Studying the interaction between silica nanoparticles and metals by spectrophotometry

The optical absorption spectra of water silica sols containing nanoparticles (NPs) of metals (Ag, Pd, Fe, and Pt) are investigated. Silica sols are obtained from natural hydrothermal solutions via membrane concentration (ultrafiltration). Water sols of silica with specific sizes, pH values, ζ potentials of SiO₂ NP surfaces, and low concentrations of SiO₂ NPs are used. Plasmon resonance in optical absorption spectra is used to study the interaction between silica and metal NPs. Parameters of plasmon resonance (position, height, and half-width of optical absorption bands), from which the degree of interaction is assessed, are determined. Relationships between the optical properties of the surfaces of nanoparticle-size silica particles, the method of their production, and the effect of adsorbed metal particles on these properties are established.     

Identification of proton-transfer pathways in human carbonic anhydrase II

We investigate the probable proton-transfer pathways from the surface of human carbonic anhydrase II into the active site cavity through His-64 that has been widely implicated as a key residue along the proton-transfer path. A recursive analysis of hydrogen-bonded clusters in the static crystallographic structure shows that there is no complete path through His-64 in either of its experimentally detected conformations. Side chain conformational fluctuation of His-64 from its outward conformation toward the active site is found to provide a crucial dynamic connectivity needed to complete the path coupled to local reorganization of the protein structure and hydration. The energy and free energy barriers along the detected pathway have been estimated to derive the mechanism of His-64 rotation toward the active site. We also investigate a dynamical connectivity map that highlights networks of disordered water molecules that may promote a direct (and probably transient) access of the solvent to the active site. Our studies reveal how such solvent access channels may be related to the putative proton shuttle mediated by His-64. The paths thus identified can be potentially used as reaction coordinates for further studies on the molecular mechanism of enzyme action.     

Fluoroaromatic-fluoroaromatic interactions between inhibitors bound in the crystal lattice of human carbonic anhydrase II

Intermolecular interactions of eleven different fluoroaromatic inhibitors are probed within the scaffolding of the crystal lattice of Phe-131-->Val carbonic anhydrase II. The degree and pattern of fluorine substitution on the inhibitor benzyl ring modulate its size, shape, and electronic character. In turn, these properties affect the geometry of intermolecular interactions between the fluoroaromatic rings of two different inhibitor molecules bound in the crystal lattice, as determined by X-ray crystallography. Depending on the degree and pattern of fluorine substitution, we observe a face-to-face (aromatic-aromatic) interaction, an atom-to-face (carbonyl-aromatic) interaction, or no interaction at all. These interaction geometries are analyzed with regard to van der Waals, electrostatic, and possible charge-transfer effects. For the aromatic-aromatic interactions investigated in this study, with aromatic ring quadrupoles specifically "tuned" by the degree and pattern of fluorination, the structural results suggest that London forces and charge-transfer complexation dominate over weakly polar electrostatic interactions in the association of aromatic ring pairs.     

Free-solution interaction assay of carbonic anhydrase to its inhibitors using back-scattering interferometry

Back-scattering interferometry (BSI) is a label-free, free-solution, small-volume technique used for characterizing binding interactions, which is also relevant to a growing number of biosensing applications including drug discovery. Here, we use BSI to characterize the interaction of carbonic anhydrase enzyme II with five well-known carbonic anhydrase enzyme II inhibitors (± sulpiride, sulfanilamide, benzene sulfonamide, dansylamide, and acetazolamide) in the presence of DMSO. Dissociation constants calculated for each interaction were consistent with literature values previously obtained using surface plasmon resonance and fluorescence-based competition assays. Results demonstrate the potential of BSI as a drug-screening tool which is fully compatible with DMSO and does not require immobilization or labeling, therefore allowing binding interactions to be characterized in the native state. BSI has the potential for reducing labor costs, sample consumption, and assay time while providing enhanced reliability over existing techniques.     

A thermodynamic study of nickel ion interaction with bovine carbonic anhydrase II molecule

A thermodynamic study on the interaction of bovine carbonic anhydrase II (CAII) with nickel ions was performed by using isothermal titration calorimetry (ITC) at 27 °C in Tris buffer solution at pH = 7.5. The enthalpies of Ni²⁺ + CAII interaction are reported and analysed in terms of the new solvation theory. It was indicated that there are three identical and non-cooperative sites for Ni²⁺. The binding of a nickle ion is exothermic with dissociation equilibrium constants of 81.306 and 99.126 μM at 27°C and 37°C, respectively. The binding of nickel ions can cause some changes in the stability of the enzyme at low and high Ni²⁺ concentrations.     

