James N. Herron

Micellar delivery of doxorubicin and its paramagnetic analog, ruboxyl, to HL-60 cells: effect of micelle structure and ultrasound on the intracellular drug uptake

The effect of Pluronic P-105 micelle structure and ultrasound on the uptake of two anthracycline drugs, doxorubicin and its paramagnetic analogue, ruboxyl, by HL-60 cells was investigated. Pluronic micellization was studied over the temperature range of 25-42 degrees C using the EPR and fluorescence spectroscopy. In the presence of Pluronic P-105 at concentrations corresponding to unimers (or loose aggregates), drug uptake by HL-60 cells was enhanced, apparently due to the effect of the polymeric surfactant on cell membrane permeability. At Pluronic concentrations corresponding to the formation of dense micelles with hydrophobic cores, drug uptake was substantially decreased. However, insonation with 70 kHz ultrasound enhanced the intracellular uptake of drugs encapsulated in dense Pluronic micelles. These findings may provide for developing a new technique of drug targeting by encapsulating the drug in micelles to prevent unwanted interactions with healthy cells and focusing ultrasound on a tumor to enhance drug uptake at the tumor site.

Biosensing based upon molecular confinement in metallic nanocavity arrays

We describe the basis for an affinity biosensor platform in which enhanced fluorescence transduction occurs through the optical excitation of molecules located within metallic nanocavities. These nanocavities are about 200 nm in diameter, are arranged in periodic or random two-dimensional arrays, and are fabricated in 70 nm thick gold films by e-beam lithography using negative e-beam resist. The experimental results show that both periodic and randomly placed metallic nanocavities can be used to enhance the fluorescence output of molecules within the cavities by about a factor of ten. In addition, the platform provides isolation from fluorescence produced by unbound species, making it suitable for real-time detection. Finally, we demonstrate the use of the platform in the real-time detection of 20-base oligonucleotides in solution.

Analytical Approaches to the Study of Monoclonal Antibody Stability

The stability of two purified monoclonal antibodies, MN12 and WT31, was investigated. The monoclonal antibodies were incubated for 32 days at different pH values (ranging from 3.0 to 10.0) at 4 and 37°C. Various analytical methods were used to assess changes in physicochemical properties of the proteins. The monoclonal antibodies were more susceptible to degradation at 37°C than at 4°C. At low pH irreversible precipitation occurred. Decomposition of the proteins was enhanced at increasing pH values in the alkaline range. This was concluded from mouse IgG-specific and antigen-specific enzyme-linked immunosorbent assays, flow cytometry, analytical gel permeation chromatography, sodium dodecyl sulfate–polyacrylamide gel electrophoresis, isoelectric focusing, and immunoblotting. No substantial change in the apparent affinity constant of MN12 was observed, as determined by an affinity enzyme-linked immunosorbent assay. Fluorescence spectra, fluorescence polarization values, and fluorescence quenching parameters of MN12 and WT31 were not substantially affected, indicating that no major irreversible conformational changes had occurred. It was concluded that each of the techniques used has only limited value for stability assessment of monoclonal antibodies and, hence, that the application of several analytical techniques is essential to gain insight into monoclonal antibody stability.

A search for site-filling ligands in the Mcg Bence-Jones dimer: crystal binding studies of fluorescent compounds.

In trigonal crystals grown in 1.9 M ammonium sulfate buffered at pH 6.2, the Mcg light-chain (Bence-Jones) dimer has a highly aromatic binding cavity accessible to a wide range of hydrophobic and aromatic ligands. A search was made for site-filling ligands by diffusing compounds into the crystals and determining their locations, orientations and relative occupancies by difference Fourier analysis at 2.7-A resolution. 1-Anilinonaphthalene-8-sulfonate, a small ligand in comparison with the rest of the series, initially occupied a site in the main binding cavity. With time, however, this ligand changed its position to the deep binding pocket beyond the floor of the main cavity. The original binding site remained vacant, despite the presence of a large excess of ligand in the soaking solution. Ligands increasing in size from fluorescein to bis(N-methyl)acridine (lucigenin) to dimers of carboxytetramethylrhodamine were found to bind with stringent stereospecificity in the main cavity, but the mode of binding was different in each case. The dimer of the 6-isomer of carboxytetramethylrhodamine, in which the two carboxyl groups are in para positions on the phenyl moiety, proved to be an effective site-filling ligand. The differences in the binding properties of dimers of 5- and 6-carboxytetramethylrhodamine led to an explanation for isomeric discrimination in the binding site. There were extensive conformational changes in the binding cavity to accommodate the ligands, particularly 6-carboxytetramethylrhodamine. The second and third hypervariable loops proved very flexible, and moved in ways to expand the binding site. The side chains of key tyrosine and phenylalanine residues in the site were also highly mobile. Their orientations adjusted to optimize complementarity with the ligands. These conformational adjustments are consistent with the tenets of a limited neo-instructive theory of ligand binding.

