Interaction of the Physiological Tripeptide Glutathione with Colloidal

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Interaction of the Physiological Tripeptide Glutathione with Colloidal Alumina Particles Maike M. Schmidt,†,‡,§ Yvonne Koehler,†,‡ Ludmilla Derr,§ Laura Treccani,§ Kurosch Rezwan,§ and Ralf Dringen*,†,‡ †

Center for Biomolecular Interactions Bremen, University of Bremen, PO Box 330440, D-28334 Bremen, Germany Center for Environmental Research and Sustainable Technology, University of Bremen, PO Box 330440, D-28334 Bremen, Germany § Faculty of Production Engineering, Advanced Ceramics, University of Bremen, Am Biologischen Garten 2, D-28359 Bremen, Germany ‡

S Supporting Information *

ABSTRACT: Understanding of the molecular interactions of alumina particles with biomolecules is fundamental for a variety of biotechnological processes. To study the interactions of polypeptides with alumina particles, we have investigated the adsorption and desorption behavior of the physiologically relevant tripeptide glutathione (GSH, γ-glutamylcysteinylglycine) onto colloidal α-alumina particles (CPs). The adsorption of GSH to positively charged alumina particles was rapid, increased proportionally to the concentration of CPs, and shifted the isoelectric point of the CP to a less alcaline pH. Desorption of particle-bound GSH was achieved by increasing the ionic strength after adding salt to the suspension, suggesting that adsorption of GSH to alumina is governed by electrostatic interactions. The presence of negatively charged and GSHstructurally related molecules such as glutamate, γ-glutamylcysteine, γ-glutamylglutamate, or methyl-S-GSH prevented the binding of GSH to the positively charged alumina surface in a concentration dependent manner, while positively charged and net-uncharged molecules and GSH esters did not affect GSH adsorption to alumina CPs. These data suggest that exclusively electrostatic interaction via the carboxylate groups of GSH governs its binding to alumina particles.

1. INTRODUCTION The adsorption and interaction of amino acids, peptides, and proteins onto solid metal oxide surfaces are fundamental phenomena that are highly relevant to technological fields such as biomedicine, biotechnology, materials chemistry, engineering, and food manufacturing. The understanding of the mechanistic principles of protein adsorption is a key to the design of biomedical1 and biotechnological applications2,3 and is also required for a better risk assessment of man-made nanomaterials.4,5 Because of its outstanding chemical, mechanical,6 and thermal stability,7 alumina-based materials (such as colloidal particles, porous membranes, and coatings) are used and considered for several life science applications such as biocatalysis, purification applications, and drug delivery systems8−15 as well as for technological applications.2,16 Because of the relevance of the protein−surface interaction in all these fields, a number of experimental and theoretical studies have aimed to characterize the protein adsorption behavior.17−21 However, since proteins are highly heterogeneous in composition and structure and since their properties change strongly by modulation of the protein environment,20,22 the models discussed to describe the mechanistic principles of protein−surface interaction differ significantly or are even © 2012 American Chemical Society

contradictory. To lower this complexity, small peptides have been used as model systems to study the interaction of proteins with alumina surfaces.19 Work on the adsorption of peptides to metal oxide surfaces has been focused predominantly on silica (SiO2) and titania (TiO2) surfaces,19,23−29 while only little is known so far on the adsorption of peptides to alumina surfaces. Peptides have been reported to interact by electrostatic interactions between basic amino acids and an anionic sapphire surface.2 For colloidal α-alumina particles (CPs), we have recently demonstrated that also the oxidized peptide glutathione disulfide (GSSG) binds by electrostatic interactions to the particles.30 The tripeptide glutathione (γ-glutamylcysteinylglycine, GSH; Figure 1) is a physiologically highly relevant peptide.31 In addition to its application in medicine, cosmetic, and food industries,32 GSH has gained special interest for nanotechnological applications.33,34 Because of its affinity for metals,35,36 GSH has been already employed to synthesize monolayer-protected metal nanoparticles33 for biotechnological Received: August 6, 2012 Revised: October 4, 2012 Published: October 10, 2012 23136

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Figure 1. Structural formulas of GSH and of related amino acids, dipeptides, and derivatives.