Theoretical improvement of the specific inhibitor of human carbonic anhydrase VII

The selectivity of a known arylsulfonamides inhibitor for two isozymes II and VII of human carbonic anhydrases (hCAs) was studied by homology modeling, molecular docking and molecular dynamics methods. The results show that the selectivity of the inhibitor for two isozymes is due to the different side chain lengths between N67 of hCA II and Q64 of hCA VII. One more methene group in the side chain of Q64 of hCA VII makes it possible to form the hydrogen bond with the bromide atom of the known inhibitor. From the point of view, the modification to the known inhibitor was performed to obtain an inhibitor with higher selectivity. The complex conformations of the new designed inhibitor and two isozymes designate the formation of the hydrogen bond between the newly added group (hydroxypropyl group) and Q64 of hCA VII but N67 of hCA II. The results of the binding free energy from the MM/PBSA approach also prove the selectivity improvement of the new inhibitor in comparison with the known inhibitor. The work will help the design of the isozyme-specific inhibitors of hCA VII.     

Production of active human carbonic anhydrase II in E. coli

cDNA encoding human carbonic anhydrase II has been isolated and its nucleotide sequence determined. Expression of the isolated carbonic anhydrase gene in Escherichia coli from a plasmid containing the tac promoter yielded an active enzyme at a level of about 1% of total protein.     

The binding of human carbonic anhydrase II by functionalized folded polypeptide receptors

Several receptors for human carbonic anhydrase II (HCAII) have been prepared by covalently attaching benzenesulfonamide carboxylates via aliphatic aminocarboxylic acid spacers of variable length to the side chain of a lysine residue in a designed 42 residue helix-loop-helix motif. The sulfonamide group binds to the active site zinc ion of human carbonic anhydrase II located in a 15 A deep cleft. The dissociation constants of the receptor-HCAII complexes were found to be in the range from low micromolar to better than 20 nM, with the lowest affinities found for spacers with less than five methylene groups and the highest affinity found for the spacer with seven methylene groups. The results suggest that the binding is a cooperative event in which both the sulfonamide residue and the helix-loop-helix motif contribute to the overall affinity.     

Structure of a 129Xe-cryptophane biosensor complexed with human carbonic anhydrase II

Cryptophanes represent an exciting class of xenon-encapsulating molecules that can be exploited as probes for nuclear magnetic resonance imaging. The 1.70 A resolution crystal structure of a cryptophane-derivatized benezenesulfonamide complexed with human carbonic anhydrase II shows how an encapsulated xenon atom can be directed to a specific biological target. The crystal structure confirms binding measurements indicating that the cryptophane cage does not strongly interact with the surface of the protein, which may enhance the sensitivity of 129Xe NMR spectroscopic measurements in solution.     

Analysis of human carbonic anhydrase II: docking reliability and receptor-based 3D-QSAR study

The ability of Gold software to predict the binding disposition of carbonic anhydrase (CA) inhibitors was evaluated using CA II as a case study. The best procedure was subsequently used for docking almost 300 CA II ligands, and the best poses were used as an alignment tool for the development of a 3D quantitative structure-activity relationship (QSAR) study. Evaluation of the resulting 3D-QSAR model allowed us to indicate the ligand properties and residues important for CA II inhibition. Since CAs are an important target involved in many pathologies such as glaucoma, obesity, and tumors, the results obtained could accurately predict the binding affinity of newly designed CA II inhibitors. Furthermore, it is reasonable that this strategy could be profitably used also for the investigation of other CAs.     

Immobilization of human carbonic anhydrase on gold nanoparticles assembled onto amine/thiol-functionalized mesoporous SBA-15 for biomimetic sequestration of CO2

A biocatalyst was synthesized by immobilizing human carbonic anhydrase onto gold nanoparticles assembled over amine/thiol-functionalized mesoporous SBA-15. The physicochemical properties of the functionalized mesoporous SBA-15 were obtained by XRD, BET, FE SEM, HR TEM, EDS, and zeta potential analysis. The biocatalytic performance was studied for para-nitrophenyl acetate (p-NPA) hydrolysis. The kinetic parameters K(m) were found to be 22.35 and 27.75 mM, and K(cat)/K(m) values were 1514.09 and 1612.25 M(-1) s(-1) for HCA immobilized on gold nanoparticles assembled on amine/thiol-functionalized mesoporous SBA-15 (HCA/Au/APTES/SBA-15 and HCA/Au/MPTES/SBA-15), respectively. These HCA/Au/APTES/SBA-15 and HCA/Au/MPTES/SBA-15 were investigated for biocatalytic hydration of CO(2) and its precipitation as CaCO(3). The amount of CaCO(3) precipitated over HCA/Au/MPTES/SBA-15 was nearly the same as that precipitated over free HCA. Storage stability and reusability studies suggested that HCA/Au/MPTES/SBA-15 retained its activity even after 20 days storage at 25 °C and 20 recycling runs. The present results demonstrate that HCA/Au/MPTES/SBA-15 and HCA/Au/APTES/SBA-15 are highly efficient potential nanobiocatalysts for industrial-scale CO(2) sequestration.     

