Mechanism of Myoglobin Molecule Adsorption on Silica: QCM, OWLS and AFM Investigations (original) (raw)
Related papers
2003
Slab optical waveguide (SOWG) spectroscopy was used to observe the adsorption behavior of three important heme proteins, namely cytochrome c, myoglobin and hemoglobin, in a quartz surface. Using prism-coupled polychromatic visible light propagated into a quartz waveguide by internal total reflection, the real-time monitoring of evanescent wave absorption revealed a strong dependence of the protein-surface interaction on the protein concentration, the solution pH and the ionic strength. For the three proteins studied, the absorbance-bulk concentration ratio was higher at low bulk concentrations, and decreased at higher concentrations. For cytochrome c and myoglobin, the absorbance approached a limiting value, but buffered hemoglobin surprisingly did not show any indication of forming a signal plateau. Moreover, the slow introduction of protein into the solution lessened the total adsorbed amount per unit area. These observations suggested a possible conformational transition of the protein molecules at the quartz surface after adsorption. For a bulkier protein, hemoglobin, adsorption onto the quartz surface was enhanced in the presence of a phosphate buffer, while the opposite effect was observed for the smaller cytochrome c and myoglobin molecules. The results of pH studies concurred with the electrostatic interactions predicted from the isoelectric data of proteins and the quartz surface.
Journal of Molecular Catalysis B: Enzymatic, 2007
Myoglobin has been immobilized onto different ordered mesoporous silicates. The effect of the pH on the adsorption, leaching and activity was studied. The results showed that the maximum amount of protein was adsorbed at a pH 6.5, just below the protein isoelectric point (7-7.3). There was no effect of increasing ionic strength on the adsorption profile at different pH values. The adsorption is rationalized in terms of local electrostatic forces acting between the enzyme and the silica surface as well as hydrophobic interactions close to the protein isoelectric point, whereas at low pH the global charges give rise to protein-protein repulsion and at high pH enzyme-silica repulsion. Higher amounts of immobilized myoglobin were leached at a pH 4, while lower amounts were leached at pH 6.5. The catalytic activity of myoglobin immobilized onto SBA-15 showed optimal activity at a pH 6.5 in comparison to a pH of 5 for the free form.
The nano-bio interface mapped by oxidative footprinting of the adsorption sites of myoglobin
Analytical and bioanalytical chemistry, 2014
Oxidative footprinting has been used to study the structure of macromolecular assemblies such as protein-protein and protein-ligand complexes. We propose a novel development of this technique to probe the protein corona that forms at the surface of nanoparticles in any biological medium. Indeed, very few techniques allow studying this interface at the molecular and residue level. Based on hydroxyl radical-mediated oxidation of proteins and analysis by nanoscale liquid chromatography coupled to tandem mass spectrometry (nanoLC-MS/MS), two sites of adsorption of myoglobin on silica nanoparticles are identified. This method gives new insights in the understanding of protein adsorption on nanomaterials.
Journal of Physical Chemistry B, 2005
The hydroxide ion catalyzed isomerization of 5-androstene-3,17-dione (1) to 4-androstene-3,17-dione (2) proceeds through the formation of an intermediate dienolate ion (1-). This dienolate ion has been observed in the ultraviolet spectrum ( A, , , ca. 256 nm) during the isomerization reaction. Rate constants for the formation of the dienolate ion and both its reversion to reactant (1) and its conversion to product (2) in aqueous solution were measured. In addition, the rate of exchange of the C-6 protons of 2 in D20/MeOD was determined. These results enable a complete description of the reaction profile to be made, including all rate constants and the pK, values for 1 (12.7) and 2 (16.1). The possible relevance of these results to the mechanism of action of the enzyme 3-oxo-A5-steroid isomerase is briefly discussed.
Steric Considerations in the Covalent Binding of Myoglobin to Thin Films
Myoglobin is shown to bind to mixed thin films of docosyl mesylate and methyl docosanoate. Myoglobin can be attached to films containing docosyl mesylate; however, films containing only methyl docosanoate exhibit only minimal myoglobin attachment. This indicates that myoglobin is specifically attached to the docosyl mesylate sites on the film surface. Surface coverage of myoglobin is dependent on the percentage of docosyl mesylate in the film. A maximum myoglobin coverage of approximately 1.2 × 10 12 myoglobin/cm 2 is estimated for films composed of between 22% and 30% docosyl mesylate.
Mechanistic and Structural Features of Protein Adsorption onto Mesoporous Silicates
The Journal of Physical Chemistry B, 2002
The adsorption of cytochrome c onto a range of different mesoporous silicates (MPS) was studied. The materials used, templated using both cationic and nonionic surfactants, have average pore-size diameters in the range from 28 to 130 Å. Cytochrome c was found to bind to all MPS investigated, with the pore diameter of the material, which was measured by N 2 gas adsorption, being crucial to mesopore penetration. The adsorption of a range of proteins with isoelectric points between 1 and 10 was investigated. For adsorption to occur, the surface charges of the protein and of the MPS must be complementary, in addition to the requirement that the pore diameter be sufficiently large. Pepsin at pH 6.5, for example, is negatively charged and does not adsorb onto cyano-modified silicate whereas subtilisin, which is of a similar size and bears an overall positive charge, is adsorbed. Using resonance Raman spectroscopy, cytochrome c was observed to occur in both high spin and low spin states, in contrast to that in solution, where the protein is predominantly in the low spin state. The presence of the high spin state may account for the enhanced peroxidative activity of the adsorbed protein.
