Binding of a highly de-N-acetylated chitosan to Japanese pheasan lysozyme as measured by H-1-NMR spectroscopy

Binding of a highly de-N-acetylated chitosan to Japanese pheasant lysozyme (JPL), which differs from hen egg white lysozyme (HEWL) by nine amino acid substitutions (including Arg114 --> His), was investigated by IH-NMR spectr oscopy. The profile of the one-dimensional spectrum of JPL is essentially i dentical to that of HEWL. Using two-dimensional spectra of JPL and HEWL, se veral aromatic and aliphatic proton resonances of JPL were assigned by comp arison. When a highly de-N-acetylated chitosan (number-average degree of po lymerization, about 18; degree of acetylation, 0.04), where the N-acetylate d units are predominantly surrounded by de-N-acetylated units (a monoacetyl ated chitosan), was added to the JPL solution, the NMR signals were clearly affected in Trp28 C5H and Ile98 gamma CH, as in the case of binding to HEW L. The dissociation constant of the monoacetylated chitosan evaluated from the NMR signal responses was calculated to be 0.23 +/- 0.05 mM (-31.5 kJ/mo l), which is similar to that of HEWL (0.11 +/- 0.02 mm, -33.3 kJ/mol). Thus , the Arg --> His substitution of the 114th amino acid, which participates in sugar residue binding at the right-sided subsite F, did not significantl y affect the chitosan binding. In addition, the C2H signal of His114 of JPL was not affected by the chitosan binding. These results suggest that the m onoacetylated chitosan binds to subsites E and F through the left-sided bin ding made.