Variability in the carbazole assay for N-desulfonated/N-acylated heparin derivatives.
The carbazole assay is commonly employed to quantify heparin and other uronic acid-containing polysaccharides. Heparin-derived standard curves are often employed to quantify solutions of various natural and unnatural heparin structures that have different levels of sulfate substitution, different levels of N-sulfo and N-acetyl groups, and other structural changes as a consequence of reducing molecular weight. Recent studies in our laboratory have focused on chemically modified heparin derivatives comprised of structurally diverse N-acyl moieties substituted into heparin in place of N-sulfo groups. We report here that although differing degrees of 2-N-sulfo-, 2-N-acetyl- or 2-amino-d-glucosamine residues within heparin do not affect signal intensity in the carbazole assay, replacing N-sulfo groups in heparin with structurally diverse N-acyl moieties affords products that display significant variation in the assay. The structure of different N-acyl groups, and to a lesser extent the degree of N-acylation by individual N-acyl groups, is shown to variably alter signal intensity in the carbazole assay even though content and structure of uronic acid residues is unaltered.
Hattan
Christopher M
CM
Division of Medicinal and Natural Products Chemistry, University of Iowa, Iowa City, IA 52242, United States.
Kerns
Robert J
RJ
D000818:Animals; D002227:Carbazoles / Q000737:chemistry*; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002621:Chemistry / Q000379:methods*; D005944:Glucosamine / Q000737:chemistry; D006493:Heparin / Q000031:analogs & derivatives / Q000737:chemistry*; D007067:Iduronic Acid / Q000737:chemistry; D008956:Models, Chemical; D008969:Molecular Sequence Data; D013552:Swine; D014574:Uronic Acids / Q000737:chemistry
D002227:Carbazoles; D014574:Uronic Acids; C041514:carbazole; D007067:Iduronic Acid; D006493:Heparin; D005944:Glucosamine