High resolution structures of the 4–4-20 Fab-fluorescein complex in two solvent systems: effects of solvent on structure and antigen-binding affinity

Three-dimensional structures were determined for three crystal forms of the antigen binding fragment (Fab) of anti-fluorescein antibody 4-4-20 in complex with fluorescein. These included 1) a triclinic (P1) form crystallized in 47% (v/v) 2-methyl-2,4-pentanediol (MPD); 2) a triclinic (P1) form crystallized in 16% (w/v) poly(ethylene glycol), molecular weight 3350 (PEG); and 3) a monoclinic (P21) form crystallized in 16% PEG. Solvent molecules were added to the three models and the structures were refined to their diffraction limits (1.75-A, 1.78-A, and 2.49-A resolution for the MPD, triclinic PEG, and monoclinic PEG forms, respectively). Comparisons of these structures were interesting because 4-4-20 exhibited a lower antigen-binding affinity in 47% MPD (Ka = 1.3 x 10(8) M-1) than in either 16% PEG (Ka = 2.9 x 10(9) M-1) or phosphate-buffered saline (Ka = 1.8 x 10(10) M-1). Even though the solution behavior of the antibody was significantly different in MPD and PEG, the crystal structures were remarkably similar. In all three structures, the fluorescein-combining site was an aromatic slot formed by tyrosines L32, H96, and H97 and tryptophans L96 and H33. In addition, several active site constituents formed an electrostatic network with the ligand. These included a salt link between arginine L34 and one of fluoresceins enolate oxygen atoms, a hydrogen bond between histidine L27d and the second enolic group, a hydrogen bond between tyrosine L32 and the phenylcarboxylate group, and two medium range (approximately 5 A) electrostatic interactions with lysine L50 and arginine H52. The only major difference between the triclinic MPD and PEG structures was the degree of hydration of the antigen-combining site. Three water molecules participated in the above electrostatic network in the MPD structure, while eight were involved in the PEG structure. Based on this observation, we believe that 4-4-20 exhibits a lower affinity in MPD due to the depletion of the hydration shell of the antigen-combining site.

Femtomolar sensitivity using a channel-etched thin film waveguide fluoroimmunosensor

A dual channel, evanescent fluoroimmunoassay format is used to detect femtomolar analyte concentrations (i.e. less than 1 part per trillion [w/w]) on an etched channel siliconoxynitride thin film integrated optical waveguide. Two assays are used to demonstrate the dose-response behaviour of the sensor: (1) a direct assay of a fluorescently-labeled protein ligand binding to an immobilized protein receptor, and (2) an indirect sandwich assay of a non-fluorescent protein ligand binding to an immobilized protein receptor, as detected by the binding of a fluorescently-labeled secondary receptor protein. A red-emitting cyanine dye (Cy-5), which minimized background fluorescence and scatter losses of the waveguide, was used in both assays. To our knowledge, this is the first report of femtomolar sensitivity in an immunosensing instrument.

