Article pubs.acs.org/JPCB
Conformation and Activity of Glucose Oxidase on Homogeneously Coated and Nanostructured Surfaces A. Seehuber and R. Dahint* Applied Physical Chemistry, University of Heidelberg, Im Neuenheimer Feld 253, 69120 Heidelberg, Germany S Supporting Information *
ABSTRACT: Protein unfolding and loss of protein function upon surface contact is a major problem in biotechnology and biomedicine. Using glucose oxidase (GOx) as a model protein, we investigated the impact of surface chemistry, topography, and confinement on enzyme activity, conformation, and affinity. A particular focus lay on the question whether the conformation of surface-bound proteins can be stabilized by embedding nanoscale adsorption sites, here in the form of monodisperse gold nanoparticles (AuNPs), into a protein-repelling matrix material. It was found that on homogeneous surfaces, GOx activity is generally lower than that in its native state and strongly affected by surface chemistry. Loss of activity is related to an increasing amount of β-sheets in the GOx secondary structure and a corresponding reduction of α-helical elements. In contrast, on AuNP surfaces, the effect of surface chemistry is negligible, and the amount of adsorbed protein only depends on particle size. The low activity of GOx on all nanostructures studied is again accompanied by an increase of β-sheet and a reduction of α-helical secondary structure. The major cause for protein unfolding on AuNPs thus seems to be the curvature of the surface. In addition, the data suggest that unfavorable orientation of the adsorbed enzyme also contributes to the loss of activity.
1. INTRODUCTION Surface-bound biomolecules have gained growing attention in the field of biotechnology and biomedicine. Important examples include heterogeneous immunoassays1 and biosensors2,3 for the specific detection of dissolved antigens, DNA and peptide chips for genome analysis4,5 and the analysis of enzymatic pathways,6 the replication of cell membranes by immobilized lipid films,7 or the control of cell adsorption onto surfaces by adhesion proteins.8 Moreover, nonspecifically adsorbed proteins significantly affect the biocompatibility of implants.9 Particular attention is also paid to immobilized enzymes as they are widely used in the industrial synthesis of drugs and food additives.10,11 By attaching them to a solid support, they are easier to handle, and thermal stability or solvent resistance is frequently enhanced.12 However, upon immobilization, those enzymes very often suffer from a significant loss in activity compared to the native enzyme in solution due to unfolding processes. In order to provide the desired function of biomoleculederivatized surfaces, not only the amount and type of adsorbed biomolecules is crucially important but also their molecular © XXXX American Chemical Society
conformation. For this reason, the adsorption behavior of proteins to artificial surfaces and its impact on protein structure have been major topics of research for more than 30 years, with continuously increasing importance in the fields of biotechnology, biomedicine, and process control. While already at an early stage it could be proven that protein interaction with hydrophilic materials is weaker than that with hydrophobic substrates,13,14 subsequent studies addressed more complex surface properties including topography. For example, in the case of protein adsorption onto phase-separated mixed films, a significant impact of surface nanostructures on the protein layer was found when the lateral dimensions of the structure corresponded to the molecular length scale of the adsorbed molecules.15 Rechendorff et. al studied the absorption behavior of fibrinogen on corrugated tantalum films depending on the root-mean-square (rms) roughness.16 They found higher protein adsorption for higher rms values and attributed this Received: February 23, 2013 Revised: May 15, 2013
A
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1-yl)undecane-1-thiol (PyUDT) as an example for a coating with aromatic end groups. In order to elucidate the conformational changes that GOx undergoes upon adsorption, we employed in situ attenuated total reflection infrared (ATR-IR) spectroscopy. The most useful spectral feature to determine protein secondary structure is the amide I band that shows its peak maximum at around 1650 cm−1 and is mainly due to the peptide bond’s CO stretching vibration. Upon application of Fourier selfdeconvolution (FSD), the amide I band shows a distinct fine structure that can be assigned to the particular protein secondary structure elements α-helix, β-sheet, turns, and bends as well as random coiled fractions.29,30 The changing percentages of the secondary structure elements α-helix and βsheet were correlated to the enzyme’s activity, which was photometrically determined by measuring its reaction rate as a function of substrate concentration according to Michaelis− Menten theory. To convert such obtained values to the more meaningful specific enzyme activity, the absolute amounts of surface-bound GOx were gathered from the results of an enzyme-linked immunosorbent assay (ELISA). For comparison, we also characterized the native enzyme in solution, as well as GOx adsorbates on homogeneously coated surfaces, in terms of activity, secondary structure, and mass density. To coat the nonstructured surfaces, we used the same thiols as those for functionalizing the AuNPs.
