Heparin acid hydrolysis

The identification of L-iduronic acid as the major glycuronic acid constituent of heparin proved to be a much slower process than the identification of the amino sugar residue. Although this compound was detected in acid hydrolyzates of heparin116117 and heparin oligosaccharides,118 its yield was usually poor, because of the drastic conditions used for the acid hydrolysis (which are known to lead to extensive destruction of uronic acid).119120 Also, L-iduronic acid escaped detection as L-idose in the hydrolyzates of carboxyl-reduced heparin, probably because L-idose is readily converted into 1,6-anhydro-L-idose under the usual hydrolytic conditions. 

Examination of early -n.m.r. spectra of heparin and of chemically modified heparins121 prompted a reinvestigation of N,0-desulfated, carboxyl-reduced heparin, leading to the isolation of substantial amounts of L-iditol pentaacetate.121,122 In addition, improved conditions for the acid hydrolysis of heparin and carboxyl-reduced heparin gave increased recoveries of L-iduronic acid and 1,6-anhydro-L-idose, respectively.123 These findings confirmed the L-enantiomeric designation of the iduronic acid, and established that it is the main uronic acid in heparin. 

Both positional linkages (uronic acid to hexosamine and hexosamine to uronic acid) were established as being (1 — 4) by structural analysis of the previously mentioned (see Section IV), crystalline disaccharides containing D-glucuronic acid, isolated from an acid hydrolyzate of carboxyl-reduced heparin.128-129 Further evidence was obtained from the structure of the D-glucuronic acid-containing counterpart of disaccharides 6 and 8, obtained as minor products from pig-mucosal heparin following nitrous acid deamination,1110 136-138 and acid hydrolysis followed by N-acetylation,130 respectively. 

Although the glycosidic bonds of uronic acid residues are usually more resistant to acid hydrolysis than those of neutral polysaccharides,218 these linkages in heparin are more readily cleavable then those between the hexosamine and the uronic acid residues. Disaccharides obtained by exhaustive hydrolysis of heparin with 0.5 MHC1 at 80° contained hexu-... 

Heparosine. A sulfated disaccharide has been isolated308 by partial, acidic hydrolysis of heparin, and it has been formulated as 4-0-(2-amino-2-deoxy-a-v-glucopyranosyl 6-sulfate)-D-glucuronic acid. Heparosine and its W-acetyl amide are amorphous. An amorphous disaccharide prepared by a similar procedure, was, however, believed309 to be a 2-amino-2-deoxy-(D-glucosyluronic acid)-D-glucose. 

Considerable information concerning the structure of heparin has been obtained from hydrolytic studies. Controlled, acid hydrolysis is a valuable... 

Little work has been published concerning the isolation of oligosaccharides following graded, acidic hydrolysis of heparin. Wolfrom and co-workers have studied the partial, acid hydrolysis of a carefully purified heparin. (Purification was effected through the benzidine salt and the neutral barium salt was fractionally precipitated from water. The less-soluble fraction, after conversion to the barium acid salt, had a N S ratio of 2 5 and 5 sulfate groups per tetrasaccharide period, consumed 1 mole of periodate per tetrasaccharide period, and was shown to be homogeneous by electrophoresis and by countercurrent distribution. The molecular... 

Using this procedure, Consden and Stanier were able to show that a hydrolyzate of Group A hemolytic streptococci contained hexosamine, rhamnose, ribose, and glucose and, also, that a hydrolyzate of purified, human fibrin contained uronic acid, hexosamine, mannose, and galactose. A similar combination of techniques has been employed by Woodin in a study of the composition of a comeal mucopolysaccharide. It is of interest that the conditions of acidic hydrolysis necessary for releasing the n-glucos-amine moiety from heparin also result in destruction of the concomitantly released n-glucuronic acid. Traces of the intact uronic acid may be revealed by zone electrophoresis of the hydrolyzates. ... 

It is very difficult to remove the last traces of such material from heparin, yet this can be pharmacologically active. The histamine found in heparin after acid hydrolysis, as in the determination of normal blood histamine, probably arises from such peptides. Heparinoids prepared from natural... 

Labelle, J. L. Briere, N. Staining of nuclear basic proteins without deoxyribonucleic acid hydrolysis, using heparin and alcian blue. Acta Histochem. 1971, 41, 338-348. 

Tables Deposition of collagen-glycosaminoglycan conjugates on poly(octadecen-a/f-maleic anhydride)-coated substrates. Fibrillogenesis and immobilization of fibrillar collagen (1.2 mg/ml) was performed for 2 h at 37 ° C in the presence of heparin and hyaluronic acid (0.4, 1.2 and 5.0 mg/ml, respectively). Thickness of the layers was determined ellip-sometrically using the refractive index of 1.6035 for the dried collagen layer. The collagen amount was determined by amino acid-based HPLC analysis after acidic hydrolysis of surface-bound collagen...

---