A Specific Fluorometric Assay for Hexosamines in Glycosaminoglycans, Based on Deaminative Cleavage with Nitrous Acid

Glycosaminoglycan (GAG) hexosamines were measured after deacetylation (2 h acid hydrolysis), deaminative cleavage by nitrous acid, and coupling of the 2,5-anhydro sugars thus produced with 3,5-diaminobenzoic acid (DABA) to give a fluorescent product. Analyses were performed after the addition of an aliquot of potassium acetate solution to the acid hydrolysates, to adjust the pH. Results on a series of GAGs were compared with an Elson-Morgan (E-M) procedure. Our method is faster, more convenient, 10-20 times more sensitive, and always gave higher figures for hexosamine content. In particular it avoids long, destructive hydrolysis in concentrated strong acid and the complications of the Moggridge-Neuberger effect. Results agreed with calculations and titrimetric data. Using internal standards, our method is tolerant of cetylpyridinium chloride, peptides, and salts. A simple fluorometer easily handled less than 1 μg GAG per sample in 1.0 ml volume. Results are available in 4-5 h. The method is suitable for automation. A strongly polycationic environment, as in chitosan, markedly slowed nitrous acid deamination, possibly because of Donnan-type exclusion from the domain of the polycation of the cationic nitrosonium species. Whereas all other α- and β-hexosaminides gave yield-hydrolysis time profiles that peaked in ∼1 h in the DABA method, the E-M profiles, particularly for 2-sulfamato-α-hexosaminides, took longer and/or achieved lower optimal yields. All the usually encountered forms of the 2-amino-2-deoxy group (free, acetylated, sulfamato) are readily assayed using the same reagents, with appropriate prefluorometric stages.