title: Reversible subunit dissociation of tumor necrosis factor during hydrophobic interaction chromatography.
abstract: Hydrophobic interaction chromatography (HIC) of recombinant tumor necrosis factor (TNF) results in reversible dissociation of the quaternary protein structure yielding separation of trimer and monomer peaks. Gel electrophoresis, size exclusion, fluorescence polarization and rechromatography were used to identify the trimeric and monomeric species. Relative amounts of these peaks varied as a function of temperature and column contact time. When TNF was chromatographed in the presence of partially proteolyzed [14 kilodalton (kD)] TNF, two additional peaks, identified as the 14-kD monomer and heterotrimer of TNF and the 14-kD fragment, appeared. Rechromatography of this heterotrimer produced TNF monomer and 14-kD peaks establishing that the multiple peak pattern in HIC was due to quaternary dissociation. Incubating TNF in denaturants prior to non-denaturing size-exclusion chromatography resulted in apparent protein unfolding. However, free, undenatured monomer was not observed. We conclude that TNF is most likely a trimer, which is tightly held together by hydrophobic forces, and that the tertiary structure of the monomer is stabilized through this subunit association. The hydrophobicity of the sorbent surface mediates reversible dissociation of the trimers to monomers through hydrophobic stabilization of the monomeric tertiary structure. After elution, the TNF monomers reassociate to form the native TNF trimer.
authors.affiliation: Analytical Development Department, Cetus Corporation, Emeryville, CA 94608.
authors.forename: M G
authors.initials: MG
authors.lastname: Kunitani
authors.forename: R L
authors.initials: RL
authors.lastname: Cunico
authors.forename: S J
authors.initials: SJ
authors.lastname: Staats
mesh_terms: D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D008970:Molecular Weight; D012997:Solvents; D013050:Spectrometry, Fluorescence; D013696:Temperature; D014409:Tumor Necrosis Factor-alpha / Q000032:analysis*
chemical_list: D012997:Solvents; D014409:Tumor Necrosis Factor-alpha