ARTICLE pubs.acs.org/Langmuir
Interaction of Biological Molecules with Clay Minerals: A Combined Spectroscopic and Sorption Study of Lysozyme on Saponite Cliff T. Johnston,*,† Gnanasiri S. Premachandra,† Tamas Szabo,‡ Joyce Lok,† and Robert A. Schoonheydt§ †
Birck Nanotechnology Center, Purdue University, 915 West State Street, West Lafayette, Indiana 47907-2054, United States Department of Physical Chemistry and Materials Science, University of Szeged, H-6720 Szeged, Hungary § Centre for Surface Science and Catalysis, K.U.Leuven, Kasteelpark Arenberg 23, B-3001 Leuven, Belgium ‡
bS Supporting Information ABSTRACT: The interaction of hen egg white lysozyme (HEWL) with Naand Cs-exchanged saponite was investigated using sorption, structural, and spectroscopic methods as a model system to study clay protein interactions. HEWL sorption to Na- and Cs-saponite was determined using the bicinchoninic acid (BCA) assay, thermogravimetric analysis, and C and N analysis. For Na-saponite, the TGA and elemental analysis-derived sorption maximum was 600 mg/g corresponding to a surface coverage of 0.85 ng/ mm2 with HEWL occupying 526 m2/g based on a cross-sectional area of 13.5 nm2/molecule. HEWL sorption on Na-saponite was accompanied by the release of 9.5 Na+ ions for every molecule of HEWL sorbed consistent with an ion exchange mechanism between the positively charged HEWL (IEP 11) and the negatively charged saponite surface. The d-spacing of the HEWL Na-saponite complex increased to a value of 4.4 nm consistent with the crystallographic dimensions of HEWL of 3 3 4.5 nm. In the case of Cs-saponite, there was no evidence of interlayer sorption; however, sorption of HEWL to the “external” surface of Cs-saponite showed a high affinity isotherm. FTIR and Raman analysis of the amide I region of the HEWL saponite films prepared from water and D2O showed little perturbation to the secondary structure of the protein. The overall hydrophilic nature of the HEWL Na-saponite complex was determined by water vapor sorption measurements. The clay retained its hydrophilic character with a water content of 18% at high humidity corresponding to 240 H2O molecules per molecule of HEWL.
’ INTRODUCTION Interest in the interaction of biomolecules with inorganic surfaces has significantly increased in recent years. Hybrid organic inorganic materials that combine biological concepts with inorganic material chemistry are emerging at a rapid pace in a wide range of disciplines that span from biotechnology, materials science, and nanomedicine.1 7 One class of inorganic materials that continues to attract attention is the group of 2:1 phyllosilicates known as smectites. Smectites are among the most abundant naturally occurring nanomaterials on earth on a surface area basis.8 Fundamental particles of smectite are sheet or lathe-like with a thickness of 1 nm and aspect ratios that range from 20 to 1000 and surface areas that approach 750 m2/g.9 These surfaces are also reactive resulting from isomorphous substitution in the clay lattice with charge densities that range from 0.3 to 0.6 charge defects per O10(OH)2 unit resulting in a negatively charged surface, which is compensated mainly by inorganic cations in the clay interlayer (e.g., Ca2+, Mg2+, Na+, K+).10 These exchangeable cations have large enthalpies of hydration and are surrounded by clusters of H2O molecules, which impart a hydrophilic character to the surface. The interlayer siloxane surface of smectite itself has a low affinity for H2O, and recent studies have shown that smectites exchanged with weakly hydrated cations (e.g., Cs+) have a surprisingly high affinity of r 2011 American Chemical Society
hydrophobic compounds such as nitroaromatics, pesticides, and dibenzo-p-dioxin.11,12 This dual hydrophobic hydrophilic character9,13 has been attributed to account for the high affinity for certain classes of organic compounds with smectites, which include metal ligand complexes,14,15 cationic dyes,16 and cationic surfactants.17 Although not fully understood or recognized, this dual hydrophobic/hydrophilic nature is thought to influence the interaction of proteins with smectites. The interaction of proteins with clay minerals has been studied over the past 70 years18 30 with emphasis in the past 10 years on the development of novel hybrid structural and functional biomaterials applied to biosensing, biocatalysis, and drug delivery.2 Hen egg white lysozyme (HEWL) has been used as a model protein to study clay protein interactions because of its high affinity for smectites and ability to intercalate into the clay interlayer.21 25,28 Sorption of HEWL can exceed values of 2000 mglysozyme/gclay and is accompanied by interlayer expansion to d-spacings of 4.4 nm.21,22,28 HEWL is a “hard”, globular protein with a MW of 14 300 Da. The high affinity of HEWL Received: August 12, 2011 Revised: October 12, 2011 Published: November 02, 2011 611