Using covalent dimers of human carbonic anhydrase II to model bivalency in immunoglobulins

This paper describes the development of a new bivalent system comprising synthetic dimers of carbonic anhydrase linked chemically through thiol groups of cysteine residues introduced by site-directed mutagenesis. These compounds serve as models with which to study the interaction of bivalent proteins with ligands presented at the surface of mixed self-assembled monolayers (SAMs). Monovalent carbonic anhydrase (CA) binds to benzenesulfonamide ligands presented on the surface of the SAM with K(d)(surf) = 89 nM. The synthetic bivalent proteins--inspired by the structure of immunoglobulins--bind bivalently to the sulfonamide-functionalized SAMs with low nanomolar avidities (K(d)(avidity,surf) = 1-3 nM); this difference represents a ~50-fold enhancement of bivalent over monovalent association. The paper describes dimers of CA having (i) different lengths of the covalent linker that joined the two proteins and (ii) different points of attachment of the linker to the protein (either near the active site (C133) or distal to the active site (C185)). Comparison of the thermodynamics of their interactions with SAMs presenting arylsulfonamide groups demonstrated that varying the length of the linker between the molecules of CA had virtually no effect on the rate of association, or on the avidity of these dimers with ligand-presenting surfaces. Varying the point of attachment of the linker between monomeric CA's also had almost no effect on the avidity of the dimers, although changing the point of attachment affected the rates of binding and unbinding. These observations indicate that the avidities of these bivalent proteins, and by inference the avidities of structurally similar bivalent proteins such as IgG, are unexpectedly insensitive to the structure of the linker connecting them.     

Interactions between carbonic anhydrase and its inhibitors revealed by gel electrophoresis and circular dichroism

Structural properties, and especially the differential stability, of complexes between carbonic anhydrase (CA) and three sulfonamide inhibitors, acetazolamide, dorzolamide and methazolamide, were investigated by spectroscopic and electrophoretic techniques. These included denaturant gradient gel electrophoresis either across a urea or a steady-state transverse sodium dodecyl sulfate (SDS) gradient. Acetazolamide, the smallest and most hydrophilic of the sulfonamides, forms the most stable complex in the presence of urea, whereas dorzolamide, with a bulky and hydrophobic structure, is most stable against the effects of SDS. At pH 7.4, complexes with dorzolamide show minimal changes in mobility across the SDS gradient, as if unaffected by the detergent, both in the presence and in the absence of excess ligand in the gel. When bound to both acetazolamide and methazolamide, on the other hand, CA displays an increase in mobility above 0.05% SDS, lower in the presence than in the absence of excess ligand. The finding of a distinct pattern for the unliganded enzyme, however, suggests the complexes can still retain the ligand, although binding of the surfactant changes their charge density. Under saturating conditions and in the presence of SDS, the surface charge of all complexes is much lower than for unliganded, denatured CA. Circular dichroism (CD) spectra clearly indicate that the increase in secondary structure and the decrease in tertiary structure brought about in CA by the presence of low concentrations of SDS are largely prevented by complexing with the inhibitors. These observations point out peculiar properties of each CA inhibitor, of potential value in the definition of their biological activities and also in the potential development of novel antagonist molecules.     

Chemical rescue of enzymes: proton transfer in mutants of human carbonic anhydrase II

In human carbonic anhydrase II (HCA II), the mutation of position 64 from histidine to alanine (H64A) disrupts the rate limiting proton transfer (PT) event, resulting in a reduction of the catalytic activity of the enzyme as compared to the wild-type. Potential of mean force (PMF) calculations utilizing the multistate empirical valence bond (MS-EVB) methodology for H64A HCA II yields a PT free energy barrier significantly higher than that found in the wild-type enzyme. This high barrier, determined in the absence of exogenous buffer and assuming no additional ionizable residues in the PT pathway, indicates the likelihood of alternate enzyme pathways that utilize either ionizable enzyme residues (self-rescue) and/or exogenous buffers (chemical rescue). It has been shown experimentally that the catalytic activity of H64A HCA II can be chemically rescued to near wild-type levels by the addition of the exogenous buffer 4-methylimidazole (4MI). Crystallographic studies have identified two 4MI binding sites, yet site-specific mutations intended to disrupt 4MI binding have demonstrated these sites to be nonproductive. In the present work, MS-EVB simulations show that binding of 4MI near Thr199 in the H64A HCA II mutant, a binding site determined by NMR spectroscopy, results in a viable chemical rescue pathway. Additional viable rescue pathways are also identified where 4MI acts as a proton transport intermediary from the active site to ionizable residues on the rim of the active site, revealing a probable mode of action for the chemical rescue pathway.     