effect to a change in the geometrical arrangement of the surface-bound proteins. Nowadays, such topographic studies have been extended to the interaction between proteins and nanoparticles as the utilization of nanoparticles is no longer restricted to materials sciences and biosensing but has found its way to in vivo applications in nanomedicine.17,18 Polymeric and magnetic nanoparticles may be used for drug delivery and cell imaging.19,20 Local heating effects induced by plasmon excitation in gold nanoparticles (AuNPs) have even been shown to facilitate the photothermal therapy of cancer.21 However, unfolding of nanoparticle-adsorbed proteins may trigger defense actions of the body and cause hazardous effects. It is, therefore, of high relevance to investigate the impact of surface structure, topography, and chemistry on the adsorption behavior of biological matter and to elucidate the underlying structure−function relationship. For instance, Lundqvist et al.22 as well as Vertegel et al.23 investigated the behavior of proteins adsorbed onto SiO2 nanoparticles and observed a correlation between protein conformation and the size of the nanoparticles; the smaller the particles, the more of the protein’s native structure that could be retained. On the other hand, some studies prove misfolding and aggregation of proteins promoted by the interaction with nanoparticles.24,25 According to above results, one may speculate that it should be possible to precisely control the structure of protein adsorbates if the lateral dimensions of nanoscaled adsorption sites are chosen carefully. If, in addition, those adsorption sites are embedded into a protein-resistant matrix material, an unfolding of the biomolecules beyond the borders of the adsorption sites should be prevented. Following this concept, the adsorption behavior of single protein molecules and potential rearrangements of their structure can be investigated as a function of the size of the adsorption sites. Moreover, the preparation of heterogeneous nanopatterns on solid substrates offers a comparatively simple way to further modify the chemical character of the adsorption sites. For example, a gold pattern on a silicon support can be selectively functionalized via the self-assembly of thiolates, whereas silicon specifically reacts with silanes or positively charged polyelectrolytes. In our study, we adsorbed submonolayers of AuNPs onto positively charged surface coatings on silicon supports and embedded them into a protein-repelling matrix of poly(acrylic acid)−poly(ethylene glycol) (PAA-PEG2000).26 Those nanoparticle arrays served as nanoscale adsorption sites for the specific deposition of a designated model protein, glucose oxidase (GOx). GOx, extracted from Aspergillus niger, is a 131 kDa homodimeric enzyme whereupon each subunit has a size of about 7 × 7 × 22 nm.27 It catalyzes the oxidation of β-Dglucose and is of great industrial interest as it is used as a preservative in food and particularly as a biosensor for glucose on test strips for Diabetes patients.28 The conformation and enzymatic activity of adsorbed GOx were investigated as a function of the AuNP’s diameter and surface properties. To mimic a broad spectrum of possible surface-chemical properties, we coated the AuNPs with the following functionalized thiols prior to protein adsorption: (i) 11-amino-1-undecanethiol (AUDT), which is supposed to render the substrate surface positively charged at neutral pH, (ii) 12-mercaptododecanoic acid (MDDA) to introduce negatively charged moieties, (iii) 1-dodecanethiol (DDT) as a representative of an uncharged and hydrophobic species, and (iv) 11-(1H-pyrol-
2. EXPERIMENTAL METHODS 2.1. Materials. 1-Dodecanethiol (DDT), AUDT, 11-(1HPyrol-1-yl)undecan-1-thiol (PyUDT), MDDA, poly(allylamine hydrochloride) (PAH, MW ≈ 15000 g/mol), gold(III)chloride trihydrate, tannic acid, 2,2′-azino-di(3-ethylbenzothiazoline)-6sulfonic acid diammonium salt (ABTS), phosphate buffered saline (PBS), bovine serum albumin (BSA; ≥98% purity), GOx (type II-S, lyophilized powder, 15−50 kU/g), and horseradish peroxidase (HRPO; type II, lyophilized powder, salt free, 150− 250 kU/g) were purchased from Sigma-Aldrich (Steinheim, Germany). Potassium phthalate buffer solution (pH 4.0) was obtained from VWR International (Fontenau-sous-Bois, France), NaCl and 1 M NaOH solution were from Baker (Deventer, Netherlands), trisodiumcitrate dihydrate and 96% acetic acid were from Merck (Darmstadt, Germany), D2O was from Deutero (Karlsruhe, Germany), 38% deuteriumchloride solution, 30% sodiumdeuteriumoxide solution, and 96% acetic acid D4 were from