HIGH-PERFORMANCE HYDROPHOBIC INTERACTION CHROMATOGRAPHY OF PROTEINS (HPLC)

JODI LYNN FAUSNAUGH, Purdue University

Abstract

A new weakly hydrophobic, high-performance liquid chromatography stationary phase was developed for the separation of native proteins based on relative differences in hydrophobicity. Starting with a covalently bound, hydrophilic polyimine matrix, stationary phases were synthesized through acylation with alkyl anhydrides and acid chlorides. Proteins in aqueous buffers were induced to bind hydrophobically to the stationary phase by the use of high salt concentrations in the mobile phase. Elution was achieved either isocratically or by decreasing the salt concentration in a linear gradient. The static loading capacity for each stationary phase was determined using a hemoglobin binding assay. Capacities normally ranged between 35 and 55 mg of hemoglobin per gram of silica. Selectivity in high-performance hydrophobic interaction chromatography (HIC) can be easily manipulated through mobile phase and stationary phase variables. Stationary phase variables include ligand chain length and ligand density. Protein retention increased concomitantly with both ligand density and chain length. Mobile phase variables that influenced retention include salt type, salt concentration and pH. Since proteins are not denatured in HIC, enzymes eluted from the column retained enzymatic activity. Recovery of the enzymatic activity of (alpha)-amylase, (beta)-glucosidase, lactic dehydrogenase, (alpha)-chymotrypsin and lysozyme each exceeded 85%. This was significantly greater than recovery from a reversed-phase column. Protein recovery of (alpha)-chymotrypsinogen-A, conalbumin and lysozyme each exceeded 80% from an HIC column. Proteins with a hydrophilic exterior had shorter retention times on an HIC column than did proteins with more hydrophobic exteriors. The HIC column was able to resolve closely related proteins which differ by as little as one amino acid residue. Chromatographic retention of related bird lysozymes deviated only when amino acid substitutions occurred on the surface of lysozyme opposite the catalytic cleft. This area may constitute a contact surface area and extends from residue 41 to 102 and from residue 75 to the (alpha)-helical region starting with residue 89. Hemoglobin variants were much more difficult to resolve than the lysozymes.

Degree

Ph.D.

Subject Area

Biochemistry

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