Zinc solid-state NMR spectroscopy of human carbonic anhydrase: implications for the enzymatic mechanism

The pH dependence of the (67)Zn solid-state nuclear magnetic resonance spectroscopy of human carbonic anhydrase (CAII) has been investigated to characterize the nature of the fourth ligand. CAII, through the Zn(2+)-bound hydroxide, catalyzes the deceptively simple reaction: CO(2) + H(2)O <==> HCO(3)(-) + H(+). The accepted mechanism for CAII would predict that water would be bound to the Zn(2+) at pH 5 and hydroxide would be bound at pH 8.5. The measured values for the electric field gradient (EFG) or quadrupole coupling constant (Cq) for CAII are independent of pH within the limits of the experimental error, i.e., 9.8 +/- 0.2 MHz. The EFG interaction has been predicted by ab initio electronic structure calculations for water and hydroxide bound to the zinc, including various levels of hydrogen bonding. After comparing the predicted Cq's with the experimental values, we conclude that the species present from pH 5-8.5 is the hydroxide form. The NMR data presented here is not consistent with the accepted mechanism for CAII. We show that the NMR data is consistent with an alternative mechanism of CAII.     

Size-dependent interaction of silica nanoparticles with different surfactants in aqueous solution

The size-dependent interaction of anionic silica nanoparticles with ionic (anionic and cationic) and nonionic surfactants has been studied using small-angle neutron scattering (SANS). The surfactants used are anionic sodium dodecyl sulfate (SDS), cationic dodecyltrimethyl ammonium bromide (DTAB), and nonionic decaoxyethylene n-dodecylether (C(12)E(10)). The measurements have been carried out for three different sizes of silica nanoparticles (8, 16, and 26 nm) at fixed concentrations (1 wt % each) of nanoparticles and surfactants. It is found that irrespective of the size of the nanoparticles there is no significant interaction evolved between like-charged nanoparticles and the SDS micelles leading to any structural changes. However, the strong attraction of oppositely charged DTAB micelles with silica nanoparticles results in the aggregation of nanoparticles. The number of micelles mediating the nanoparticle aggregation increases with the size of the nanoparticle. The aggregates are characterized by fractal structure where the fractal dimension is found to be constant (D ≈ 2.3) independent of the size of the nanoparticles and consistent with diffusion-limited-aggregation-type fractal morphology in these systems. In the case of nonionic surfactant C(12)E(10), micelles interact with the individual silica nanoparticles. The number of adsorbed micelles per nanoparticle increases drastically whereas the percentage of adsorbed micelles on nanoparticles decreases with the increase in the size of the nanoparticles.     

Effective interaction between charged nanoparticles and DNA

We investigate the effective interaction mediated by salt ions between charged nanoparticles (NPs) and DNA. DNA is modeled as an infinite cylinder with a constant surface charge in an implicit solvent. Monte Carlo simulations are used to compute the free energy of the system described in the framework of the primitive model of electrolytes, which accounts for excluded volumes of salt ions. A mean-field Poisson-Boltzmann theory also allows us to compute the free energy and provides us with explicit formulae for its main characteristics (position and depth of the minimum). We intend here to identify the physical parameters that have a major impact on the NP-DNA interaction, in an attempt to evaluate physico-chemical properties which could play a role in genotoxicity or, which could be exploited for therapeutic use. Thus, we investigate the influence on the effective interaction of: the shape of the nanoparticle, the magnitude of the nanoparticle charge and its distribution, the value of the pH of the solution, the magnitude of Van der Waals interactions depending on the nature of the constitutive material of the NP (metal vs. dielectric). We show that for positively charged concave NPs the effective interaction is repulsive at short distance, so that it presents a minimum at distance from the DNA. This short-range repulsion is specific to indented particles and is a robust property that holds for a large range of materials and charge densities.     

A spectroscopic study on the interaction between ferric oxide nanoparticles and human hemoglobin

Magnetic nanoparticles can promote many attractive functions in biomedicine, which may contribute to the prevention of human disease but also may be potentially harmful. In the present study, the interaction of Fe₂O₃ nanoparticles with human hemoglobin (Hb) was studied by fluorescence, circular dichroism and UV/vis spectroscopies. Fluorescence data revealed that the fluorescence quenching of Hb by Fe₂O₃ nanoparticles was the result of the formed complex of Fe₂O₃ nanoparticles-Hb. Binding constants and other thermodynamic parameters were determined at three different temperatures. The hydrophobic interactions are the predominant intermolecular forces to stabilize the complex. Circular dichroism studies did not show any changes in the content of secondary structure of hemoglobin after Fe₂O₃ nanoparticles treatment. This study provides important insight into the interaction of Fe₂O₃ nanoparticles with hemoglobin, which may be a useful guideline for further using of these nanoparticles in biomedical applications.