title: Purification of serum amyloid A and its isoforms from human plasma by hydrophobic interaction chromatography and preparative isoelectric focusing.
abstract: The present work was aimed at isolating human serum amyloid A, (SAA), an acute-phase protein mainly complexed to high density lipoproteins, directly from human plasma without sequential ultracentrifugation of lipoproteins and subsequent delipidation of the apolipoprotein moiety. Hydrophobic-interaction fast-protein liquid chromatography on Octylsepharose, using stepwise gradient elution profiles under dissociating conditions, followed by fast-protein liquid-gel permeation chromatography on a Superdex TM75 column revealed a higher than 95% purity of isolated SAA. Further purification of SAA from coeluting apolipoproteins C and A-II was achieved by preparative isoelectric focusing between pH5-7 using a Rotofor apparatus. Separation of the main SAA isoforms, SAA1 (pI 6.5) and SAA1 des-Arg (pI 6.0, lacking the N-terminal arginine), was achieved by anion-exchange fast-protein liquid chromatography on a Fractogel EMD DEAE 650-S column. The purity of the SAA1 and SAA1 des-Arg isoforms, thus isolated, was checked by immunochemical techniques and amino acid analysis. With the described method various SAA isoforms can be isolated, purified and separated directly from human plasma/serum without prior ultracentrifugation.
authors.affiliation: Department of Internal Medicine, Philipps University, Marburg/Lahn.
authors.forename: E
authors.initials: E
authors.lastname: Malle
authors.forename: H
authors.initials: H
authors.lastname: Hess
authors.forename: G
authors.initials: G
authors.lastname: Münscher
authors.forename: G
authors.initials: G
authors.lastname: Knipping
authors.forename: A
authors.initials: A
authors.lastname: Steinmetz
mesh_terms: D015153:Blotting, Western; D002845:Chromatography; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007122:Immunoelectrophoresis; D007525:Isoelectric Focusing; D000685:Serum Amyloid A Protein / Q000302:isolation & purification*
chemical_list: D000685:Serum Amyloid A Protein