Carl Roth (Karlsruhe, Germany), and 30% hydrogen peroxide solution was from AppliChem (Darmstadt, Germany). Rabbit anti-GOx Ig (0.8 mg/mL, polyclonal) was purchased from Thermo Scientific (Rockford, U.K.), and goat anti-rabbit IgG conjugated with horseradish peroxidase (HRPO-AB; 0.8 mg/mL, polyclonal) was from Jackson Immuno Research (West Grove, U.S.A.). The synthesis of PAA-PEG2000 can be gathered from earlier published work,26 and AuNPs were prepared according to the citrate reduction method described in the literature.31,32 All chemicals were used without further purification. One-side optically polished silicon (100) wafers were purchased from Silicon Materials (Landsberg am Lech, Germany), and hemispherical silicon (100) ATR crystals with a diameter of 25.4 mm were from NewEra Enterprises (Vineland, U.S.A.). Deionized water was purified with a Milli-Q plus system by Millipore (Eschborn, Germany). 2.2. Sample Preparation. Gold-coated substrates were prepared by evaporating at first a 5 nm layer of titanium as an adhesion promoter and subsequently a 100 nm layer of gold B
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onto silicon wafers at a pressure of approximately 10−7 mbar. All substrates were cut into pieces of about 2 × 2 cm2 prior to sample preparation and treated with ozone for 1 h to remove any adsorbed organic compounds. Ozone was generated by the irradiation of air with a mercury vapor lamp (NIQ 40/18 from Heraeus, Hanau, Germany) at λ = 254 nm and 10 W. 2.2.1. Preparation of Thiol Films. DDT, PyUDT, AUDT, and MDDA were dissolved in pure ethanol at a concentration of 1 mM. Freshly cleaned gold-coated silicon substrates were kept in those solutions overnight, carefully rinsed with ethanol afterward, and blown dry with nitrogen. Submonolayers of AuNPs (see below) were kept in thiol solutions for only 1 h to avoid a possible lift-off of the particles. 2.2.2. Preparation of Polyelectrolyte Films. PAH and PAAPEG2000 were dissolved in water at a concentration of 1 mg/ mL. The 1:1 mixtures of 1 aliquot of the accordant stock solution and potassium phthalate buffer were deposited on the substrates and remained there overnight at room temperature. After formation of the respective polyelectrolyte layer, the substrates were carefully rinsed with water and blown dry with nitrogen. 2.2.3. Preparation of AuNP Submonolayers. AuNP solutions were deposited onto PAH-coated silicon substrates overnight at room temperature to adsorb the particles. Subsequently, the samples were thoroughly rinsed with water and blown dry with nitrogen. Afterward, the substrates were incubated with PAA-PEG2000 to embed the AuNPs into a protein-resistant matrix. The AuNPs were coated with thiols prior to protein adsorption. 2.2.4. Adsorption of GOx. GOx was dissolved in 100 mM acetate/100 mM NaCl buffer at pH 5.5 and a concentration of 1 mg/mL. Next, the solution was filtrated using a syringe filter (cellulose membrane) with a pore size of 0.2 μm to remove possible aggregates and undissolved matter. Each substrate was put into a small plastic Petri dish and covered with acetate buffer for approximately 5 min. An equal amount of protein solution was added and allowed to settle on the substrates for 1 h at room temperature. Subsequently, each Petri dish was flushed with a huge amount of water to avoid a Langmuir− Blodgett-like transfer of proteins at the air−water interface when the samples were removed. The samples were then washed 3 × 10 min in PBS on a shaker, rinsed again with water, and kept under acetate buffer in the refrigerator until they were used. 2.3. Characterization of the Films and Protein Adsorbates. 2.3.1. FT-IR Spectroscopy. Infrared spectra were recorded on a Vertex 70 FT-IR spectrometer (Bruker Optics, Ettlingen, Germany) at a resolution of 4 cm−1 in the range of 800−4000 cm−1. The spectrometer is equipped with a liquid-nitrogen-cooled mercury cadmium telluride (MCT) detector, a variable angle reflection accessory (type A 513, Bruker Optics, Ettlingen, Germany) set to 45° for ATR-IR spectroscopy, and a homemade flow cell with an internal volume of about 0.3 mL for in situ measurements. In order to reduce absorption of water vapor in the spectral ranges of 3000−3500 and 1300−1800 cm−1, the sample compartment was continuously purged with dry air. To ensure similar conditions inside of the sample compartment, each measurement was started with a delay of 30 min after mounting the sample. All spectra were recorded by accumulating 500 single scans. As the H2O bending vibration fully overlaps with the amide I band, we used D2O as a solvent for all IR measurements.
