Purification and substrate specificity of heparitinase I and heparitinase II from Flavobacterium heparinum. Analyses of the heparin and heparan sulfate degradation products by 13C NMR spectroscopy.
The purification of two heparitinases and a heparinase, in high yields from Flavobacterium heparinum was achieved by a combination of molecular sieving and cation-exchange chromatography. Heparinase acts upon N-sulfated glucosaminido-L-iduronic acid linkages of heparin. Substitution of N-sulfate by N-acetyl groups renders the heparin molecule resistant to degradation by the enzyme. Heparitinase I acts on N-acetylated or N-sulfated glucosaminido-glucuronic acid linkages of the heparan sulfate. Sulfate groups at the 6-position of the glucosamine moiety of the heparan sulfate chains seem to be impeditive for heparitinase I action. Heparitinase II acts upon heparan sulfate producing disulfated, N-sulfated and N-acetylated-6-sulfated disaccharides, and small amounts of N-acetylated disaccharide. These and other results suggest that heparitinase II acts preferentially upon N,6-sulfated glucosaminido-glucuronic acid linkages. The total degradation of heparan sulfate is only achieved by the combined action of both heparitinases. The 13C NMR spectra of the disaccharides formed from heparan sulfate and a heparin oligosaccharide formed by the action of the heparitinases are in accordance to the proposed mode of action of the enzymes. Comparative studies of the enzymes with the commercially available heparinase and heparitinase are described.
Nader
H B
HB
Departamento de Bioquímica, Escola Paulista de Medicina, São Paulo, Brazil.
Porcionatto
M A
MA
Tersariol
I L
IL
Pinhal
M A
MA
Oliveira
F W
FW
Moraes
C T
CT
Dietrich
C P
CP
D002240:Carbohydrate Sequence; D002247:Carbon Isotopes; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004187:Disaccharides / Q000032:analysis; D005417:Flavobacterium / Q000201:enzymology*; D007527:Isoenzymes / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008969:Molecular Sequence Data; D011133:Polysaccharide-Lyases / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity
D002247:Carbon Isotopes; D004187:Disaccharides; D007527:Isoenzymes; D011133:Polysaccharide-Lyases; C021349:heparitinsulfate lyase