Use of dansyl N-acetyl glucosamine as substrate for chitin synthetase activities

Abstract

The biophysical properties of fungal cell walls are determined by the composition, level and structural arrangement of their polysaccharides. A small amount of glucosamine in the cell wall (1–3%) forms chitin, which is a β(1 → 4) linear homopolymer of N-acetylglucosamine (GlcNAc). Chitin synthetase activity is often analysed by radioactivity using [ 14C] GlcNAc. In this study, a fluorescent method was developed for assessing chitin synthetase activity using N-dansyl N-acetyl glucosamine as chitin synthetase substrate. The technique is based on the preparation of the fluorophore N-acetyl glucosamine and its subsequent decrease due to its polymerisation by chitin synthetase. The resulting decrease of fluorescence can be measured by spectrofluorometery. This method is suitable and adaptable for chitin synthetase assays. Results are parallel to those obtained by radioactivity method described in literature.