concanavalin-a and iminodiacetic-acid

Topics: Adsorption; Cellulose; Chelating Agents; Chromatography; Chromatography, Affinity; Concanavalin A; Copper; Glucose Oxidase; Glycoproteins; Imino Acids; Ions; Ligands; Metals; Powders; X-Ray Diffraction

Immobilized lectin chromatography can be employed for glycoprotein enrichment, but commonly used columns have limitations of yield and resolution. To improve efficiency and to make the technique applicable to minimal sample material, we have developed a nanoscale chelating Concanavalin A (Con A) monolithic capillary prepared using GMA-EDMA (glycidyl methacrylate-co-ethylene dimethacrylate) as polymeric support. Con A was immobilized on Cu(II)-charged iminodiacetic acid (IDA) regenerable sorbents by forming a IDA:Cu(II):Con A sandwich affinity structure that has high column capacity, as well as stability. When compared with conventional Con A lectin chromatography, the monolithic capillary enabled the better reproducible detection of over double the number of unique N-glycoproteins in human urine samples. Utility for analysis of minimal biological samples was confirmed by the successful elucidation of glycoprotein profiles in mouse urine samples at the microliter scale. The improved efficiency of the nanoscale monolithic capillary will impact the analysis of glycoproteins in complex biological samples, especially where only limited material may be available.

Topics: Amino Acid Sequence; Animals; Chromatography, Affinity; Concanavalin A; Copper; Glycoproteins; Humans; Imino Acids; Mice

A new procedure has been developed to immobilize iminodiacetic acid (IDA) onto the surface of silica supports, such as LiChrospher Si-1000 and 1.5-microns nonporous silica, for use in high-performance immobilized metal affinity chromatography (HPIMAC) of proteins. This IDA immobilization method has been achieved through the synthesis of a new silylation reagent, 1-(iminodiacetic acid di-tert-butylester)-3-glycidoxy-propyltrimethoxysilane (IDA-silane). Various modified silicas of different ligand densities have been prepared by using mixtures between 1 and 100% of the IDA-silane diluted with the corresponding 3-glycidoxy-propyltrimethoxysilane (GLYMO-silane). Frontal analysis was used with the IDA-Cu(II)-Concanavalin-A and IDA-Cu(II)-lysozyme systems to evaluate the capacity and the association constants for these HPIMAC sorbents. With these metal chelate sorbents the specific binding capacity per unit area increased continuously with the ligand density for the nonporous sorbents but reached a maximum at about 50% of the maximum ligand coverage for the porous sorbents. The association constant for the chelate-protein complex was highest for both concanavalin-A (Con-A) and lysozyme (HEWL) at the highest ligand density and decreased with lower ligand density. These observations have been evaluated in terms of the accessibility of histidine residues on the surface of the two test proteins and their ability to act as binding sites for the copper ions in the coordination complex. The experimental data indicate that both steric and conformational effects result in multiple classes of binding phenomena with Con-A and HEWL at high ligand concentrations. These experimental results provide a useful guideline for the design of silica-based sorbents for application in the HPIMAC of proteins.

Topics: Adsorption; Chelating Agents; Chromatography, Affinity; Concanavalin A; Copper; Evaluation Studies as Topic; Imino Acids; Ligands; Muramidase; Proteins; Silicon Dioxide

The utilisation of organosilanes to introduce active chemical groups onto zirconia surfaces, suitable for the subsequent immobilisation of proteins or other biomimetic ligands, is described. Two different types of porous zirconia-based particles with nominal pore diameters of 160 and 1000 A pore size were modified with two different affinity ligands. In the first case, methods to immobilise iminodiacetic acid-Cu(II) and its application in Cu(II) immobilised metal ion affinity chromatography (IMAC) were established. In the second series of experiments, concanavalin A was immobilised and the interaction of this lectin with the enzyme horseradish peroxidase examined. For both systems, adsorption isotherms were recorded as batch experiments. In each case, the experimental results could be fitted to langmuirean type adsorption isotherms, indicating that under the chosen conditions only one type of interaction is present, with nonspecific interactions with the support surface playing an insignificant role. These studies document the potential of surface modified zirconia particles for the immobilisation of chemical ligands or proteins for use in biospecific affinity chromatography and immobilised enzyme bioreactors.

Topics: Adsorption; Chelating Agents; Chromatography, Affinity; Chromatography, High Pressure Liquid; Concanavalin A; Horseradish Peroxidase; Imino Acids; Myoglobin; Particle Size; Porosity; Spectrophotometry, Ultraviolet; Thermodynamics; Zirconium

Concanavalin A (Con A) was immobilized via metal interactions on macroporous, microparticulate silica support having covalently bound iminodiacetic acid functions (IDA-silica) chelated with Cu(II) at the surface. The amount of copper and of Con A in the column could readily be controlled by the conditions used for chelating the metal by IDA-silica and for immobilization of the lectin. The retention behavior of columns packed with the stationary phase did not change under a wide range of elution conditions, indicating no loss of immobilized lectin. However, the Con A proper could readily be removed from the column at pH 3.0 or together with Cu(II) by perfusion with EDTA at neutral pH. Columns containing Con A immobilized by this technique exhibited dual retention behavior for proteins, glycoproteins, and carbohydrates according to the pertinent glycan-lectin or protein-metal interactions. The glycoproteins, peroxidase and alpha 1-acid glycoprotein, were retained by the Con A moiety and eluted with eluents containing competing sugars, whereas the proteins, beta-lactoglobulin, alpha-chymotrypsinogen A, and ribonuclease A and B were retained by the chelated copper and were eluted and separated with eluents containing sodium chloride or borate. Binding constants of glycosides on the immobilized Con A were evaluated chromatographically and found to be one-third to two-thirds those reported in the literature on the basis of experiments in free solution.

Topics: Carbohydrates; Chromatography, Affinity; Chromatography, High Pressure Liquid; Concanavalin A; Copper; Glycoproteins; Imino Acids; Kinetics; Proteins; Silicon Dioxide