The binding of opioid peptides to the Mcg light chain dimer: Flexible keys and adjustable locks☆

Enkephalins and beta-casomorphins (opioid peptides) were found to bind in a variety of conformations to a human light chain (Bence-Jones) dimer from a patient (Mcg) with amyloidosis. The peptides were diffused into crystals of the protein and their positions, relative occupancies and modes of binding were determined at 2.7 A resolution by difference Fourier analyses. Collectively, the opioid peptides occupied practically all of the available space in the concave, internal parts of the binding region, as well as flat or convex external surfaces around the rim of the binding cavity. Suitable ligands ranged in size from four to seven residues. As many as five residues could be accommodated inside the binding region, and there was space for at least four residues on the external surfaces. External binding was influenced by solvent effects and local packing interactions among adjacent protein molecules in the crystal lattice. In the enkephalin series the presence of amino-terminal tyrosine was necessary, but not sufficient for binding. [Met]-enkephalin, a pentapeptide, showed two different modes of binding in overlapping subsites. In one subsite, preferred over the second in a ratio of 1.3:1.0, the side chain of amino-terminal tyrosine penetrated through the floor of the main cavity to lodge in the deep binding pocket about 20 A from the entrance. The remainder of the peptide spanned the length of the main cavity in an extended conformation. In the second subsite the amino end was restricted to the main cavity and the peptide backbone turned abruptly upward at residue 3 to interact with external surfaces. An (Arg-6, Phe-7) heptapeptide extension of [Met]-enkephalin entered the deep pocket and assumed an extended conformation in the main cavity like the pentapeptide. Its last two residues flattened against the external surfaces. [Leu]-enkephalin and its analogues displayed a combination of internal and external binding like [Met]-enkephalin in its secondary subsite. Enkephalin analogues with D-amino acids in position 2 generally adopted conformations which were more convoluted than those in the L-isomers. Moreover, external interactions tended to be more prominent in the D-derivatives. The beta-casomorphin-7 heptapeptide penetrated into the deep pocket and traversed the main cavity in as extended a conformation as the presence of two proline residues would permit. On removal of the ligand there was an unexpected hysteresis effect involving permanent structural alterations in the walls of the binding region. beta-casomorphins-4 and -5 were bound in the main cavity with the carboxyl ends protruding from the entrance.(ABSTRACT TRUNCATED AT 400 WORDS)

Physical methods to characterize pharmaceutical proteins

Application of Fluorescence Spectroscopy for Determining the Structure and Function of Proteins W. Jiskoot, et al. Structural Information on Proteins from Circular Dichroism Spectroscopy: Possibilities and Limitations M. Bloemendal, W.C. Johnson, Jr. Fourier Transform Infrared Spectroscopy Investigations of Protein Structure E.A. Cooper, K. Knutson. Two, Three, and Four-dimensional Nuclear Magnetic Resonance (NMR) Spectroscopy of Protein Pharmaceuticals D.G. Vander, Velde et al. Thermodynamic Strategies for Rational Protein and Drug Design K.P. Murphy, E. Freire. Chromatographic Techniques for the Characterization of Proteins J.J.M. Hothuis, R.J. Driebergen. Capillary Electrophoresis of Proteins T.A.A.M. van de Goor. Applying Genetic Engineering to the Structural Analysis of Proteins P.T. Hamilton. Index.

Properties of Immobilized PEG Films and the Interaction with Proteins

Poly(ethylene oxide), or as it is frequently denoted in the literature, poly-(ethylene glycol) (PEG), is a nonionic, water-soluble polymer widely used for stabilizing colloids in food and paints and in formulating pharmaceuticals and cosmetics. The reason for the extensive use of this polymer is that it acts as a dispersant and yet is inert, e.g., it does not interfere adversely with other functional ingredients in the dispersion.

Comparison of site-specific coupling chemistry for antibody immobilization on different solid supports

Abstract Silica is an important chromatographic support for high-performance affinity chromatography due to its high mechanical stability. However, silica is very different from traditional gel chromatographic materials, such as agarose, dextran, and polyacrylamide, with respect to many chemical and physical properties. Thus, it is expected that different immobilization techniques must be used for orienting the immobilized ligand on the surfaces of the two substrates. In this study, a site-specific coupling chemistry for immobilization of antibodies on modified silica surfaces and on agarose gels was investigated. The effects of substrates on the orientations of immobilized native and partially denatured antibodies was deduced. An important conclusion is that non-covalent interactions (physical adsorption) dominate the orientation of immobilized antibodies on silica surfaces.