HS/HP digesting enzymes from bacteria
Heparansulfate (HS) and heparin (HP) are digested with some enzymes from bacteria. The enzymes, termed heparinase or heparitinase, depolymerize HS/HP 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 HS/HP and preparation of extensive oligosaccharides. In this protocol, digestion patterns of HS/HP with some heparinases are shown by gel filtration chromatography.
• Heparan sulfate from pig aorta (Seikagaku Corp., Tokyo, Japan)
• Heparin from pig intestine (Merck Millipore, Billerica, MA)
• Heparinase I (Sigma-Aldrich, St. Louis, MO and Funakoshi Co., Ltd. Tokyo, Japan) or heparinase (Seikagaku Corp.) from Flavobacterium heparinum
• Heparinase II (Sigma-Aldrich and Funakoshi Co., Ltd.) or heparitinase II (Seikagaku Corp.) from Flavobacterium heparinum
• Heparinase III (Sigma-Aldrich and Funakoshi Co., Ltd.) or heparitinase I (Seikagaku Corp.) from Flavobacterium heparinum
Superdex Peptide 10/300 column (GE Healthcare, Little Chalfont, UK)
LC-20 series HPLC system (Shimadzu Corp., Kyoto, Japan)
Enzymatic digestions of HS/HP
Incubate HS/HP (10 μg) in a total volume of 50 μL buffer containing 50 mM Tris-HCl, pH 7.2, 10 mM CaCl2, and the enzymes at 37°C for 1 h.
The used enzymes were (1) heparinase I (heparinase, 1 mU), (2) heparinase II (heparitinase II, 1 mU), (3) heparinase III (heparitinase I, 1 mU), and (3) mixed enzymes of heparinase I (0.4 mU), heparinase II (0.2 mU), and heparinase III (0.4 mU).
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 heparansulfate and heparin on a Superdex peptide column
(A) Heparan sulfate and (B) heparin were digested with heparinase I (hepatinase), heparinase II (heparitinase II), heparinase III (heparitinase I), and the mixture of the three enzymes. The digests were chromatographed on a Superdex peptide column using 0.2M 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, 22, and 23 indicate non-sulfated, di-sulfated, and tri-sulfated ∆disaccharides, respectively.