title: Investigation of the solubility and the potentials for purification of serum amyloid A (SAA) from equine acute phase serum--a pilot study.
abstract: BACKGROUND
  Serum amyloid A (SAA) is useful as a diagnostic marker of systemic inflammation in horses, but only heterologous assays based on non-equine calibration and standardization are available for measurements of equine SAA. More accurate measurements could be obtained using purified species-specific SAA in native conformation for assay calibration and standardization. Further knowledge about the biochemical properties of SAA would facilitate a future production of native species-specific calibration material Therefore, the aim of the study was an investigation of the solubility and potentials for purification of equine SAA based on biochemical properties.Freeze dried equine acute phase serum was dissolved in 70% 2-propanol, 8 M urea, and milli-Q water, respectively. Supercritical fluid extraction (SFE), size-exclusive chromatography (FPLC-SEC), and preparative isoelectric focusing (IEF) were performed in the attempt to purify. Immunostaining of IEF blots were used for isoform-specific detection of SAA in the preparations and purity was assessed by silverstained SDS-PAGE.
  
  
  RESULTS
  SAA was soluble in 70% 2-propanol, 8 M urea and Milli-Q water. SAA was not separated in the lipophilic or ampipathic fractions following SFE. SAA was included in a FPLC-SEC-fraction of 237 kDa, despite the molecular weight known to be much smaller, suggesting binding to other serum constituents. SAA precipitated following separation of other serum proteins by preparative IEF.
  
  
  CONCLUSIONS
  No effective purification of SAA was achieved in the present study, but findings important for future investigations were made. The study suggested that SAA is not exclusively hydrophobic, but appears less hydrophobic when interacting with other serum components. These results suggest more complex aspects of solubility than previously believed, and indicate potentials for purification of native SAA.
authors.affiliation: Department of Veterinary Clinical and Animal Sciences, University of Copenhagen, Groennegaardsvej 3, ground floor, Frederiksberg C 1870, Denmark. mic@sund.ku.dk
authors.forename: Michelle B
authors.initials: MB
authors.lastname: Christensen
authors.forename: Jens Christian
authors.initials: JC
authors.lastname: Sørensen
authors.forename: Stine
authors.initials: S
authors.lastname: Jacobsen
authors.forename: Mads
authors.initials: M
authors.lastname: Kjelgaard-Hansen
mesh_terms: D000210:Acute-Phase Reaction / Q000097:blood* / Q000662:veterinary*; D000818:Animals; D002138:Calibration; D002850:Chromatography, Gel; D006736:Horses; D057927:Hydrophobic and Hydrophilic Interactions; D007249:Inflammation / Q000097:blood* / Q000662:veterinary*; D007525:Isoelectric Focusing; D010865:Pilot Projects; D000685:Serum Amyloid A Protein / Q000302:isolation & purification*; D016622:Silver Staining; D012995:Solubility
chemical_list: D000685:Serum Amyloid A Protein