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Purification and Characterization of Hamster Serum Amyloid A Protein (SAA) by Cholesteryl Hemisuccinate Affinity Chromatography
Author(s) -
NIEWOLD TH. A.,
TOOTEN P.C.J.
Publication year - 1990
Publication title -
scandinavian journal of immunology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.934
H-Index - 88
eISSN - 1365-3083
pISSN - 0300-9475
DOI - 10.1111/j.1365-3083.1990.tb02784.x
Subject(s) - chemistry , affinity chromatography , hamster , serum amyloid a , size exclusion chromatography , apolipoprotein b , ultracentrifuge , chromatography , lipoprotein , serum amyloid a protein , biochemistry , cholesterol , microbiology and biotechnology , biology , immunology , enzyme , inflammation
High‐density lipoprotein (HDL) apolipoprotein was separated from hamster serum by cholesteryl hemisuccinate atlinity chromatography (CHAC) in comparison with the density‐gradient ultracentrifugation (DGUC), The apolipoprotein recovery from scrum by CHAC was 70% and by DGUC 80%, This disadvantage is compensated for by the ease of purification by CHAC, a method particularly suited for the processing of large amounts of serum. From the acute‐phase HDL CHAC fraction, apo SAA was isolated by gel filtration. Using isoelectrofocusing, two‐dimensional gel electrophoresis, und titration curve, four isotypes of hamster apo SAA were identified and characterized In the acute‐phase serum, one of the isotypes was predominant (apo SAA 1 ), In serum of amyloidotic animals, the relative contribution of apo SAA l was considerably lower, suggesting selective removal of the latter during amyloidogencsis and possibly its deposition in hamster AA amyloid. Furthermore, the affinity chromatography method was modified with gradient elution of affinity‐bound material. By this method HDL apolipoprotein was separated into three subclasses. Apo SAA was shown to associate with two different subclasses. In acute‐phase serum most of the apo SAA 1 was found in the subclass with the lowest affinity for the cholesteryl beads, whereas thc latter was depressed in amyloidotic serum, suggesting that the amyloidogenicity ofa particular apo SAA isotype is determined by its cholesteryl‐binding properties.