dx.doi.org/10.1021/la203161n | Langmuir 2012, 28, 611–619
Langmuir for smectites is attributed to its isoelectric point (IEP) of 11.4 that is attracted to the negatively charged clay surface at essentially all pH ranges of interest.31 Recent studies have exploited HEWL as a type of molecular probe to study clay surfaces. Layer-by-layer (LbL) assembly methods were used recently to produce smectite protein complexes using protamine, papain, and HEWL.32 All three of these positively charged proteins showed a high affinity for the Na-smectite. In related work, HEWL has been used in conjunction with surface force apparatus (SFA) studies and with AFM to investigate the adsorption of positively charged lysozyme onto negatively charged mica surfaces.33 36 In the SFA experiments, the surface forces between parallel sheets of mica have been examined in the presence of HEWL under a variety of conditions. Mica is a 2:1 phyllosilicate, similar to smectites, but characterized by a higher layer charge and by nonexpanding interlayers. SFA studies of HEWL mica complexes reveal a hard wall repulsion at 3 nm, and another “force wall” at 6 nm, corresponding to 1 and 2 layers of HEWL between the mica sheets.33 Although the general behavior of HEWL interactions with clay minerals is known including its use as a model probe in SFA and LbL studies, a detailed molecular understanding of HEWL smectite complexes has not been reported. For example, clayinduced changes in protein secondary and tertiary structure of α-chymotrypsin,37 bovine serum albumin,38 flagellar FliF protein ring,39 protamine and papain,32,40 and ovin prion protein have been reported, but only limited information is known about clayinduced changes in the secondary and tertiary structure of HEWL on clay surfaces. In an earlier LbL study, we investigated the structure of HEWL on saponite, a trioctahedral smectite with isomorphic substitution restricted to the tetrahedral sheet. Interlayer expansion of Na-saponite was achieved by HEWL with a d-spacing of 3.56 nm for the 15-layer complex.32 ATR-FTIR spectra of the (HEWL/saponite)15 complex were minimally perturbed upon deuteration, indicating that HEWL did not unfold, spread, or denature on the clay surface and behaves as a “hard” protein.32 Protein sorption mechanisms in the LbL construction process are presumably somewhat different from what occurs in an aqueous clay suspension as alternating layers of clay and protein are deposited or self-assembled. In the LbL method, protein sorption occurs on “external” clay surfaces. There was no evidence of water in the LbL films, and the d-spacing of 3.56 nm was less than the 4.4 nm value reported from batch sorption to smectite in aqueous suspension24,25 where (1) two clay interfaces area present when interlayer sorption occurs and (2) excess water is present throughout the process. The objectives of this study were to investigate the molecular mechanisms of HEWL sorption to Na- and Cs-exchange saponite from aqueous suspension. In the case of Na-exchanged saponite, interlayer sorption is expected to occur, whereas interlayer sorption on Cs-saponite is anticipated to be absent or limited. Strongly hydrated Na+ ions function as molecular props that permit access to the large molecules, including proteins. In contrast, Cs+ ions are weakly hydrated, and Cssaponite does not swell in the presence of water. HEWL clay complexes have been the subject of recent atomistic molecular dynamics simulations, providing insight into the sorption process of HEWL to an “external” clay surface, including changes to the HEWL structure and alignment of sorbed HEWL molecules.41,42 Of interest here was to investigate the structural changes to HEWL based on analysis of the amide I amide II region. Finally, little is known about the hydrophilic nature of clay protein
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complexes, which has important implications for supported enzymes. From our earlier LbL study of HEWL saponite, there was no evidence of water in the LbL films.32 Given the approximately globular structure of HEWL and the sheet-like structure of saponite, there is certainly “free space” in the clay interlayer, which could accommodate water molecules.