Prior to each sample spectrum, we recorded a reference spectrum with only acetate buffer solution in the flow cell. Following the reference single-channel spectrum, we recorded an absorbance spectrum of the reference without opening the sample compartment. Therewith, we obtained a bare spectrum of the remaining water vapor in the sample compartment, which was used to manually subtract residual water vapor bands from all sample spectra. Next, the GOx solution was injected by means of a syringe. After an adsorption time of 1 h at room temperature, the flow cell was flushed with 40 mL of PBS and subsequently 10 mL of D2O to remove all of the protein solution. Finally, the D2O was replaced by acetate buffer, and an absorption spectrum of the adsorbed GOx was recorded. After water band subtraction, all spectra were baseline corrected and slightly smoothed (5−9 points adjacent averaging) if necessary. To determine protein secondary structure, the spectra were deconvoluted in the range of 1600−1700 cm−1 assuming a full width at half-maximum of 13 cm−1 for the original, unresolved components. The relative areas of the individual components of the deconvoluted spectra were resolved by an iterative curve fitting procedure that assumed Gaussian band envelopes for the deconvoluted components. 2.3.2. Scanning Electron Microscopy. To evaluate the quality of AuNP submonolayers, images were taken on a Leo 1530 scanning electron microscope (SEM; Zeiss, Jena, Germany) at an accelerating voltage of 5 kV. 2.3.3. ELISA Quantification of GOx Adsorbates. Absolute amounts of adsorbed GOx were quantified by means of an ELISA. Briefly, we used a primary antibody (anti-GOx) specific for GOx and a secondary antibody that was specific for antiGOx and conjugated with HRPO (HRPO-AB). The amount of adsorbed GOx was then indirectly measured by monitoring the enzymatic conversion of H2O2 in the presence of an oxidationsensitive dye (ABTS). The detailed procedure is provided in the Supporting Information. 2.3.4. Determination of the Enzymatic Activity of GOx. Because the enzymatic conversion of glucose by GOx is not directly observable by means of photometry, the reaction has to be coupled to the conversion of H2O2 in the presence of ABTS, which is catalyzed by HRPO. The catalytic activity of GOx was assayed at 15 different substrate concentrations ranging from 1 to 500 mM of glucose. The obtained data were evaluated according to the Michaelis−Menten theory, which yields the catalytic activity A and the Michaelis−Menten constant KM of GOx. Note that the latter value is inversely proportional to the affinity of GOx to its substrate. Specific catalytic activities are obtained if the absolute amount of enzyme involved is taken into account. The detailed procedure is given in the Supporting Information.
