Analysis of chitin structure by nuclear magnetic resonance spectroscopy and chitinolytic enzyme digestion

Solid-state 13C-NMR analysis of chitin prepared from cuticle of the tobacco hornworm, Manduca sexta (L.), and of crab yielded spectra that demonstrate a high degree of chemical homogeneity (greater than 95%) for the preparations. The chemical shifts of the well-resolved carbon signals from both samples matched closely those of the monomeric unit 2-acetamido-2-deoxy-D-glucopyranoside (GlcNAc). Chromatographic analysis of products from the digestion of chitin by the binary chitinase system (endo splitting chitinase and exo splitting beta-N-acetylglucosaminidase) isolated from M. sexta molting fluid showed that the major product from both chitin preparations is GlcNAc. Also detected was a minor product (product U) that had a chromatographic retention time on the carbohydrate analysis column intermediate between those of chitin penta- and hexasaccharides. Gel filtration chromatography of U indicated that U had an apparent molecular weight intermediate between that of GlcNAc and of N,N'-diacetylchitobiose. Cation-exchange chromatography of U after acid hydrolysis revealed the presence of glucosamine only. Derivatization with trinitrobenzenesulfonate showed the presence of a free amino group in U. Solution proton and carbon NMR spectroscopy were used to identify U as a N-monoacetylchitobiose [O-beta-D-2-amino-2-deoxyglucopyranosyl- (1----4)-2-acetamido-2-deoxy-beta-D-glucopyranose] with the residue at the nonreducing end deacetylated. These studies showed that chitin prepared from alkali- and heat-treated insect or crab cuticle contains trace levels of deacetylated residues that are released as a dead-end product, N-monoacetylchitobiose, after digestion by the binary enzyme system.