The purpose of this study was to determine the differential digestion of two different batches of branded enoxaparin (Aventis, USA), three generic versions of enoxaparin (GlandPharma, India; Lazar, Argentina; and Biochemie, Brazil), dalteparin (Pfizer, USA) and tinzaparin (Leo, USA) by heparinase-I and heparinase-II. Heparinase-I (Ibex Tech., Montreal, Canada) and heparinase-II (Ram Sasisekharan, MIT, Cambridge, MA) were isolated from Flavobacterium heparinum. The substrate specificity of these enzymes has been inferred from the reducing and non-reducing terminal structures of the di and oligosaccharides formed by digesting heparin. Heparinase-I cleaves the glucosaminidic linkage in GlcN (N-sulfate) a 1–4 IdceA (2-sulfate) and endures C-6 sulfation of hexosamine unit. More susceptibility of polymers such as heparin than oligomers to this enzymatic depolymerization indicates the size dependency of this enzymatic activity.

Heparinase-II cleaves all the glucosaminidic linkages in heparin independent of O-and/or N-sulfation as well as the type of uronic acid residue. The non-sulfated substrates are somewhat resistant to this enzyme. The glucosaminidic linkage adjacent to a 3-O-sulfated GlcN residue and the innermost glucosaminidic linkage right next to the glycosaminoglycan-protein linkage region of proteoglycan are resistant to this enzymatic activity (Sugahara et al., 1994, Glycobiology 4, 535–544). In this study, several low molecular weight heparins (LMWHs) produced from different depolymerization methods of unfractionated heparin were digested with heparinase-I and heparinase-II to determine the differential digestion of these two enzymes.

Eight different LMWHs with average molecular weight (MW) ranging from 3425 to 6281 Da (in UV detection at 205nm) were prepared at a concentration of 10mg/ml in 0.3M Na2SO4. Each LMWH was incubated with these enzymes (1.0 U/mL) separately for 30 minutes at 37° C in the presence of calcium

Following the incubation, the samples were heated at 100° C to inactivate enzymatic activity. The molecular weight profiling of these samples was determined by using a gel permeation chromatography-high performance liquid chromatography (GPC-HPLC) system with UV detection at 205nm. A narrow range calibration method comprised of oligosaccharides and polysaccharides was used to determine the relative molecular profile of the native and digested products.

The LMWHs were digested to LMW oligosaccharides with average MW of 1510± 275 Da with heparinase-I and 3071± 1044 Da with heparinase-II. The extent of digestion observed with all the LMWHs was more with heparinase-I than heparinase-II. This may be due to the different specific binding sites available for these enzymes and the requirement of sulfation at different positions in GlcN/uronic acid residues. All the LMWHs were equally digested into oligosaccharides (di, tetra and hexa) with heparinase-I. However heparinase-II resulted in the formation of only hexa, octa and decasaccharides.

These results show that the LMWHs were more susceptible to heparinase-I than heparinase-II. The possible reason for the less susceptibility of these compounds to heparinase-II is likely due to the oligosaccharide composition and degree and pattern of sulfation in GlcN/uronic acid residues of these compounds. The heparinase-I and heparinase-II digestion can therefore be used in the profiling of these agents.

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