water and autoclaved to regenerate terminal hydroxyl groups on the particle surface. The CP suspension was deagglomerated for 15 min by ultrasound treatment and subsequently incubated with GSH under the conditions indicated for the individual experiments. The CPs used have an average diameter of 159 ± 7 nm and a specific surface area of 12.8 ± 0.14 m2/g.30 All incubations of alumina CPs with GSH were performed at room temperature and in the presence of a large molar excess of dithiotreitol (DTT, 5 mM for adsorption/desorption experiments and 50 mM for IEP measurements) to prevent oxidation of GSH to GSSG during the incubation. Precipitation of the alumina CPs was prevented by permanent mixing of the samples during incubation by using a rotator drive STR4 (Staffordshire, USA) at a speed of 13 rpm (adsorption/ desorption experiments) or a horizontal shaker (Unimax 1011, Heidolph Instruments, Germany) (IEP measurements). To quantify the amount of CP-bound GSH, the samples (containing the given concentrations of Al2O3-CPs and GSH in 1 mL total volume) were centrifuged for 10 min at 12 100g, washed once with 1 mL of pure H2O, and centrifuged again. The supernatant was removed, and the CP pellet was dispersed in 1 mL of 10 mM KOH (pH 11.7). After a further centrifugation step (10 min at 12 100g), the KOH supernatant was used to quantify the amount of GSH liberated from the CPs. As previously shown for GSSG,30 application of 10 mM KOH completely prevents the binding of GSH to alumina CPs and completely desorbs bound GSH from CPs (data not shown). The effects of various charged or uncharged molecules that are structurally related to GSH (Figure 1) on the adsorption of GSH to Al2O3-CPs was studied by incubation of 30 μM GSH with 0.1 vol % CP suspension in the absence or presence of the substances given in Table 1 or Figure 8 in concentrations of up to 1000 μM for 60 min. The amount of CP-adsorbed GSH was

applications and for synthesis of thiolate-protected gold nanocrystals for photovoltaic purposes.33,34 In addition to the thiol group, GSH bears one amino group and two carboxylic acid groups (Figure 1), thus having multiple options to interact by covalent and/or electrostatic bonds with material surfaces.37,38 Here, we report that GSH binds rapidly and reversibly to αalumina CPs in a process that is strongly affected by the pH, by the concentration of unbound GSH, by the ionic strength of the solution, and by the presence of negatively charged structurally related molecules. These data demonstrate that electrostatic attraction between the carboxylate groups of GSH and the alumina surface is responsible for the binding of GSH to alumina CPs.

2. EXPERIMENTAL SECTION 2.1. Materials. High purity α-alumina CPs (Al2O3-CPs; >99.99% wt, Taimei, TM-DAR, Lot. No. 7553) were purchased from Krahn Chemie (Germany). Dithiothreitol (DTT), nicotinamide adenine dinucleotide phosphate (NADPH), and sulfosalicylic acid were obtained from Applichem (Germany). 2-Vinylpyridine, 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB), ethylenediamine tetraacetic acid (EDTA), GSH, and Trizma base were obtained from Sigma-Aldrich (Germany). Glutathione reductase was purchased from Roche Diagnostics (Germany) and ammonium sulfate and sodium sulfate from Riedel de Häen (Germany). All other chemicals were obtained from Fluka (Switzerland) or Merck (Germany) at analytical grade. 2.2. Methods. 2.2.1. Experimental Incubation of Alumina CPs with GSH. The preparation of aqueous Al2O3-CP suspensions was performed as previously described.30 Briefly, α-Al2O3 powder was calcinated at 400 °C for 4 h to eliminate any contaminating organic compounds. An aqueous 1 vol % stock suspension of CPs was prepared in double deionized 23137

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persion Technology, USA). After incubation of the CP suspension with GSH under the conditions described in the legend of Figure 4, the suspensions were titrated using 1 M KOH or 1 M HCl to measure the ζ-potential as a function of pH. 2.2.4. Analysis of Binding Data and Statistical Analysis. Information on the analysis of binding data is provided as Supporting Information. All experimental data are presented as means ± SD of values derived from at least three independent experiments. Analysis of significance between the means of groups of data was performed by one-way analysis of variance (ANOVA) followed by the Bonferroni posthoc test. The precondition of this analysis is Gaussian distribution/normality of the data within one experimental group. The level of significance compared to controls is indicated in Table 1 by ***p < 0.001; p > 0.05 was considered as not significant.

