Abraham M. Lenhoff

Porous silica via colloidal crystallization

Microstructured porous silicas have potential applications in catalysis, separations, coatings, microelectronics and electro-optics, but methods for producing materials with uniform submicrometre pores have not been available. We have now developed a method in which modified colloidal crystals are used as templates for silica polymerization. This method yields products with highly uniform and structured pores of tuneable size in the submicrometre region.

Materials: A class of porous metallic nanostructures

Colloidal crystals are ordered arrays of particles in the nanometre-to-micrometre size range. Useful microstructured materials can be created by replicating colloidal crystals in a durable matrix that preserves their key feature of long-range periodic structure. For example, colloidal crystals have been used to fabricate structures from inorganic oxides, polymers, diamond and glassy carbon, and semiconductor quantum dots, and some structures have photonic properties or are patterned on different hierarchical length scales. By using colloidal crystals as templates, we have synthesized a new class of metallic materials with long-range nano-scale ordering and hierarchical porosity.

PROTEIN INTERACTIONS IN SOLUTION CHARACTERIZED BY LIGHT AND NEUTRON SCATTERING : COMPARISON OF LYSOZYME AND CHYMOTRYPSINOGEN

The effects of pH and electrolyte concentration on protein-protein interactions in lysozyme and chymotrypsinogen solutions were investigated by static light scattering (SLS) and small-angle neutron scattering (SANS). Very good agreement between the values of the virial coefficients measured by SLS and SANS was obtained without use of adjustable parameters. At low electrolyte concentration, the virial coefficients depend strongly on pH and change from positive to negative as the pH increases. All coefficients at high salt concentration are slightly negative and depend weakly on pH. For lysozyme, the coefficients always decrease with increasing electrolyte concentration. However, for chymotrypsinogen there is a cross-over point around pH 5.2, above which the virial coefficients decrease with increasing ionic strength, indicating the presence of attractive electrostatic interactions. The data are in agreement with Derjaguin-Landau-Verwey-Overbeek (DLVO)-type modeling, accounting for the repulsive and attractive electrostatic, van der Waals, and excluded volume interactions of equivalent colloid spheres. This model, however, is unable to resolve the complex short-ranged orientational interactions. The results of protein precipitation and crystallization experiments are in qualitative correlation with the patterns of the virial coefficients and demonstrate that interaction mapping could help outline new crystallization regions.

Pore size distributions of cation-exchange adsorbents determined by inverse size-exclusion chromatography

The pore dimensions, pore size distributions, and phase ratios were determined for a set of cation-exchange adsorbents using inverse size-exclusion chromatography (ISEC). The adsorbents examined represent a diverse set of materials from Pharmacia, TosoHaas, BioSepra, and EM Industries, which are widely used for protein purification. The ISEC was carried out using dextran standards with relative molecular masses of 180-6,105,000. This technique provided a comparative characterization of the accessible internal pore surface area, as a function of solute size, for the adsorbents tested. Adsorbent preparation strategies in which polymers are generated in situ or grafted onto base materials were found to have significant effects on pore dimensions and phase ratios.

Molecular Origins of Osmotic Second Virial Coefficients of Proteins

The thermodynamic properties of protein solutions are determined by the molecular interactions involving both solvent and solute molecules. A quantitative understanding of the relationship would facilitate more systematic procedures for manipulating the properties in a process environment. In this work the molecular basis for the osmotic second virial coefficient, B22, is studied; osmotic effects are critical in membrane transport, and the value of B22 has also been shown to correlate with protein crystallization behavior. The calculations here account for steric, electrostatic, and short-range interactions, with the structural and functional anisotropy of the protein molecules explicitly accounted for. The orientational dependence of the protein interactions is seen to have a pronounced effect on the calculations; in particular, the relatively few protein-protein configurations in which the apposing surfaces display geometric complementarity contribute disproportionately strongly to B22. The importance of electrostatic interactions is also amplified in these high-complementarity configurations. The significance of molecular recognition in determining B22 can explain the correlation with crystallization behavior, and it suggests that alteration of local molecular geometry can help in manipulating protein solution behavior. The results also have implications for the role of protein interactions in biological self-organization.

Rapid measurement of protein osmotic second virial coefficients by self-interaction chromatography.

Weak protein interactions are often characterized in terms of the osmotic second virial coefficient (B(22)), which has been shown to correlate with protein phase behavior, such as crystallization. Traditional methods for measuring B(22), such as static light scattering, are too expensive in terms of both time and protein to allow extensive exploration of the effects of solution conditions on B(22). In this work we have measured protein interactions using self-interaction chromatography, in which protein is immobilized on chromatographic particles and the retention of the same protein is measured in isocratic elution. The relative retention of the protein reflects the average protein interactions, which we have related to the second virial coefficient via statistical mechanics. We obtain quantitative agreement between virial coefficients measured by self-interaction chromatography and traditional characterization methods for both lysozyme and chymotrypsinogen over a wide range of pH and ionic strengths, yet self-interaction chromatography requires at least an order of magnitude less time and protein than other methods. The method thus holds significant promise for the characterization of protein interactions requiring only commonly available laboratory equipment, little specialized expertise, and relatively small investments of both time and protein.

