CS/DS digesting enzymes from bacteria

Authors：

Category

Glycosaminoglycans

Protocol Name

Authors

Sugiura, Nobuo
Institute for Molecular Science of Medicine, Aichi Medical University

KeyWords

gel filtration dermatan sulfate lyase chondroitin sulfate

Reagents

●	Substrates: • 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.)
●	Enzymes: • 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)

Instruments

●	Superdex Peptide 10/300 column (GE Healthcare, Little Chalfont, UK)
●	LC-20 series HPLC system (Shimadzu Corp. Kyoto, Japan)

Methods

Enzymatic digestions of CS/DS

1)	Incubate CS/DS (10 μg) with each chondroitinase (10 mU) in a total volume of 50 μL buffer. The reaction conditions were (1) in 50 mM Tris-HCl buffer, pH 8.0 at 37°C for 1 h for chondroitinase ABC, (2) in 50 mM phosfate buffer, pH 6.0 at 37°C for 1 h for chondroitinase ACII, and (3) in 50 mM Tris-HCl buffer, pH 8.0 at 30°C for 1 h for chondroitinase B, respectively.	Comment 0

2)	Terminate the reactions with heating at 100°C for 1 min.	Comment 0

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.

Comment 0

Notes

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.

Copyrights

Attribution-Non-Commercial Share Alike
This work is released underCreative Commons licenses

Date of registration：2014-04-23 15:30:01