The staff (and food) of The Collegiate was also an integral part of my graduate school experience. The estimated diametral distributions for the SAS batch of glass microspheres as a function of the interval. SEM image of the 20-10-70 SLS composition of glass 144 microspheres used for the protein adsorption study.
Ribbon representation of the structure of HSA as 145 determined by He and Carter (PDB file 1UOR). Ramachandran plot of dihedral angles of BPTI before 188 and after 20 ps of simulated adsorption.
Introduction
The electrostatic contribution to the change in free energy that occurs during the interaction of a sphere of radius R and a flat plate is given by. However, Norde has noted that protein adsorption can still occur even under conditions where strong electrostatic repulsion should exist.22 This observation is attributed to the overwhelming influence of hydrophobic forces associated with nonpolar groups and hydrogen bonds. These electron donor and acceptor components can then be expressed in terms of their contribution to the surface.
These electric charges develop due to hydrogen association/dissociation reactions that occur between the protein and/or glass and the surrounding aqueous medium.27-29 Hydrogen bonding can occur due to the presence of hydroxyl species, such as silanol groups, on the glass surface. Brash, "The Vroman Effect in Tube Geometry: Effect of Flow on Protein Adsorption Measurements," J.
Techniques for Characterizing the Adsorption of Proteins at Solid-Liquid Interfaces
RAY PHOTOELECTRON SPECTROSCOPY
The amount of protein adsorbed onto a surface can be determined from the ratio of the N and C signals, as given by. Although the quantitative determination of the amount of adsorbed protein is unlikely to be greatly affected by drying, studies of protein orientation should therefore be carefully examined. The efficiency and accuracy of the electrophoretic separation process can be improved by unfolding the proteins in a sample.
This unfolding or denaturation of the polypeptide can be most easily achieved by boiling the proteins in the presence of a surfactant. The nonpolar segment of the anionic species binds to the denatured protein, and the polar sulfate group is directed outward into the solution. The faceplate is actually made up of a polyacrylamide gel (shown in yellow) sandwiched between two plastic sheets (shown in grey).
The first half of the electrical circuit is formed from the cathode to the anode via the polyacrylamide gel which is saturated with an electrolyte solution (a tris-glycine buffer solution is common). However, it is often the function of the adsorbed protein that is really of interest. The intensity of the reflected infrared radiation will also depend on the possible presence of absorbing species such as proteins at the solid-liquid interface.
This indicated that even after the shortest exposure time of 5 seconds, proteins were irreversibly adsorbed to the surface of the germanium crystal despite extensive rinsing. A second disadvantage of ATR-FTIR is the generally irrelevant surface chemistry of the reflection element. The first method uses the intrinsic fluorescent properties of the amino acids tryptophan (Trp) and tyrosine (Tyr).
Production and Surface Area Estimation of Glass Microspheres
A Micromeritics Accupyc 1330 pycnometer was used to determine the density of each batch of glass microspheres. The specific surface area, SS, of each batch of glass microspheres was simply determined by The rationale for choosing an interval size of 10 µm to create a diametrical distribution of glass microspheres is shown in Figure 3.21.
Distribution data for 20-80 SS glass microspheres: (a) Cumulative normal probability plot and (b) Approximate diametric distribution and lognormal model fit. Distribution data for 15-5-80 SAS glass microspheres: (a) Cumulative normal probability plot and (b) Approximate diametric distribution and lognormal model fit. Distribution data for 15-5-80 SLS glass microspheres: (a) Cumulative normal probability plot and (b) Approximate diametric distribution and lognormal model fit.
Distribution data for 30–70 SS glass microspheres: (a) Cumulative normal probability plot and (b) Estimated diametrical distribution and lognormal model fit. Distribution data for 25-5-70 SAS glass microspheres: (a) Cumulative normal probability plot and (b) Estimated diametrical distribution and lognormal model fit. Distribution data for 15-15-70 SAS glass microspheres: (a) Cumulative normal probability plot and (b) Estimated diametric distribution and lognormal model fit.
Distribution data for Pyrex® glass microspheres: (a) Cumulative normal probability plot and (b) Estimated diametral distribution and normal model fit. The estimated diametral distributions for the 15-15-70 SAS batch of glass microspheres as a function of the interval size used to generate the percentage frequency histograms. Comparison of (a) initial and (b) smoothed percent frequency, surface area, and volume distributions for 15-5-80 SLS glass microspheres.
Effect of Glass Corrosion on the Detection of Proteins by Silver Staining
The result of this ion exchange reaction is an increase in the pH of the solution. Corrosion samples were prepared by weighing between 15.0 and 15.5 mg of glass microspheres into a 1.5 mL microfuge tube. The pH of the various mixtures at 25 °C was also determined using a calomel electrode saturated with KCl.
Finally, the reaction of uncorroded glass microspheres with LSB resulted in a greater increase in the pH of LSB in each case. It should also be noted that the "craters" present on the surface of the microsphere in Figure 4.3(a) are actually internal pores that are initially present inside the 30-70 SS samples and are later revealed during dissolution.
