Hyaluronic acid oligosaccharides

Authors：

Category

Glycosaminoglycans

Protocol Name

Authors

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

KeyWords

hyaluronic acid chondroitinase fluorescence

Reagents

●	Chondroitinase ABC (a conventional preparation) from Proteus vulgaris (Seikagaku Corp., Tokyo, Japan)
●	Chondroitinase AC-II from Arthrobacter aurescens (Seikagaku Corp.)
●	Unsaturated chondro-disaccharide kit (D kit) (for HPLC) (Seikagaku Corp.)
●	2-Cyanoacetamide (Sigma-Aldrich, St. Louis, MO)

Instruments

●	Carbonex column (4.6 mm i.d. x 100 mm) (Tonen Co., Ltd., Tokyo, Japan)
●	Speed Vac Concentrator (Thermo Fisher Scientific Inc., Waltham, MA)
●	HPLC system (L-2000 Series: Hitachi High-Tech Science Corporation, Tokyo, Japan)

Methods

HPLC analysis of unsaturated disaccharide from HA

1)	Incubate HA (1-250 ng) with a mixture of chondroitinases ABC and AC-II (5 mIU each) in a total volume of 15 μL of 50 mM Tris-acetate buffer (pH 8.0) at 37˚C for 3 h.	Comment 0

2)	Terminate the reactions by boiling at 100˚C for 1 min.	Comment 0

3)	Dry the sample by Speed Vac.	Comment 0

Add 10 μL of distilled water, then inject 8 μL of the sample into the postcolumn HPLC system. The flow diagram and HPLC conditions are shown in Fig. 1. Typical chromatogram is shown in Fig. 2.

Comment 1

Figure & Legends

Fig. 1. Flow diagram of the postcolumn HPLC system for the determination of unsaturated disaccharides from HA

A Carbonex column (4.6 mm i.d. x 100 mm) was eluted at 40˚C with 50 mM sodium phosphate buffer (pH 11.0) in 3.5% acetonitrile at a flow rate of 0.5 mL/min. The column eluate is then mixed with 0.5% 2-cyanoacetamide and 1.0% NaOH supplied by the double-plunger pump (flow rate, 0.25 mL/min), and reacted at 110˚C to form fluorescence products. Reaction coil, 0.5 mm i.d. x 10 m; cooling coil, 0.25 mm i.d. x 2 m; detection, excitation 335 nm, emission 395 nm.

Fig. 2. Typical chromatogram of unsaturated disaccharides derived from HA and CS/DS

Sample size (8 μL, 10 ng of each sugar). Peaks: 1, ΔDi-4S; 2, ΔDi-0S; 3, ΔDi-6S; 4, ΔDi-HA.

This figure was originally published in Anal Sci. Mada A, Toyoda H. et al. "Utility of a carbon column for high-performance liquid chromatographic separation of unsaturated disaccharides produced from glycosaminoglycans" 1992, 8(6):793-7. © The Japan Society for Analytical Chemistry.

Copyrights

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

Date of registration：2014-05-13 09:40:18