Structured metallic films for optical and spectroscopic applications via colloidal crystal templating

áeñ = 2.22 in the infiltrated one (reducing the contrast at the same time). After inversion the mean dielectric constant decreases to áeñ = 1.41 and, accordingly, the L pseudogap energy shifts upwards (see Fig. 5). The peak width is a function of both the dielectric contrast and the filling factor of the structure. Bare opals present a contrast eSiO2/eair = 2.1 that shifts to epolymer/eSiO2 = 1.24 when infiltration takes place. So, the pseudogap width is largely decreased, as is observed in both the experiment and band structure calculation. When inversion occurs, the dielectric contrast is increased up to epolymer/eair = 2.6. It has to be noticed that, although bare and inverse opal have similar values of the refractive index contrast, inverse opals show a much broader pseudogap than the direct opal structure which reflects the fact that inverse structures are more powerful scatterers (see Fig. 5A). When the sample is tilted with respect to normal incidence, the k vector ceases to be collinear with C±L. For a given direction (tilt angle), at some point of the energy scan, k crosses the Bragg plane and a reflection is obtained. Since L is the closest (to C) point of the Bragg plane, tilting increases both the wavevector length and the energy for which reflection occurs. This pseudogap energy position, can be followed along the L±U (or L±K or L±W) line in the Brillouin zone. In Figure 5C experimental data are superimposed on the band structure diagram by using Snells law with an average refractive index for calculating the internal angle (with respect to the C±L direction). The theory gives a good account of the behavior of the pseudogap position. In summary, we have obtained and optically analyzed polymer inverse opals with a long-range order. Their photonic crystal behavior has been studied both experimentally and theoretically, and a good agreement between band-structure calculations and experiments was found. From a fundamental point of view, they can be regarded as model systems, where studying the effect of topology and dielectric contrast is possible. Regarding their potential applications, they can be used to modify the emission properties of luminescent species, such as dyes, that can easily be incorporated into the polymer. Polymer inverse opals offer, in turn, the interesting possibility of being used as matrices to obtain new spherical colloidal particles, whose shape cannot be controlled otherwise, from different materials.

Why is the osmotic second virial coefficient related to protein crystallization

Abstract A molecular basis is presented for characterizing the osmotic second virial coefficient, B 22 , of dilute protein solutions, which provides a measure of the nature of protein–protein interactions and has been shown to be correlated with crystallization behavior. Experimental measurements of the second virial coefficient of lysozyme and bovine α -chymotrypsinogen A were performed by static light scattering, as a function of pH and electrolyte concentration. Although some of the trends can be explained qualitatively by simple colloidal models of protein interactions, a more realistic interpretation based on protein crystallographic structures suggests a different explanation of experimental trends. The interactions accounted for are solute–solute excluded volume (steric), electrostatic and short-range (mainly van der Waals) interactions. The interactions depend strongly on orientation, and this profoundly affects calculated second virial coefficients. We find that molecular configurations in which complementary surfaces are apposed contribute disproportionately to the second virial coefficient, mainly through short-range interactions; electrostatic interactions play a secondary role in many of these configurations. Thus molecular recognition events can play a role in determining the solution thermodynamic properties of proteins, and this provides a plausible basis for explaining the observed relationship with crystallization behavior.

Comparison of protein adsorption isotherms and uptake rates in preparative cation-exchange materials

Adsorption isotherms and effective diffusivities of lysozyme in a set of six preparative cation-exchange stationary phases were determined from batch uptake data in a stirred vessel. Both a pore diffusion and a homogeneous diffusion model were used in estimating diffusivities, with the isotherms fitted to a non-Langmuirian analytical isotherm equation. The capacities inferred from the isotherms are found to be correlated with the surface area accessible to lysozyme, the effective surface concentrations obtained being in agreement with values measured by different methods in various non-chromatographic systems. The pore diffusivities show systematic trends with protein and salt concentration, and effects of pore size and connectivity are also evident. Some trends in the homogeneous diffusivities are quite different to those in the pore diffusivities, but these differences largely disappear when the homogeneous diffusivities are rescaled to account for adsorption equilibrium behavior. Additional information is required to elucidate further the mechanisms of coupled diffusion and adsorption in stationary phases.

Determinants of protein retention characteristics on cation-exchange adsorbents

There are currently a large number of commercially available strong and weak cation-exchange adsorbents for preparative protein purification, typically prepared by coupling charged ligands to a mechanically rigid porous bead. Because of the diverse chemical nature of the base matrix (carbohydrate, synthetic polymer, inorganic) and the coupling and ligand chemistry, cation-exchange adsorbents from different suppliers can differ substantially in chemical surface properties and physical structure. The differences in chemical properties can be in ionic capacity, hydrophobicity, the presence of hydrogen bond donors/acceptors, and the nature of the charged functional groups. In order to probe the effects of these factors on protein affinity, the isocratic retention of a set of model proteins was examined on a set of cation-exchange adsorbents to obtain a quantitative assessment of retention differences between adsorbents. Two adsorbent factors were found to be the dominant determinants of overall protein retention: the anion type and the adsorbent pore size distribution. Protein retention on strong cation-exchangers was found to be greater than that on corresponding weak cation-exchangers. Protein retention was increased on adsorbents with pore size distributions that include significant amounts of pore space with dimensions similar to those of the protein solute.