Chondroitin sulfate oligosaccharides

Authors：

Category

Glycosaminoglycans

Protocol Name

Authors

Kinoshita-Toyoda, Akiko
Laboratory of Bio-analytical Chemistry, College of Pharmaceutical Sciences, Ritsumeikan University

KeyWords

chondroitinase gel filtration hyaluronidase

Reagents

●	Sheep testicular hyaluronidase (EC 3.2.1.35) (Sigma-Aldrich, St. Louis, MO)

Instruments

●	Bio-Gel P-10 resin (Bio-Rad Laboratories, Hercules, CA)
●	Sephadex G-25 fine resin (GE Healthcare, Little Chalfont, UK)
●	Speed Vac Concentrator (Thermo Fisher Scientific Inc., Waltham, MA)

Methods

Chondroitin sulfate oligosaccharides

1)	Incubate chondroitin sulfate E (100 mg) with hyaluronidase (10 mg, approximately 15,000 National formulary units) in a total volume of 2 mL of 50 mM sodium phosphate buffer, pH 6.0, containing 150 mM NaCl at 37˚C.	Comment 1

2)	Withdraw an aliquot of 2 μL of the sample at 1 h intervals to monitor the reaction.	Comment 0

3)	Mix the aliquot with H₂O (1 mL) and boil at 100˚C for 2 min.	Comment 0

4)	Add 50 μL of 2.5% cetrimide containing 2% NaOH to an aliquot of 25 μL of the mixture and measure the absorbance at 400 nm.	Comment 0

5)	Add 4 mg (approximately 6,000 National formulary units) of the additional enzyme into the reaction mixture and incubate to complete the digestion.	Comment 0

6)	Add 0.44 mL of 30% trichloroacetic acid and centrifuge at 2500 rpm for 10 min.	Comment 0

7)	Wash the precipitate with 0.5 mL of 5% trichloroacetic acid.	Comment 0

8)	Extract the combined supernatant with 6 mL ether.	Comment 0

9)	Centrifuge at 1000 rpm for 3 min and remove the ether phase.	Comment 0

10)	Repeat step 8 and 9 (4 times).	Comment 0

11)	Remove the ether phase completely.	Comment 0

12)	Neutralize the aqueous phase with 1 M Na₂CO₃.	Comment 0

13)	Concentrate the neutralized solution by Speed Vac to a final volume of less than 2 mL.	Comment 0

14)	Fractionate the sample by gel filtration chromatography on a Bio-Gel P-10 column (Fig. 2).	Comment 1

15)	Desalt the subfractions by gel filtration chromatography on a Sephadex G-25 fine column.	Comment 1

16)

Dry up the flow-through fraction by Speed Vac.

Comment 0

Figure & Legends

Fig. 1. Action of hyaruronidase and chondroitinase

Testicular hyaluronidase cleaves the N-acetyl-D-galactosaminidic linkages in CS chains in a hydrolytic fashion to yield tetra-, hexa-, octa-, deca-, and polysaccharides with glucuronic acid at the nonreducing ends as major products. In contrast, bacterial chondroitinase is a lyase that cleaves N-acetyl-D-galactosaminidic linkages in an eliminative fashion to give unsaturated disaccharides as major products. G: N-acetyl-D-galactosamine, U: glucuronic acid, ΔU: unsaturated uronic acid.

Fig. 2. Gel filtration column chromatography of the hyaluronidase digest of Squid cartilage chondroitin sulfate E on a Bio-Gel P-10 column

Squid cartilage chondroitin sulfate E was exhaustively digested with sheep testicular hyaluronidase. The digest was fractionated on a Bio-Gel P-10 column (1.6 cm i.d. x 95 cm) using 1 M NaCl, 10% ethanol as the eluent. Fractions (2 mL) were monitored by absorbance at 210 nm. The sizes of oligosaccharides are indicated.

This figure was originally published in J Biol Chem. Kinoshita A. et al. "Novel tetrasaccharides isolated from squid cartilage chondroitin sulfate E contain unusual sulfated disaccharide units GlcA(3-O-sulfate)beta1-3GalNAc(6-O-sulfate) or GlcA(3-O-sulfate)beta1-3GalNAc" 1997, 272(32):19656-65. © the American Society for Biochemistry and Molecular Biology.

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Date of registration：2014-04-24 15:50:49