3. RESULTS AND DISCUSSION 3.1. Enzymatic Activity of Native GOx in Solution. Provided that O2 acts as the electron-accepting species, GOx exhibits its optimum enzymatic activity at about pH 5.5.28 At this pH, we assayed the catalytic activity of GOx at five different enzyme concentrations in the range of 10−1000 ng/mL. The detailed results for the determined specific activities A as well as the Michaelis−Menten constants KM are provided in the Supporting Information. Surprisingly, the enzymatic activity drops from 216 ± 3 to 100 ± 5 U/mg with increasing enzyme concentration. This behavior might be ascribed to the formation of protein C
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1011 molecules/cm2) of surface-bound GOx. Presuming that the average adsorption area occupied by one GOx molecule in its native conformations is roughly 195 nm2,27 and considering a maximum surface coverage of 54.7% for a random sequential protein adsorption process,33 full monolayer coverage should correspond to approximately 61 ng/cm2 (2.8 × 1011 molecules/ cm2). Hence, GOx adsorbs to less than a monolayer on DDT and MDDA, while we observe slight multilayer formation on AUDT and PyUDT. For comparison, nonspecific adsorption is limited to 3−6 ng/cm2. The latter values have been subtracted from the overall response to obtain the actual mass density of surface-bound GOx. 3.2.2. Enzymatic Activity. The determined specific enzymatic activities of all GOx adsorbates on homogeneously coated surfaces are summarized in Table 1. Compared to the native enzyme in solution at c(GOx) = 100 ng/mL (A = 141 ± 3 U/mg), the enzymatic activity of adsorbed GOx was preserved by 60% on the positively charged AUDT coating. On the uncharged hydrophobic DDT-coated surface, the activity dropped to 49%, on the negatively charged MDDA surface, it dropped to 35%, and on the aromatic PyUDT surface, it dropped to only 9% of the reference value. As the enzyme is slightly negatively charged at pH 5.5, one may at first glance expect that only moderate changes in the proteins’ conformation are necessary to form attractive interactions with a positively charged surface, so that most of the enzyme’s activity should be preserved. Yet, GOx exhibits only 66 (10%) acidic amino acids per subunit. Therefore, the conformational deformation of GOx on AUDT still has to be rather pronounced to establish strong interactions between the COOH moieties of the acidic amino acids and the NH2 groups of the surface coating. As a result, the enzyme looses 40% of its native catalytic activity. If GOx is kept under physiological conditions, that is to say, in aqueous solution, hydrophobic side chains will be located in its interior to avoid unfavorable contact with water molecules. Consequently, in the case of hydrophobic interactions, the redirection of hydrophobic residues toward the substrate surface must come along with a significant structural reorientation within the protein molecule. On the other hand, GOx exhibits 238 (37%) hydrophobic amino acids per subunit. This may explain why the catalytic activity drops by only 11% more on DDT than that in the case of GOx on AUDT. Finally, each GOx subunit consists of only 56 (9%) basic amino acids, which will result in an even more distinctive perturbation of the protein’s conformation if they are directed toward the negatively charged MDDA surface. Indeed, the measured enzymatic activity on MDDA decreases to only 35% of the reference value.
aggregates in the high-concentration regime. Consequently, those aggregated enzymes would not be accessible for the substrate anymore. On the other hand, KM likewise drops from 22 ± 2 to 8 ± 4 mM with increasing enzyme concentration, indicating that the substrate affinity of GOx enhances at high enzyme concentrations. We, therefore, assume that the protein molecules stabilize themselves in their native conformation at elevated enzyme concentrations, whereas at low concentrations, partially unfolded enzyme molecules contribute to the catalytic reaction. 3.2. GOx on Homogeneously Coated Surfaces. 3.2.1. Quantification. To obtain reference values for the enzymatic activity and secondary structure of adsorbed GOx on nonstructured surfaces, four different homogeneous thiolate coatings on gold were prepared, and the enzyme was adsorbed to these surfaces. In order to calculate the specific activity of the adsorbed GOx, the absolute amount of surface-bound enzyme must be determined. We, thus, measured the coverage of GOx on each surface coating by ELISA experiments. To evaluate the potential contribution of nonspecifically adsorbed antibodies to the overall response, we prepared in each case reference samples that were initially incubated with BSA instead of GOx. A detailed description of the procedure and a brief consideration of potential sources of error in the ELISA quantification can be gathered from the Supporting Information. According to Figure 1, the least amount of GOx, which is 51 ± 5 ng/cm2 ([2.4 ± 0.2] × 1011 molecules/cm2), can be found
Figure 1. ELISAs were performed to determine the absolute amounts of adsorbed GOx. To account for nonspecifically adsorbed antibodies, control samples with BSA (hatched white columns) were prepared and compared to the GOx-coated samples (gray columns).
