michaelis−Menten constants (Km) for the aqueous enzymes increased in the following order: native GOx (30 ± 1 mM) < cGOx (34 ± 4 mM) < S1−cGOx conjugate (37 ± 5 mM), suggesting a small reduction in the substrate binding affinity possibly via mass transfer resistance after cationization and polymer surfactant complexation. As the focus of our work was based on exploiting the structural anisotropy of the dimeric protein rather than an investigation of solvent-free enzymatic activity, the catalytic efficiencies of the S1−cGOx solvent-free solid or liquid states were not determined. Lyophilization of an aqueous solution of S1−cGOx for 48 h produced a light yellow low-density powder (Figure 1a) with a water content of
