Unsaturated disaccharides in the enzymatic digests of heparan sulfates

High performance liquid chromatography was performed by an ion-pair reversed-phase method of six standard unsaturated disaccharides derived from heparan sulfate and heparin. Separation of delta Di-GlcNAc, delta Di-GlcN(2S), delta Di-GlcNAc(6S), delta Di-GlcN(2,6- or 2,2'-diS) and delta Di-GlcN(2,6,2'-triS) was achieved on a column of Jasco SC-02 with 10 mM tetrabutylammonium phosphate (pH 7.0) containing 30 or 47% methanol as a mobile phase. delta Di-GlcN(2,6-diS) and delta Di-GlcN(2,2'-diS) were separated on the same column with 35 mM triethylamine phosphate (pH 5.3). Four preparations (BL-1.0-1, BL-1.0-2, BL-1.0-3, and BL-1.25-1) separated from crude bovine lung heparan sulfate, a standard bovine lung heparan sulfate (BL-ST), bovine kidney heparan sulfate 1.0 M Fr and 1.25 M Fr (BK-1.0 and BK-1.25), and porcine kidney heparan sulfate 1.0 M Fr (PK-1.0) were digested with a mixture of heparinase, and heparitinases 1 and 2. The resulting foregoing unsaturated disaccharides in the digests were analyzed by the above HPLC procedures. The proportions of the unsaturated disaccharides in the digests of BL-1.25-1 and BL-ST were similar, but those of the others differed from each other. It is noteworthy that delta Di-GlcNAc plus delta Di-GlcNAc(6S) in the digest of BL-1.0-1 was approximately 95% of the total unsaturated disaccharides. Small amounts of delta Di-GlcN (2,6,2'-triS) were found in all the samples. It was found that delta Di-GlcN(2,2'-diS) was a prominent component in the disulfated unsaturated disaccharides from BL-1.25-1 and BK-1.25.