CS/DS digesting enzymes from bacteria
Chondroitin sulfate (CS) and dermatan sulfate (DS) are digested with some enzymes from bacteria. The enzymes, termed chondroitinases or chondroitin lyases, depolymerize CS/DS chains via a β-elimination reaction, which generates an unsaturated 4,5-bond on the uronic acid residue at the site of cleavage. The enzymes are useful for quantification on structure analysis of CS/DS and preparation of extensive oligosaccharides. In this protocol, digestion patterns of CS/DS with some chondroitinases are shown by gel filtration chromatography.
• Chondroitin sulfate A from whale cartilage (Seikagaku Corp., Tokyo, Japan)
• Chondroitin sulfate C from shark cartilage (Seikagaku Corp.)
• Chondroitin sulfate D from shark fin cartilage (Seikagaku Corp.)
• Chondroitin sulfate E from squid cartilage (Seikagaku Corp.)
• Dermatan sulfate from pig kidny (Seikagaku Corp.)
• Chondroitinase ABC from Proteus vulgaris (Seikagaku Corp. and Sigma-Aldrich, St. Louis, MO)
• Chondroitinase ACII from Atrobactor aurescens (Seikagaku Corp., Sigma-Aldrich, and Wako Pure Chemical Industries Ltd., Osaka, Japan)
• Chondroitinase B from Flavobacterium heparinum (Seikagaku Corp., Sigma-Aldrich, and Funakoshi Co., Ltd. Tokyo, Japan)
Superdex Peptide 10/300 column (GE Healthcare, Little Chalfont, UK)
LC-20 series HPLC system (Shimadzu Corp. Kyoto, Japan)
Enzymatic digestions of CS/DS
Incubate CS/DS (10 μg) with each chondroitinase (10 mU) in a total volume of 50 μL buffer.
Terminate the reactions with heating at 100°C for 1 min.
Inject 35 μL of the reactions into the gel filtration HPLC system using 0.2M NaCl as developing buffer at a flow rate of 1 mL/min, monitored by measuring UV absorbance at a wavelength of 220 nm.
Seikagaku Corp. discontinued the enzymes in 2011.
Figure & Legends
Fig. 1. Gel filtration patterns of digest products of chondroitin sulfates on a Superdex Peptide column
(A) Chondroitin sulfate A, (B) chondroitin sulfate C, (C) chondroitin sulfate D, (D) chondroitin sulfate E, and (E) dermatan sulfate were digested with chondroitinase ABC, AC II, and B. The digests were chromatographed on a Superdex Peptide column using 0.2 M NaCl as the eluent. The elutions were monitored by absorbance at 220 nm. The molecular weights of polysaccharides (20k, 12k, and 6k) and the sizes of oligosaccharides (10, 8, 6, and 4) of hyaluronan standards are indicated. The signs of 20, 21, and 22 indicate non-sulfated, mono-sulfated, and di-sulfated ∆disaccharides, respectively.