Isolated GAG samples were incubated with chondroitin lyase ABC (10 m-units, Seikagaku Corporation, Tokyo, Japan) and chondroitin lyase ACII (5 m-units, Seikagaku Corporation) at 37°C for 10 h and the enzymatic products were recovered by centrifugal filtration as described above, but at 13,000 × g. CS/DS disaccharides that passed through the filter were freeze-dried for future analysis. GAGs remaining in the retentate were collected by reversing the filter and spinning at 13,000 × g, followed by incubation with 10 m-units of heparin lyases I, II, and III at 37°C for 10 h. The products were recovered by centrifugal filtration and the HS/HP disaccharides collected and freeze-dried for LC-MS analysis. Cloning, overexpression in Escherichia coli, and purification of the recombinant heparin lyase I (EC 4.2.2.7), heparin lyase II (no EC assigned), and heparin lyase III (EC 4.2.2.8) from Flavobacterium heparinum were all performed as previously described [31 (link),32 (link)].
Chondroitin lyase acii
Manufactured by Seikagaku
Sourced in Japan
Chondroitin lyase ACII is an enzyme that catalyzes the cleavage of chondroitin sulfate, a type of glycosaminoglycan. It is used in the analysis and characterization of chondroitin sulfate and related compounds.
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Lab products found in correlation
2 protocols using chondroitin lyase acii
1
Chondroitin and Heparin Lyase Digestion
2
Glycosaminoglycan Disaccharide Analysis
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The recovered GAGs were next completely depolymerized using polysaccharide lyases. Polysaccharide lyases cleave the glycosidic linkages between hexosamine and uronic acid residues in glycosaminoglycans, converting these polysaccharides into disaccharides containing unsaturated uronic acid residues (ΔUA) at their non-reducing ends. Chondroitin lyase ABC (5 m-units, Seikagaku, Japan) and chondroitin lyase ACII (2 m-units, Seikagaku) in 10 μl of 0.1% BSA were added to a ~5 μg GAG sample in 25 μl 50 mM Tris solution containing 60 mM sodium acetate at pH 8.0, and incubated at 37° C for 10 h. The enzymatic products were recovered by centrifugal filtration at 12,000 × g. CS/DS disaccharides that passed through the filter were freeze-dried for LC-MS analysis. GAGs remaining in the retentate were collected by reversing the filter and spinning at 12,000 × g, followed by incubation with 10 m-units of heparin lyase I, II, and III (Seikagaku) at 35° C for 10 h. The products were recovered by centrifugal filtration using a YM-10 spin column, and the disaccharides were collected in the flow-through and freeze-dried.
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