’ MATERIALS AND METHODS Lysozyme, a protein made of chicken-egg white purchased from Sigma-Aldrich (MW 14 600 with an isoelectric point of 11.4), was stored at 4 °C and used for this study. Saponite was obtained from the Source Clays Repository of the Clay Minerals Society. It was saturated with Na+ by repeated exchange with 1 M NaCl solutions followed by dialysis with water until the water tested Cl -free using AgNO3. The particle size fraction of 0.5 2.0 μm was obtained by centrifugation. The resulting 0.5 2.0 μm saponite dispersion was freeze-dried and stored as a powder at room temperature. After the 0.5 2.0 μm fraction of the Na-exchanged saponite was obtained by centrifugation, a portion of the clay was washed with a 0.1 M CsCl solution, followed by washing and freezedrying to obtain Cs+-exchanged saponite. Sorption experiments were conducted in 25 mL glass (Corex) screw cap centrifuge tubes with two replications. Aliquots of a lysozyme stock solution and deionized distilled water were added to 12.5 mg of Na-saponite in the tubes to reach a final volume of 15 mL with lysozyme concentrations ranging from 0 to 2000 μg mL 1. The tubes were shaken overnight (16 h) in a reciprocating shaker. The tubes were centrifuged at 6000 rpm for 20 min, and 7 mL of the supernatant was removed for analysis of pH, lysozyme, and Na content. The saponite lysozyme complexes were redispersed with the remaining supernatant to make self-supported clay films. The aqueous clay protein dispersion was passed through a 0.45 μm Supor-450 hydrophilic polyethersulfone membrane (47 mm diameter, made by Pall Life Sciences) on a Millipore holder under vacuum. The resulting saponite lysozyme deposits on the filter were allowed to airdry and were removed from the filter by running the filter and clay deposit over a knife edge. Lysozyme concentration in the supernatant was determined using the BCA Protein Assay Kit (Pierce). An adsorption isotherm was developed by plotting lysozyme sorbed (mg g 1 saponite) against the equilibrium concentration in the supernatant. Na+ concentration in the supernatant was measured using an atomic adsorption spectrophotometer-6800 (Shimadzu). Acetylene/air flame and a Na lamp were used with a Shimadzu auto sampler (model ASC-1600). Infrared spectra of the self-supported clay films were obtained on a Perkin-Elmer GX2000 Fourier transform infrared (FTIR) spectrometer equipped with deuterated triglycine (DTGS) and mercury cadmium telluride (MCT) detectors, an internal wire grid IR polarizer, a KBr beam splitter, and a sample bench purged with dry air. The unapodized resolution for the FTIR spectra was 2.0 cm 1, and a total of 64 scans were collected for each spectrum. The FTIR spectrum of lysozyme crystals was obtained in KBr pellets corresponding to 2 mg sample in approximately 248 mg of spectral grade KBr. Postprocessing of the FTIR spectra was restricted to baseline correction, curve fitting, and integration using GRAMS/32 program (Galactic Software). A set of self-supported clay films were used in thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) analysis after taking the FTIR spectra. The clay films were lightly ground into a powder and weighed in 70 μL ceramic crucibles. The crucibles were placed in the thermogravimetric analyzer (model TGA/SDTA851e, Mettler Toledo, OH) and ran in the temperature range of 25 1000 °C at an increment rate of 20 °C per minute in a N2 atmosphere. For DSC analysis, powder samples were weighed into 40 μL aluminum sample pans. Sample pans were sealed and placed in the DSC (model DSC 30 612
dx.doi.org/10.1021/la203161n |Langmuir 2012, 28, 611–619
Langmuir
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determinations and amount of protein sorbed. At low protein concentrations of