on DDT, and with 56 ± 3 ng/cm2 ([2.6 ± 0.1] × 1011 molecules/cm2), only slightly more enzyme adsorbs to MDDA. On AUDT and PyUDT, we quantified 81 ± 8 ng/cm2 ([3.7 ± 0.4] × 1011 molecules/cm2) and 83 ± 3 ng/cm2 ([3.8 ± 0.1] ×
Table 1. Specific Activity of GOx on Homogeneously Coated and on AuNP-Structured Surfaces homogeneous surface
nanostructured surface
KM [mM]
A [U/mg]
n/a
n/a
A [U/mg] dAuNP
coating AUDT DDT MDDA PyUDT
12 19 18 8
± ± ± ±
4 2 6 2
85 69 49 12
± ± ± ±
11 ± 2 nm 9 7 3 1
12 13 14 20
± ± ± ± D
1 1 5 1
20 ± 3 nm 13 22 17 30
± ± ± ±
4 3 3 2
33 ± 4 nm 35 17 12 39
± ± ± ±
4 1 3 6
40 ± 5 nm 17 14 16 18
± ± ± ±
1 2 2 3
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Figure 2. Deconvolved amide I bands of GOx adsorbed on (a) AUDT, (b) DDT, (c) MDDA, and (d) PyUDT. The peak below 1620 cm−1 was attributed to aromatic side chains and, therefore, not included in the quantification of secondary structure elements.
Table 2. Secondary Structure of GOx in Solution (Native) and on Homogeneously Coated Surfaces α-helix native Coating AUDT DDT MDDA PyUDT
β-sheet
turns and bends
random coil
[%]
[cm−1]
[%]
[cm−1]
[%]
[cm−1]
[%]
[cm−1]
37
1654
21
1627, 1678
14
1669, 1693
28
1640
26 25 23 23
1655 1653 1653 1655
28 29 33 34
1628, 1630, 1632, 1630,
18 21 16 18
1665, 1663, 1665, 1665,
28 25 24 25
1642 1642 1643 1644
1677 1678 1679 1678
1691 1692 1696 1694
Figure 3. Correlation between enzymatic activity and quantitative fractions of the main secondary structure elements, the (a) α-helix and (b) βsheet, for GOx adsorbates on homogeneously coated surfaces.
structure is in general known to be stabilized by π−π interactions between opposing aromatic amino acid side chains.34 If the interaction between those amino acid pairs is
The enzyme’s behavior on the PyUDT surface is affected not only by the hydrophobic properties of this particular coating but especially by its aromatic character. Protein tertiary E
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broken up to establish π−π interactions with the aromatic moieties of the PyUDT surface coating, a dramatic loss in enzymatic activity due to a massive structural alteration is very likely. In fact, only 9% of the activity of the native protein is preserved on PyUDT. Surprisingly, the substrate affinity of the adsorbed GOx is best on PyUDT where its catalytic activity is worst. With 8 mM, the value of KM is even lower than that for GOx in solution at c = 100 ng/mL where we determined a KM of 19 ± 2 mM. Considering the slight multilayer formation of GOx on PyUDT, we assume that in this case only enzymes in the upper layer, which are not in direct contact with the surface coating, remain intact and, therefore, account for the value of KM. On AUDT, the substrate affinity (KM = 12 mM) is slightly worse than that on PyUDT but still similar to what has been determined in solution. Also on DDT and MDDA, where the enzymatic activities were significantly reduced, the values of KM (19 and 18 mM) are comparable to those found for the native enzyme in solution at moderate concentrations. 3.2.3. Secondary Structure. Deconvolved spectra of adsorbed GOx are shown in Figure 2. The quantitative fractions of secondary structure elements of native and adsorbed GOx as extracted from the IR amide I band can be seen from Table 2. The error of all percentages is about ±1− 3% (cf. Figure 3), and the deviation in wavenumbers is about ±2 cm−1. For the native enzyme in solution, we determined 37% αhelical structures and 21% β-sheets, which is in good agreement with the results of X-ray analysis by Hecht et al.35 Compared to the native structure, the fraction of α-helices decreased for all GOx adsorbates while at the same time the fraction of β-sheets increased. The evaluation of the secondary structure of GOx confirms the conclusions drawn from the measurements of enzymatic activity; a lower activity correlates with a more pronounced change in protein conformation. Hereby, the almost linear drop of activity with an increasing amount of βsheets is even more evident than the correlation between the breakup of α-helices