A comparison of different purification methods of aggrecan fragments from human articular cartilage and synovial fluid.
In the study of aggrecan fragmentation several methods to extract and purify aggrecan from cartilage and synovial fluid (SF) are used. This work compares and evaluates the effectiveness for purification of aggrecan of the most commonly used methods by the ratio of sulfated glycosaminoglycan (sGAG) to protein and by fragment analysis by Western blot. A novel method for purification of aggrecan fragments from SF by boiling (Boiled SF) is also presented. Of the sGAG extracted from cartilage by guanidinium, 66% was recovered by associative-dissociative cesium chloride density gradient centrifugation (A1D1-D3) with a 9 times higher ratio of sGAG to protein in the A1D1 fraction. Although less enriched in aggrecan, the Western blot aggrecan pattern of the guanidinium extracted sample resembled that of the combined patterns of the A1D1, A1D2 and A1D3 fractions. The recoveries of sGAG from SF purified by anion chromatography and Alcian blue precipitation were around 50%, while the recoveries were over 80% in the associative or dissociative density gradient fractions (A1 and D1) and Boiled SF. The purification compared to neat SF ranged from 9 times in boiled SF to 1800-1900 times in Alcian blue and D1 samples. To obtain reliable results when analyzing synovial fluid aggrecan fragments by Western blot, purification was necessary. The immuno-pattern of anion chromatography purified SF resembled the patterns of A1 and D1, while the pattern of Boiled SF resembled the D1 sample. This work suggests that aggrecan fragments extracted from cartilage by guanidinium need no further purification to be analyzed by Western blot, whereas aggrecan fragments in SF are best analyzed in the A1 and D1 fractions or in the Boiled SF sample.
Struglics
André
A
Department of Orthopaedics, Clinical Sciences Lund, Lund University, Lund, Sweden. andre.struglics@med.lu.se
Larsson
Staffan
S
D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D053674:Aggrecans / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D000818:Animals; D002358:Cartilage, Articular / Q000737:chemistry*; D006801:Humans; D007719:Knee Joint / Q000033:anatomy & histology; D008875:Middle Aged; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification*; D013582:Synovial Fluid / Q000737:chemistry*; D055815:Young Adult
D053674:Aggrecans; D010446:Peptide Fragments