Proteins size-exclusion chromatography

Mass of an Oligomeric Protein Size Exclusion Chromatography (SEC)... 

Understanding the action mechanism of flavoenzymes heavily relies on the combination of different chemical tools and techniques. First, it is of utmost importance to have a pure and stable (recombinant) protein. Size exclusion chromatography will provide information about the enzyme quaternary structure, and mass spectrometry can establish posttranslational modifications. For a detailed insight into the protein structure, well-diffracting crystals are needed to determine the X-ray structure. 

Duhin, P.L. Edwards, S.L. Mehta, M.S. Tomalia, D. Quantitation of non-ideal behavior in protein size-exclusion chromatography. J. Chromatogr. 1993, 635, 51. 

Examples of the application of size-exclusion chromatography to the analysis of proteins. The separation in (a) uses a single column that in (b) uses three columns, providing a wider range of size selectivity. (Chromatograms courtesy of Alltech Associates, Inc. Deerfield, IL). 

Column Si. Size-exclusion chromatography columns are generally the largest column on a process scale. Separation is based strictly on diffusion rates of the molecules inside the gel particles. No proteins or other solutes are adsorbed or otherwise retained owing to adsorption, thus, significant dilution of the sample of volume can occur, particularly for small sample volumes. The volumetric capacity of this type of chromatography is determined by the concentration of the proteins for a given volume of the feed placed on the column. 

The total stationary-phase volume required to process a given feed stream is proportional to the inlet concentration and volume of the feed. For example, for a typical inlet concentration of protein of 10 g/L, in a 100 L volume of feed, a column volume of at least 100 L is needed for size-exclusion chromatography. In comparison, an ion-exchange column having an adsorption capacity of 50 g/L would only require 20 L of column volume for the same feed. 

The packed columns of Shodex OHpak SB-800HQ series are packed with polyhydroxymethacrylate gels and are designed for use with high-resolution, high-speed aqueous size exclusion chromatography. The packed columns are best suited for the analysis of water-soluble polymers and proteins (Table 6.8). 

Peptides are much more difficult to run by size exclusion chromatography than proteins because their solubilities differ greatly. Their shapes also vary from linear to semidefined. For a three peptide standard, Mant and Hodges... 

For the classical form of size exclusion chromatography in organic solvents, packings based on highly cross-linked styrene-divinylbenzene are used. For SEC of polar polymers using polar or aqueous solvents, packings based on a polar methacrylate polymer are used. Diol-derivatized silica is used for the separation of proteins and other polar polymers. The different packings will be discussed in sections dedicated to their different application areas. 

Protein-Pak packings are designed for the size exclusion chromatography of proteins and related compounds. They are based on silica, which is deactivated with glycidylpropylsilane. The diol function prevents the interaction of the target analytes with the silica surface. However, because coverage of the silica surface is always incomplete, residual acidic silanols can interact with the analytes. For this reason, most applications are carried out with a salt concentration above 0.2 mol/liter, which eliminates the interaction of analytes with surface silanols. Protein-Pak packings are stable from pH 2 to pH 8. 

This section discusses in detail the column types that are available for the size exclusion chromatography of both polar and nonpolar analytes. It first discusses the various columns available for standard nonaqueous size exclusion chromatography. It then reviews the columns available for general size exclusion chromatography using aqueous mobile phases. Finally, it examines the columns designed for size exclusion chromatography of proteins and peptides. 

Special Considerations for the Size Exclusion Chromatography of Proteins... 

For the size exclusion chromatography of proteins on silica-hased diol packings, it is generally recommended to use fully aqueous mobile phases with a salt concentration between 0.1 and 0.3 M. In general, a phosphate buffer around pH 7 is used as the mobile phase. Under these circumstances, the tertiary structure of most proteins is preserved without difficulty and the interaction of proteins with each other is minimized. However, other inorganic buffers or combinations of buffers with organic solvents can be used without difficulties for special applications. 

Separation methods based on size include size exclusion chromatography, ultra-filtration, and ultracentrifugation (see Chapter Appendix). The ionic properties of peptides and proteins are determined principally by their complement of amino acid side chains. Furthermore, the ionization of these groups is pH-dependent. 

A. V. Lemmo and J. W. Jorgenson, Two-dimensional protein separation by mictocolumn size-exclusion chromatography-capillary zone electrophoresis , 7. Chromatogr. 633 213-220(1993). 

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