It is possible that the elevated pH of the HSA samples prepared with LSB supernatant from the 30–70 SS corrosion tests offset the acidic environment and prevented complete fixation of the protein within the polyacrylamide gel. This suggests that the pH of the LSB is the most important factor in determining the final intensity of the HSA stained band. In particular, the leaching of Na+ ions from the glass causes an undesirable increase in the pH of the surrounding LSB matrix.
The high pH of the LSB solution could neutralize the acidic irrigation medium and prevent effective gel fixation. Subsequent imaging of HSA added to the corrosion supernatant showed a gradual decrease in the intensity of the stained protein band as the pH of the LSB increased. Replacing the LSB that had reacted with the 30-70 SS glass microspheres with pure LSB resulted in increased sample pH and increased staining intensity.
Adsorption of Albumin onto Simple Multi-Component Glass Microspheres
The filter was removed from the microfuge tube and 250 µL of Laemmli Sample Buffer (LSB) was added to the top of the glass microspheres. We measured the initial masses of the microfuge tubes containing the glass microspheres and the remaining buffer solution. However, a faint band appears after 60 seconds of stirring, indicating that the adsorbed protein has been removed from the surface of the glass microspheres.
Figures 5.23 (a) and (b) show the HSA adsorption capacity of the SAS and SLS glass microspheres as a function of CaO and Al2O3 content. The replacement of Na2O with Al2O3 results in an immediate reduction in the tendency of the glass microspheres to leak Na+ ions into solution. This observation can be understood based on the role Al3+ plays in the glass network.
The formation of BO increases the bonding of the glass network and subsequently improves the chemical stability. The dissolution of glass microspheres has several important consequences from the point of view of the analysis of protein adsorption results. Aqueous corrosion of glass causes an irreversible change in the surface composition, especially in the alkali content.
These arguments can be used to explain the initial trends observed in the HSA adsorption capacities of SAS and SLS glasses. SEM image of the composition of 20-10-70 SLS glass microspheres used for the protein adsorption study. Ribbon representation of the structure of HSA as determined by He and Carter (PDB file 1UOR).
Spectra have been shifted for clarity. a) The stretching of two bands into a single band and (b) the second derivative of the refracted absorption band. SDS-PAGE analysis of HSA eluted in TBS after mechanical agitation of protein-coated 15-15-70 SLS glass microspheres for 60 and 120 s. The calibration graph is used to determine the amount of liquid retained by 50 mg of glass microspheres after filtration by centrifugation.
Amount of HSA adsorbed on (a) SAS and (b) SLS microspheres as a function of Al2O3 and CaO content. Predicted point of zero charge for the studied (a) SAS glasses and (b) SLS glasses using the model of Carre et al. Conti, “The effect of in vitro modeling conditions on the surface reactions of bioactive glasses,” J.
Papini, “Diffuse reflection spectroscopy study of granular materials and their mixtures in the mid-infrared spectral region,” Vib. Lommen, "Incorporation of alumina and boron oxide in sodium silicate glasses studied by X-ray photoelectron spectroscopy". Ionescu, "Characteristics of structure and chemical stability of microspheres and grains made from the same glass", Sprechsaal.
Patterson, "The adsorption of some proteins on hydroxylapatite and other adsorbents used for chromatographic separations," Biochim. Ishikawa, "Investigation of the particle texture dependence of protein adsorption using synthetic micrometer-sized calcium hydroxyapatite particles," Colloids Surf., B.
Methods for Analyzing Molecular Dynamics Simulations of Protein Adsorption
Similarly, an oriented hydration layer located at 2.5 Å was found along the polar region of the enkephalin molecule. A schematic diagram of the two torsion angles (otherwise known as dihedral angles) Ψ and Φ is shown in Figure 6.2. A majority of the points in the dihedral angle distribution for BPTI before simulated adsorption fall within the regions.
The remaining region of conformational space not enclosed within the thick lines corresponds to conformations that are generally very unfavorable. The percentage distribution of dihedral angles among the three regions of the Ramachandran plot is given in Table 6.1. This result may be indicative of the influence that the α-quartz surface exerts on BPTI.
One such construction is shown in Figure 6.4, in which the modulus of dihedral angle change between the native and adsorbed states after 20 ps of simulation time is plotted in a linear sequence corresponding to the amino acid residues. The observed changes in dihedral angles could possibly be explained based on the flexibility of the polypeptide chain. The figure of merit, HV, is obtained as the product of the normalized values of H and V.
Chou and Fasman investigated the structures of twelve proteins and calculated the rotational potential of the twenty amino acids using the formula. The contact map is then constructed by plotting the inter-pair distances of the amino acid sequence against itself, as given in Figure 6.7, which shows the contact map for BPTI before simulated adsorption over the α-quartz surface. The advantage of a contact map is that features of the three-dimensional protein structure are preserved as emergent patterns that indicate proximity over an extended sequence of amino acid residues.