Journal of Physical Chemistry B, 2004
This study characterizes the CO rebinding kinetics after photodissociation of horse heart myoglobin (Mb) and human R-state hemoglobin (Hb) encapsulated in wet silica gels, in the presence of various concentrations of glycerol. The geminate yield for HbCO is scarcely affected by the gel matrix, indicating that the protein can fluctuate as in a homogeneous solution. On the contrary, the geminate yield for gel-embedded MbCO is much higher than that in solution, suggesting that the gel matrix inhibits the movements of the protein. The geminate yield for both proteins increases substantially with the addition of glycerol to the bathing solution. The observed kinetics could be rationalized using a simple three-state model. Rate constants have been modeled using a modified Kramers equation, which indicated that the gel exerts an internal friction on the elementary rate constants. The rate constant for geminate rebinding, k CA , is essentially viscosity independent below ∼20 cP for both proteins. The internal friction for the ligand escape rate, k CS , is much smaller and is found to be negligible for HbCO and ∼5 cP for MbCO. The activation barrier for k CS increases with glycerol concentration in response to increased viscosity and reduced ligand solubility. The rate k SC showed a complex behavior that reflects the opposing effects of viscosity and activity arising from molecular confinement and crowding. Accordingly, the corresponding activation barriers show a biphasic behavior, with a minimum at ≈40% glycerol for HbCO and at ≈75% glycerol for MbCO. The results highlight the potential of silica gel encapsulation for in Vitro studies aimed to reproduce the crowded and confined environment experienced by proteins in ViVo. The diverse response to encapsulation of Mb and Hb could actually reflect physiologically relevant functional properties escaping detection in the diluted solutions normally used for biophysical investigations.
Journal of Biomedical Optics, 2005
Evanescent wave cavity ring-down spectroscopy (EW-CRDS) is used to observe the adsorption isotherm for hemoglobin (Hb) from controlled urine samples to assess the potential for rapid diagnosis in hemoglobinuria. The absorbance of Hb at 425 nm is monitored using an alexandrite laser-pumped, room temperature, LiF:F 2 ϩ** color-center pulsed laser. A minimum absorbance detection level of 2.57ϫ10 Ϫ4 is achieved, corresponding to a minimum detectable concentration of Hb in urea of 5.8 nM. A multilayered Hb biofilm is formed, and a minimum of eight layers are required to model the adsorption isotherm, allowing for cooperative binding within the layers and extending 56 nm into the interface. A binding constant for Hb to silica 18.23Ϯ7.58ϫ10 6 M is derived, and a binding constant for Hb to Hb in subsequent layers is determined to be 5.631Ϯ0.432 ϫ10 5 M. Stoichiometric binding coefficients of 1.530Ϯ0.981 for layer one and 1.792Ϯ0.162 for subsequent layers suggest that cooperative binding both to the silica surface and between the layers of the biofilm is important.
Protein adsorption in fused-silica and polyacrylamide-coated capillaries
ELECTROPHORESIS, 2005
The model proteins cytochrome c, myoglobin, ovalbumin, and b-lactoglobulin were investigated with regard to their adsorption properties on capillaries for electrophoresis. The model compounds were selected to cover a wide range of properties. Cytochrome c is a basic protein (isoelectric point (pI): 9.6; M r : 11.7 kDa), b-lactoglobulin is rather acidic (pI: 5.4, M r : 18.4 kDa), myoglobin was chosen as a neutral reference protein (pI: 6.8-7.4, M r : 17.8 kDa), and ovalbumin (pI: 5.1, M r : 45.0 kDa) was selected as a relatively larger analyte. First, the pH dependence of adsorption was investigated for the bare fused silica. A clear correlation to the respective pIs was noted. For myoglobin and ovalbumin, none or negligible adsorption was found above the pI, whereas strong adsorption was noted just below this parameter. Cytochrome c and b-lactoglobulin already showed distinct adsorption above their pIs. However, none of the proteins showed any significant adsorption more than one pH unit above the pIs. For linear polyacrylamide-coated capillaries, a decreased but not a complete lack of adsorption was observed. Here, pH-dependent adsorption was noted as well. Regeneration of the capillaries by rinsing with buffers containing 200 mM SDS was also investigated. This method was completely successful for myoglobin, but that too for only freshlyadsorbed protein. After a storage time of 24 h and due to the aging of the adsorbate, a sufficient regeneration was no longer possible*.
Competitive adsorption of lysozyme and myoglobin on mesostructured cellular foam silica
Microporous and Mesoporous Materials, 2015
A model of multicomponent multilayer adsorption has been developed to describe the simultaneous adsorption of lysozyme (14.3 kDa, pI=11.35) and myoglobin (17.6 kDa, pI=7.2) on mesostructured cellular foam (MCF) silica in 0.02 M potassium phosphate buffer at pH 7 and 25⁰C. The mechanism of adsorption was proposed based on batch adsorption isotherms and flow microcalorimetry (FMC) traces for the enthalpy of adsorption. Thermodynamic derivations of Guggenheim-Anderson-deBoer (GAB) and Langmuir models were used to develop the model based on the proposed mechanism. Regression analysis demonstrated the effect of each of the proposed mechanistic equations.