PubMed · 14071011
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R TERANISHI, T R MON. 1963. LARGE DIVIDED COLUMNS.. https://doi.org/10.1016/s0021-9673(01)83706-7
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Supercoiled plasmid DNA was selectively purified from its open circular form by thiophilic interaction chromatography, performed in the presence of high concentrations of water-structuring salts. To identify optimal conditions for purification, various aromatic thioether ligands were coupled to a chromatographic support and screened for their ability to separate plasmid isoforms from each other and from other host cell contaminants, including RNA, genomic DNA, protein, and endotoxins. Selectivity of the chromatographic medium depended on the structure of the ligands, with characteristics of the substituents on the aromatic ring determining the resolution between the different plasmid DNA isoforms. Optimal resolution was obtained with ligands consisting of an thioaromate, substituted with highly electronegative groups. When 2-mercaptopyridine was used as a ligand, the difference in conductivity for eluting open circular and supercoiled plasmid DNA is only 6 mS/cm. However, with 4-nitrothiophol the resolution for plasmid DNA separation on the media increased, resulting in a 20 mS/cm difference. When used in combination with a prior group separation step, these aromatic thioether ligands facilitated the isolation of highly purified supercoiled plasmid DNA, suitable for use in gene therapy and DNA vaccine applications.
Human tumor necrosis factor-alpha (TNF-alpha) is a trimeric protein consisting primarily of beta-sheet. GdnHCl-induced unfolding of TNF-alpha was investigated at room temperature by circular dichroism (CD) and size exclusion chromatography. The secondary and tertiary structure of TNF-alpha persisted up to 0.9N GdnHCl regardless of incubation time, but, in the range of 1.2 N to 2.1 N GdnHCl, there was loss of tertiary structure accompanied by the formation of an alpha-helix, as revealed by far- and near-UV CD spectra. The structural changes occurred gradually in 1.2 and 2.1 N GdnHCl, but were rapid in 1.5 and 1.8 N GdnHCl. The GdnHCl-induced state of TNF-alpha is an unfolded, alpha-helical aggregate of about 130 monomers, as shown by size exclusion chromatography. We suggest the most likely pathway for the transition from beta-sheet to alpha-helix.
Both spatial and temporal peaks that are produced by the discrete parcel model can be mathematically approximated by Gaussian functions, but the transformation from a spatial pattern to a temporal image requires a convolution treatment. A first-order convolution is given for temporal peaks under a linear isotherm, whereas a second-order convolution is proposed for those under non-linear isotherms. Numerical tests show that the peak shapes generated by the proposed temporally convoluted Gaussian equations (TCG) match perfectly with those obtained by the discrete parcel model. Although the full TCG equation may be quite complicated, it can be made easier by a recursion calculation technique, and a group of peak curves can be plotted simultaneously on computer worksheet. The results also suggest that the temporal distortion effect should be predominately considered, in addition to those known-to-exist spatial effects, for explaining the peak asymmetry.