Purification of serum amyloid A and its isoforms from human plasma by hydrophobic interaction chromatography and preparative isoelectric focusing

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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 pH 5–7 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.