A colorimetric method for determination of linkage in hexosamine-containing compounds.

The Ehrlich reagent has been used extensively for the estimation of acetylated and free hexosamines. The Morgan-Elson (1) method depends upon reaction of this reagent with a chromogen formed from N-acetylhexosamines under mildly alkaline conditions, while the Elson-Morgan (2) method depends upon the formation of chromogens resulting from the interaction of hexosamines and acetylacetone. Under certain conditions (3), the Morgan-Elson reaction permits the distinction from each other of 3-, 4-, and 6-substituted hexosamines (4, 5), since 4-substitution abolishes the reaction whiie 3-substitution enhances color production. Increased color yield has also been observed with N-acetylhyalobiuronic acid, the disaccharide obtained by the action of bacterial hyaluronidase on hyaluronic acid (“bacterial disaccharide”), and with an acetylated amino sugar isolated from a uridine nucleotide (6, 7). In a previous publication from this laboratory (7), it was demonstrated that application of the Elson-Morgan reaction to the deacetylated form of this amino sugar results in a spectrum differing from that obtained with glucosamine or galactosamine. Strange and Dark (8) have also observed this spectral shift with an amino sugar isolated from cell walls of a number of microorganisms. In a preliminary report (9), it was suggested that this amino sugar is 3-0earboxyethylhexoFamine. These facts raised the possibility that the observed spectral shift is correlated with 3-substitution of hexosamines and might be useful for the determination of structure in hexosamine-containing polysaccharides. It is the purpose of this paper to present evidence that such a correlation does exist and to indicate the applicability of this method to structural determinations.