Heparin/heparin sulfate oligosaccharides

Authors：

Category

Glycosaminoglycans

Protocol Name

Authors

Yamada, Shuhei
Department of Pathobiochemistry, Faculty of Pharmacy, Meijo University

KeyWords

heparinase gel filtration heparitinase

Reagents

●	Heparinase from Flavobacterium heparinum (Seikagaku Corp., Tokyo, Japan)
●	Heparitinases from Flavobacterium heparinum (Seikagaku Corp., Tokyo, Japan)
●	Dimethylmethylene blue dye (Sigma-Aldrich, St. Louis, MO)
●	Ba(NO₂)₂ (Nacalai Tesque Inc., Kyoto, Japan)

Instruments

●	Bio-Gel P-10 resin (Bio-Rad Laboratories, Hercules, CA)
●	Sephadex G-25 fine resin (GE Healthcare, Little Chalfont, UK)
●	Superdex^TM Peptide 10/300 GL column (GE Healthcare)
●	Speed Vac Concentrator (Thermo Fisher Scientific Inc., Waltham, MA)
●	HPLC system (LC-20 Series: Shimadzu Corporation, Kyoto, Japan)

Methods

Enzymatic digestion.

1)	Incubate Hep/HS (20 mg) with Hep lyase (heparinase, heparitinase, or a mixture of heparinase and heparitinase) (0.2 IU) in a total volume of 2 mL of 20 mM acetate-Na buffer, pH 7.0, containing 2 mM Ca(CH₃COO)₂ at 37˚C.	Comment 1

2)	Withdraw aliquots (5 μL) at 0.5- to 1- h intervals to monitor the reaction.	Comment 0

3)	Mix the aliquot with 0.01 M HCl (495 μL) and measure the UV-absorbance at 232 nm.	Comment 1

4)	Terminate the reaction by boiling at 100˚C for 2 min, when the absorbance reaches the target value.	Comment 1

5)	Fractionate the digest by gel filtration chromatography on a Bio-Gel P-10 or Superdex^TM Peptide column.	Comment 1

6)	Desalt the subfractions by gel filtration chromatography on a Sephadex G-25 fine column using distilled water as the eluent.	Comment 1

Lyophilize the flow-through fraction by Speed Vac.

Comment 0

Chemical degradation of Hep/HS. (To chemically depolymerize Hep/HS chains, nitrous acid treatment is often used.)

1)	Lyophilize Hep/HS (50 μg) in a glass tube by Speed Vac.	Comment 0

2)	Add 40 μL of 0.5 M HNO₂ (pH 1.5) reagent and incubate at room temperature for 30 min.	Comment 0

3)	Neutralize the incubation mixture with 1 M NaOH.	Comment 1

4)	Add 50 μL of 1 M NaBH₄/0.05 M NaOH and incubate at room temperature for 2 h.	Comment 1

5)	Acidify the incubation mixture with glacial acetic acid to terminate the reaction.	Comment 1

6)	Neutralize with 1 M Na₂CO₃.	Comment 0

Fractionate oligosaccharides by gel filtration chromatography on a Superdex^TM Peptide column.

Comment 1

Figure & Legends

Fig. 1. Substrate specificity of Hep lyases

The arrows indicate the glycosidic linkages cleaved by heparinase (A), heparitinase I (B), and heparitinase II (C). The hexuronate residues (marked by asterisks) are either D-glucuronic acid (GlcA) or L-iduronic acid (IdoA). C-2 NH₂ of the D-glucosamine (GlcN) and C-2 OH of the hexuronate residue should be sulfated for sensitivity to heparinase (A). C-3 OH of GlcN adjacent to the disaccharide cleavage site (marked by #) has to be free for sensitivity to heparitinases I and II (B, C). Heparitinase I recognizes nonsulfated hexuronate (C-2 OH should be free as marked by #) and prefers GlcA to IdoA as a substrate (B).

Fig. 2. Fractionation of the heparitinase digest of bovine kidney HS

Bovine kidney HS (33 mg) was digested by heparitiase until the reaction reached 60% completion. The digest was gel-filtrated on a column (1.6 cm i.d. x 95 cm) of Bio-Gel P-10 using 1.0 M NaCl/10% ethanol as the eluent. Two-milliliter fractions were collected and aliquots were monitored by measuring absorbance at 232 nm. The sizes of oligosaccharides in the resolved peaks are indicated.

This figure was originally published in Glycobiology. 4(4):535-44. 1994 "Structural studies on the oligosaccharides isolated from bovine kidney heparan sulphate and characterization of bacterial heparitinases used as substrates" Sugahara K. Yamada S. et al. Oxford University Press.

Fig. 3. Gel-filtration of the heparinase digest on Bio-Gel P-10

Hep (300 mg) was digested with heparinase. The digest was subjected to gel-filtration on a column (1.5 cm i.d. x 46 cm) of Sephadex G-25 fine using 0.25 M NH₄HCO₃/7% 1-propanol as the eluent, and separated into oligosaccharides and disaccharides (data not shown). The oligosaccharide fraction was further fractionated on a column (1.6 cm i.d. x 95 cm) of Bio-Gel P-10 using 1.0 M NaCl/10% ethanol as the eluent. Fractions (2 mL) were collected and monitored by measuring absorbance at 232 nm. The sizes of oligosaccharides in the resolved peaks are indicated.

This figure was originally published in J Biol Chem. Yamada S. et al. "Isolation of the porcine heparin tetrasaccharides with glucuronate 2-O-sulfate. Heparinase cleaves glucuronate 2-O-sulfate-containing disaccharides in highly sulfated blocks in heparin" 1995, 270(15):8696-705. © the American Society for Biochemistry and Molecular Biology.

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Date of registration：2013-12-27 15:45:37