No 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.

Immunonephelometry of apolipoprotein A-II (apo A-II) in serum requires that factors responsible for nonspecific increases of light scattering be eliminated. Complete delipidation is not necessary for immunonephelometry of apo A-II; in fact, light scattering of immunocomplexes from delipidated HDL is greater than from isolated apo A-II. Therefore, using the isolated peptide as a standard does not suffice for measurements in serum. The concentration of protein influences the development of light scattering and must be considered in standardizing this measurement.

No abstract

No abstract