Table 1. Adsorption of GSH to Colloidal Alumina Particles in Presence of Amino Acids, Dipeptides, and Glutathione Derivatives KOH-desorbed GSH net charge

compound

−1 +1 0 0 0 0 0 −1 −1 −1 −1 −1 −2

GSH GSH diethylester GSH monoethylester cysteine glutamine glycine cysteinylglycine aspartate glutamate γ-glutamylcysteine γ-glutamylglycine methyl-S-GSH γ-glutamylglutamate

nmol 11.0 9.6 10.7 11.5 11.2 11.2 10.7 2.9 2.3 2.7 2.8 2.7 0.3

± ± ± ± ± ± ± ± ± ± ± ± ±

1.2 1.8 1.0 1.2 0.4 1.0 1.2 0.5*** 0.5*** 0.7*** 0.7*** 0.2*** 0.1***

% of control 100.0 86.7 97.3 105.1 104.9 102.0 97.0 26.9 21.0 23.8 24.7 24.5 2.84

± ± ± ± ± ± ± ± ± ± ± ± ±

10.4 7.6 2.0 13.6 13.8 2.6 5.0 7.0*** 3.1*** 3.9*** 4.3*** 1.1*** 0.7***

3. RESULTS 3.1. Time- and Concentration-Dependent Adsorption of GSH to Al2O3-CPs. To determine whether GSH has the potential to adsorb to Al2O3-CPs, a 0.1 vol % CP suspension was exposed in water (pH 5.4) to GSH in concentrations ranging from 3 μM to 1000 μM. The GSH bound to the CPs was quantitatively desorbed in the presence of 10 mM KOH, as previously reported also for GSSG.30 For all concentrations of GSH applied, maximal amounts of bound GSH were determined already after 5 min of incubation, while a longer incubation of up to 120 min did not alter the amount of CPbound GSH (Figure 2). The amount of adsorbed GSH

determined as described above after desorption of GSH by KOH and is indicated as KOH-desorbed GSH. To investigate the desorption of CP-bound GSH in water, 0.1 vol % CP suspension was preincubated with GSH in concentrations of up to 1000 μM for 60 min and centrifuged for 10 min at 12 100g. The pellet was washed once with 1 mL of pure H2O and subsequently dispersed in water in the volumes indicated and incubated for up to 120 min. After a given incubation time, samples were centrifuged, and the supernatant was harvested to quantify the GSH that had been liberated from the CPs during the main incubation in water (H2O-desorbed GSH). The pellet of the centrifugation was dispersed in 1 mL of 10 mM KOH and centrifuged. The KOH supernatant was used to quantify the amount of GSH that remained CP-bound after incubation with water but was completely desorbed from the particles by KOH. To study the potential effects of mono- and divalent salts on GSH desorption from alumina CPs, 0.1 vol % CP suspension was preincubated with 1000 μM GSH in water in 1 mL total volume for 60 min. After centrifugation for 10 min at 12 100g, the pellet was washed once with 1 mL of pure H2O and subsequently dispersed in 1 mL of water or of solutions of up to 100 mM NaCl, Na2SO4, or (NH4)2SO4 in water. After an additional 60 min incubation, the samples were centrifuged, and the supernatant was harvested and used for the quantification of GSH that was liberated from the particles by salts during the main incubation. The remaining pellet was dispersed in 1 mL of 10 mM KOH. After a further centrifugation step, the supernatant was used to quantify the amount of GSH that was liberated from the CPs by KOH. 2.2.2. Quantification of GSH. GSH was quantified as described previously30 in microtiter plates according to the colorimetric enzymatic cycling method originally described by Tietze.39 This assay allows the quantification of both GSH and of its oxidation product GSSG in picomole amounts.30 Quantification of GSH and GSSG by this assay was not affected by the presence of KOH or salts in the concentrations used to desorb GSH or GSSG from alumina CPs (data not shown). 2.2.3. ζ-Potential Measurements. The ζ-potential measurements were performed in 1 vol % aqueous Al2O3 suspensions using the electroacoustic colloidal vibration current technique40 (Acoustic and Electroacoustic Spectrometer DT-1200, Dis-

Figure 2. Time-dependent adsorption of GSH to Al2O3-CPs. GSH in the indicated concentrations was incubated with 0.1 vol % Al2O3-CPs in the presence of 5 mM DTT for up to 120 min. The data shown are the amounts of GSH that had been liberated from the particles by KOH.

