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 microgram 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 alpha- and beta-hexosaminides gave yield-hydrolysis time profiles that peaked in approximately 1 h in the DABA method, the E-M profiles, particularly for 2-sulfamato-alpha-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.
Bosworth
T R
TR
Chemical Morphology, Chemistry Building, Manchester University, United Kingdom.
Scott
J E
JE
D062365:Aminobenzoates; D000818:Animals; D002417:Cattle; D002686:Chitin / Q000031:analogs & derivatives / Q000737:chemistry; D048271:Chitosan; D003315:Cornea / Q000737:chemistry; D003641:Deamination; D005470:Fluorometry / Q000379:methods* / Q000706:statistics & numerical data; D006025:Glycosaminoglycans / Q000737:chemistry*; D006493:Heparin / Q000737:chemistry; D006595:Hexosamines / Q000032:analysis*; D015394:Molecular Structure; D009077:Mucins / Q000737:chemistry; D009608:Nitrous Acid; D012680:Sensitivity and Specificity
D062365:Aminobenzoates; D006025:Glycosaminoglycans; D006595:Hexosamines; D009077:Mucins; D002686:Chitin; C017856:3,5-diaminobenzoic acid; D006493:Heparin; D048271:Chitosan; D009608:Nitrous Acid