Displaced protein characterization

Characterization of Displaced Protein. Two approaches were used simultaneously to characterize the I-thrombin displaced from heparin-PVA columns filtration on Sephadex G-200 and heparin-Sepharose affinity chromatography using the elution conditions described by Collen et al. (18) for the separation of antithrombin III from enzyme-antithrombin III complex. Radiolabelled antithrombin III displaced from the heparin-PVA column was characterized in a similar way by affinity chromatography on heparin-Sephrose. Detailed methods are presented elsewhere ( ... 

Characterization of Displaced Protein. With labelled antithrombin III, chromatography of the displaced radioactivity on heparin-Sepharose revealed that the bulk of the displaced radioactive material did not bind to heparin-Sepharose (Table II). With arvinized plasma as the displacing eluent, 65% of the antithrombin III eluted in the void volume, compared with 49% of the control I-antithrombin III (diluted in citrated plasma) that had not previously been used to inactivate thrombin the latter unbound fraction was likely labelled impurities or inhibitor modified by radiolabelling to lose its heparin affinity. With 5% (w/v) albumin used as a displacing eluent, 78% of the I-antithrombin III came out in the void volume. This increase in material that did not bind to heparin after displacement from heparin-PVA was attributed to post-complex antithrombin III, a modification of the original inhibitor resulting from the inactivation of thrombin. Neither thrombin-antithrombin III complex nor free antithrombin III were detected in the 5% (w/v) albumin displaced fractions while there was a barely detectable amount of complex (6%) and free antithrombin III (4%) in the material displaced by arvinized plasma. With the control I-antithrombin III, 25% of the radioactivity was determined to be free antithrombin III and 2% as complex. The remainder (22-27%) was not recovered from the column. 

Malmquist, G. and Lundell, N., Characterization of the influence of displacing salts on retention in gradient-elution ion-exchange chromatography of peptides and proteins, J. Chromatogr., 627, 107, 1992. 

Nearly all structurally characterized DNA polymerases are found to induce B-A transition in DNA bases adjacent to nucleotide incorporation. This induced A-conformation facilitates sufficient access for the protein to desirable contact points in the DNA minor groove. Negative base-pair displacement in the A-form... 

Displacement Chromatography in the Separation and Characterization of Proteins and Peptides... 

As outlined above displacement chromatography may find its most important uses in the analytical area. The ability to enrich trace levels of components is ideally suited to the proteomics where more powerful tools are desperately needed to address the vast concentration ranges present in order to identify trace components. The technique also offers a way to isolate large quantities of protein variants which is important for the identification and characterization of minor product-related impurities commonly associated with therapeutic proteins. 

While much has been learned about the role and selection of the operation parameters in displacement chrcmatography (60-66), little is known yet about the rules of displacer selection and the means available to control the selectivity of the separation. The paucity of well characterized displacers and the lack of knowledge of the solute adsorption isotherms hinder most strongly the wider acceptance and use of displacement chromatography. In most cases, displacer selection is still done by trial-and-error. In the majority of modem displacement chromatographic publications a reversed-phase system was used to separate small polar molecules, antibiotics, oligopeptides and small proteins (52-66). 

How rapidly diffusion occurs is characterized by the diffusion coefficient D, a parameter that provides a measure of the mean of the squared displacement x of a molecule per unit time f. For diffusion in two dimensions such as a membrane, this is given by = 4Ht. The Saffman-Delbrtlck model of Brownian motion in biologic membranes describes the relationship between membrane viscosity, solvent viscosity, the radius R and height of the diffusing species, and D for both lateral and rotational diffusion of proteins in membranes (3, 4). This model predicts for example that for lateral diffusion, D should be relatively insensitive to the radius of the diffusing species, scaling with log (1/R). 

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