depended strongly on the initial GSH concentration and increased proportional to the logarithm of the GSH concentration applied, at least for GSH concentrations above 10 μM (Figure 3a). A final saturation of the CP surface with GSH was not observed, even after application of GSH in concentrations of up to 1000 μM (Figure 3a). For the conditions used, the KOH supernatants contained exclusively GSH, while GSSG was not detectable in the KOH supernatant (Figure 3a), excluding any oxidation of particle-bound GSH to GSSG during the incubation. Variation of the concentrations of alumina CPs revealed that the amount of adsorbed GSH increased almost proportional to the amount of alumina CPs present (Figure 3b). Fitting of the data obtained for the concentration dependence of the GSH adsorption (Figure 3a) by a Temkin isotherm model for GSH concentrations larger 23138

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Figure 5. Time-dependent desorption of GSH from Al2O3-CPs; 0.1 vol % of Al2O3-CPs were preincubated with 10, 100, or 1000 μM GSH for 60 min, washed, were incubated in water for up to 120 min in the presence of 5 mM DTT, and finally exposed to 10 mM KOH. Panel a shows the amounts of H2O-desorbed GSH, panel b the amounts of GSH that remained bound to the particles during the water incubation and that were finally liberated from the CPs by KOH. For each time point, the sum of H2O- and KOH-desorbed GSH is shown in panel c.

Figure 3. Concentration-dependent adsorption of GSH to Al2O3-CPs. GSH in concentrations of up to 1000 μM was incubated for 60 min with 0.1 vol % of CPs (a) or with the indicated concentrations of CPs (b) in the presence of 5 mM DTT. In panel a, the desorbed material was analyzed for the presence of GSH and of its oxidation product GSSG.

than 10 μM revealed values of ΓT = 10.2 nmol and ΔG = −30.3 kJ/mol with a fitting correlation coefficient of 0.998. The adsorption of GSH to Al2O3-CPs was confirmed by the differences in the pH dependency of the ζ-potential curves measured for untreated and GSH-exposed Al2O3-CPs (Figure 4a). The presence of GSH lowered significantly the IEP of the

of GSH that had bound during the preincubation. From the total amount of adsorbed GSH (sum of H2O-desorbed and KOH-desorbed GSH) determined after preincubation with 10, 100, and 1000 μM GSH, a main incubation with water liberated 23%, 31%, and 41%, respectively (Figure 5b). The absence of any further change in the amounts of H2O-desorbed (Figure 5a) and KOH-desorbed GSH (Figure 5b) during a longer incubation of GSH-treated CPs demonstrated that already within 5 min an equilibrium between unbound GSH and CPbound GSH was established (Figure 5). To further confirm the establishment of an equilibrium between adsorbed and unbound GSH after incubation of alumina CPs with GSH in water, a 0.1 vol % CP solution that had been preincubated with various concentrations of GSH was exposed during a desorption phase of 60 min to different volumes of water. While the total amounts of water-desorbed GSH increased strongly by increasing the water volume (Figure 6a), the concentration of desorbed GSH in the watersupernatants of GSH-treated CPs was almost identical for all three volumes of water applied (Figure 6b). 3.3. Ionic Strength Dependent Desorption of GSH from Al2O3-CPs. To test whether electrostatic interactions between the negatively charged GSH and the positively charged surfaces of alumina CPs are responsible for the adsorption of GSH to the CPs, we incubated a 0.1 vol % CP suspension that

Figure 4. Zeta-potential curves and isoelectric points of GSH-treated Al2O3-CPs. GSH in concentrations of up to 10 mM was incubated for 60 min with 1 vol % CP suspension in the presence of 50 mM DTT. Panel a shows the zeta-potential curves for a representative experiment. The determined IEPs (b) represent means ± SD of values obtained in three independent experiments.

CPs from an initial value of 9.81 ± 0.07 (absence of GSH) to a value of 9.18 ± 0.03 that was reached after incubation of the 1 vol % Al2O3-CP dispersion with 1 mM GSH and was not further significantly (p > 0.05) lowered in the presence of 3 mM or 10 mM GSH (Figure 4b). Fitting of the absolute value of the IEP change (ΔIEP) of the CPs as a function of the concentration of GSH applied with a Langmuir isotherm revealed a saturation value of ΔIEP = 0.75 (corresponding to an IEP of 9.06) and a binding free energy ΔG = −30.1 kJ/mol. 3.2. Desorption of GSH from Al2O3-CPs. To investigate to which extent Al2O3-CP bound GSH will desorb from the CPs after removal of unbound GSH, a 0.1 vol % suspension was preincubated with 10, 100, or 1000 μM GSH for 60 min to achieve GSH adsorption. The remaining unbound GSH in the supernatant was then separated from the CPs by centrifugation, and the particles were subsequently incubated in water (at pH 5.4) for up to 120 min to investigate desorption of GSH from the CPs. Maximal amounts of desorbed GSH were already observed after the first 5 min of the main incubation (Figure 5a). The amounts of GSH that desorbed from the particles during the main-incubation depended strongly on the amount

