In the reactions used to break heparin down to mono- and oligosaccharides, androsugars are formed at two stages. The first of these is the well-known cleavage of heparin with nitrous acid to convert the N-sulfated D-glucosamines to anhydro-D-mannose residues; this reaction has been studied in detail. It is demonstrated here that only low pH (less than 2.5) reaction conditions favor the deamination of N-sulfated D-glucosamine residues; the reaction proceeds very slowly at pH 3.5 or above. On the other hand, N-unsubstituted amino sugars are deaminated at a maximum rate at pH 4 with markedly reduced rates at pH2 or pH6. At room temperature solutions of nitrous acid lose one-fourth to one-third of their capacity to deaminate amino sugars in 1 h at all pHs. A low pH nitrous acid reagent which will convert heparin quantitatively to its deamination products in 10 min at room temperature is described, and a comparison of the effectiveness of this reagent with other commonly used nitrous acid reagents is presented. It is also shown that conditions used for acid hydrolysis of heparin convert approximately one-fourth of the L-iduronosyluronic acid 2-sulfate residues to a 2,5-anhydrouronic acid. This product is an artifact of the reaction conditions, and its formation represents one of several pathways followed in the acid-catalyzed cleavage of the glycosidic bond of the sulfated L-idosyluronic acid residues.

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Abstract. Growing and confluent cultures of a rat hepatocyte cell line were labeled with 35SO42-and the heparan sulfate in the culture medium, the pericellular matrix, the nucleus, the nuclear outer membrane, and the remaining cytoplasmic pool was purified by DEAE-cellulose chromatography. The heparan sulfate in all pools from the confluent cells was bound more strongly on the DEAE-cellulose column than the corresponding pools from the growing cells. Gel filtration of each pool before and after ~-elimination showed that the heparan sulfate from the nuclear and nuclear membrane pools was composed of primarily free chains, whereas the heparan sulfate in all of the other pools was a mixture of proteoglycans and free chains. The heparan sulfate in each pool was cleaved with nitrous acid to obtain mixtures of di-and tetrasaccharides. Analysis of these mixtures showed that the structural features of the heparan sulfates in each pool were different and were altered significantly when the growing cells became confluent. The nuclear-plus-nuclear membrane pools represented 6.5% and 5.4% of the total cell-associated heparan sulfate in the growing cells and the confluent cells, respectively. The structural features of the heparan sulfate in the two nuclear pools were very similar to each other, but were markedly different from those of the heparan sulfate from the other pools or from any previously described heparan sulfate or heparin. The most unusual aspect of these structures was the high content of ~-D-glucuronosyl(2-SO4)--~D-glucosamine-N,o-(SO4)2 disaccharide units in these sequences. The mode of biosynthesis and delivery of these unusual sequences to the nucleus and the potential significance of these observations are discussed.H EPARAN sulfate proteoglycan (HSPG) ~ is a structurally variable polymer which is turned over rapidly in animal cells (2, 64,68,77). Heparan sulfate (HS) is structurally related to heparin, even though the core proteins on which the two polymers are synthesized are different (61, 67). The biosynthesis of heparin and HS appear to follow similar pathways (66,69). Heparin is synthesized as a polymer of repeating o-glucuronic acid (GlcUA)-~N-acetyl-o-glucosamine (GlcNAc) disaccharide units which then undergoes a series of maturation reactions initiated by N-deacetylation, which occurs randomly at some of the GlcNAc residues along the polymer chain, followed by N-sulfation of the resulting Dglucosamine (GlcN) residues. Further maturation, which occurs in the regions around the N-sulfo-D-glucosamine (Glc-NSO3) residues, involves additional N-deacetylation/N-sulfation at GIcNAc residues adjacent to the GlcNSO3 residues, C5 epimerization of some of the GlcUA residues to form Liduronic acid (IdoUA) residues, and o-suifation of some of the GlcNAc and GIcNSO3 residues at C6 and some of the uronic acid residues at C2 (69). The resulting polymer contains blocks of unsulfated disaccharides interspersed with blocks of disaccharides which are sulfated to varying degrees (13,48, 73). HS chains are less highly...

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Heparin was carboxyl-reduced with sodium boro[3H]hydride and converted to a mixture of oligosaccharides by treatment with nitrous acid at pH 2. The oligosaccharide mixture was aldehyde-reduced with sodium boro[3H]hydride and the mixture of products, labeled both in the hexoses formed in the carboxyl-reduction step and in the reducing sugars formed in the nitrous acid reaction, was separated and analyzed. The major product, L-idosyl 2-sulfate leads to anhydro-D-mannitol 6-sulfate (I), contained 60% of hexoses derived from the hexuronic acid residues in the original heparin. A second product, which contained 15% of the hexoses derived from the hexuronic acid residues in the original heparin, was identified as a tetrasaccharide composed of two L-idosyl 2-sulfate residues, one anhydro-D-mannitol 6-sulfate residue (the reducing end),and a hydroxymethylpentose sulfate residue formed by deamination of a disulfated D-glucosamine residue without bond cleavage. Several additional disaccharides derived from the regions of the polymer which contained D-glucuronic acid residues and lower degrees of O-sulfation were also identified among the deamination products. The oligosaccharides that were obtained accounted for 100% of the original carboxyl-reduced heparin, and paper chromatographic profiles of the oligosaccharide separations can be used as a fingerprint of the heparin preparation. The properties of I were examined in greater detail. The glycosidic bond of the L-idosyl 2-sulfate residue was found to be extremely labile to 0.1 N HCl at 100 degrees C, hydrolyzing with a t 1/2 of 18 min to give high yields of L-idose 2-sulfate and anhydro-D-mannitol 6-sulfate. L-Idofuranose was also identified as an intermediate in the conversion of L-idose 2-sulfate to L-idosan. The acid lability of the L-idosyl 2-sulfate bond in I offers a new route for the selective cleavage of carboxyl-reduced heparin.