and the declining enzymatic activity, as visualized in Figure 3. With 28%, the least amount of β-sheets can be found on the positively charged AUDT surface, while their fraction slightly increases to 29% on DDT and finally ends up at 33% on MDDA and even 34% on PyUDT. In order to elucidate the correlation between structure and activity in more detail, it is important to know the distribution of secondary structure elements within the GOx molecule. Figure 4 illustrates the crystal structure of the native enzyme and shows that the binding pocket for the substrate is mainly made up by random coiled structures and turns and bends as well as some β-sheet strands. However, in the domain where both subunits are connected via a number of carbohydrates, one can predominantly find α-helical structures. Therefore, one may assume that a partial breakup of those α-helices will severely disturb the interaction between both subunits of the enzyme. Akhtar et al. were able to show that the dimeric structure of GOx is destroyed in highly concentrated solutions of divalent cations.36 Hereby, the enzyme loses its catalytic activity almost completely. They assume that the flavin adenine dinucleotide (FAD) cofactor loses its anchoring in the monomeric enzyme and may even dissolve away from it. Hence, if the interaction of both GOx subunits is perturbed by conformational changes, the enzymatic activity will decline. The preferential formation of β-sheets as a result of adsorption to negatively charged and aromatic surfaces may
Figure 4. Secondary structure of one subunit of GOx:27,35 dark red, αhelices; dark green, β-sheets; light red, random coil; light green, turns and bends; blue, carbohydrates; and black, FAD. The binding pocket is located in the FAD region.
be explained by the fact that in a β-sheet, the side chain of every second peptide group faces the same direction, while a complete turn of an α-helix is comprised of 3.6 amino acids.37 Thus, a β-sheet structure enables more contact points between amino acid side chains and the moieties of the accordant surface coating. As GOx exhibits only a few positively charged amino acids (see section 3.2.2) that can establish attractive interactions with negatively charged surfaces, the βsheet conformation is particularly advantageous. The formation of π−π interaction between aromatic amino acids and the pyrrol rings of the PyUDT coating requires a certain alignment of the amino acid side chains, which again seems to favor a structural transition toward β-sheets. 3.3. GOx on Nanostructured Surfaces. 3.3.1. Quantification. SEM images of the AuNP monolayers onto which GOx was adsorbed are shown in Figure 5. An upper limit of 40 nm in diameter was selected as no sufficiently narrow size
Figure 5. AuNPs of different diameter, (a) 11 ± 2, (b) 20 ± 3, (c) 33 ± 4, and (d) 40 ± 5 nm, adsorbed to PAH-coated silicon substrates. F
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Table 3. Calculated Area of Adsorption Available on AuNP-Structured Surfaces and Maximum Mass Density of Adsorbed GOxa 2r [nm] Aad [nm2] scAuNP [%] AuNP/cm2 × 10−10 nmax(GOx)/AuNP mmax(GOx)/cm2 [ng] nmax(GOx)/Aad × 10−11 [cm−2] a
11 294 22 23 1 50 3.4
± ± ± ± ± ± ±
2 53 2 5 0 10 0.6
20 1100 35 11 6 143 5.5
± ± ± ± ± ± ±
3 165 1 2 1 35 1.2
33 3162 35 4 16 139 5.1
± ± ± ± ± ± ±
4 383 1 0.5 2 25 0.9
40 4712 31 2.5 24 131 5.1
± ± ± ± ± ± ±
5 589 1 0.5 3 31 0.9
Note that the calculated values do not include the factor of 0.547 for random sequential protein adsorption.
Figure 6. ELISA quantification of GOx adsorbed to thiol-coated AuNP submonolayers: (a) absolute amounts of adsorbed GOx, and (b) number of GOx molecules per particle as a function of the particle diameter 2r.
Essentially, there are three possible explanations for this result: (1) The AuNPs affect the redox behavior of the adsorbed GOx. In this case, molecular O2 could act as a redox mediator to accomplish the electron transfer between the catalytically active FAD cofactor and the AuNPs. Yet, this scenario would require a very short distance between the FAD and the particle surface (