Figure 6. Desorption of GSH from Al2O3-CPs in water. GSH in concentrations of up to 1000 μM was preincubated with 0.1 vol % of Al2O3-CPs for 60 min in the presence of 5 mM DTT followed by a 60 min incubation in 0.5, 1, or 2 mL of water (pH 5.4). The data show the amount (a) and the concentration (b) of H2O-desorbed GSH as function of the amount of GSH that remained adsorbed after the water incubation and was finally desorbed by KOH application. 23139

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Figure 7. Effects of ionic strength on the desorption of GSH from Al2O3-CPs; 1000 μM GSH was preincubated with 0.1 vol % Al2O3-CPs for 60 min in the presence of 5 mM DTT followed by a 60 min incubation in water without or with NaCl (a), Na2SO4 (b), or (NH4)2SO4 (c) in the concentrations indicated. The data show the amounts of desorbed GSH and the corresponding amounts of GSH that remained bound to the particles and were finally desorbed from the particles by KOH.

had been preincubated with 1000 μM GSH for 60 min to facilitate GSH adsorption, with NaCl, Na2SO4, or (NH4)2SO4 in concentrations of up to 100 mM (Figure 7). While a mainincubation of GSH-loaded CPs with water in the absence of salt ions desorbed only about 45% of the particle-bound GSH, the presence of salt ions enhanced in a concentration-dependent manner the desorption of GSH from the CPs (Figure 7). The presence of NaCl (Figure 7a) in concentrations of 1, 10, and 100 mM increased the desorption of GSH from the CPs to 63%, 80%, and 90% of the initially bound GSH, respectively, whereas already 0.1 mM of the divalent salts Na2SO4 (Figure 7b) or (NH4)2SO4 (Figure 7c) almost completely desorbed the particle-bound GSH. 3.4. Effects of Amino Acids, Dipeptides, and GSH Derivatives on the Adsorption of GSH to Al2O3-CPs. To test which structural related compounds may affect binding of GSH to alumina CPs, a 0.1 vol % Al2O3-CP suspension was incubated with 30 μM GSH for 60 min in the presence of a 10fold molar excess of various potential competitors of binding (Figure 1), including amino acids, dipeptides, and GSH derivatives (Table 1), and the GSH content that was liberated from the particles by KOH was determined. The potential of a compound to prevent adsorption of GSH to the CPs strongly depended on its net charge in aqueous solution (Table 1). A 10-fold molar excess of the positively charged GSH diethylester or of various net-uncharged compounds did not affect the amount of GSH bound to the CPs. In contrast, the presence of an excess of negatively charged compounds lowered the amount of adsorbed GSH by 70% to 75% (Table 1), while the −2 charged dipeptide γ-glutamylglutamate (γ-GluGlu) almost completely prevented the adsorption of GSH to the alumina CPs (Table 1). To study the concentration dependence of some of the compounds that lowered GSH adsorption to CPs, a 0.1 vol % CP suspension was incubated for 60 min with 30 μM GSH in the presence of various concentrations of glutamate, γglutamylcysteine (γ-GluCys), γ-GluGlu, or methyl-S-GSH (Figure 8). All of these compounds lowered the amount of particle bound GSH in a concentration-dependent manner. The concentrations of γ-GluGlu, γ-GluCys, glutamate, and methyl-S-GSH that lowered by 50% the amount of adsorbed GSH were 19, 82, 138, and 368 μM, respectively (Figure 8). This demonstrates that, among the tested compounds, the −2 charged dipeptide γ-GluGlu was most potent to prevent binding of GSH to alumina CPs.

Figure 8. Concentration-dependent effects of glutamate, dipeptides, and methyl-S-GSH on the adsorption of GSH to Al2O3-CPs; 30 μM GSH was incubated for 60 min with 0.1 vol % Al2O3-CPs and 5 mM DTT in the absence or the presence of glutamate, γ-glutamylcysteine (γ-GluCys), γ-glutamylglutamate (γ-GluGlu), or methyl-S-GSH in the concentrations indicated. The data shown represent the amounts of GSH that had been liberated from the particles by KOH.

4. DISCUSSION Adsorption/desorption experiments as well as ζ-potential measurements demonstrated that the physiological tripeptide GSH adsorbs quickly (