The interactions between highly de‐N‐acetylated chitosans and lysozyme from chicken egg white studied by 1H‐NMR spectroscopy

We have investigated the binding interactions between highly de-N-acetylated chitosans and lysozyme from chicken egg white by one-dimensional and two-dimensional 1H-NMR spectroscopy. A fully de-N-acetylated chitosan (fraction of N-acetylated units, FA < 0.001) induced no observable changes in the 1H chemical shifts of lysozyme. However, a chitosan with FA = 0.04, where the N-acetylated units are predominantly surrounded by de-N-acetylated units (a monoacetylated sequence), induced significant shifts of several lysozyme resonances, demonstrating a specific interaction between lysozyme and de-N-acetylated units in the chitosan. The interaction between the two positively charged molecules increased with increasing ionic strength, as expected. The dissociation constant (Kd) between lysozyme and the monoacetylated sequence was strongly dependent on pH* (pH measured in D2O), with Kd = 0.02 ± 0.01 mM at pH* 6.0, Kd = 0.11 ± 0.02 mM at pH* 4.5, and Kd≈2 mM at pH* 3, suggesting that electrostatic forces contribute to the observed binding. The complex was strikingly stable, with bound lifetimes in the range of 10−25 ms at pH* 4.5 and 328−300 K. Most lysozyme resonances that were affected by the binding were assigned, and we suggest that the monoacetylated chitosan sequence binds to the active site cleft of lysozyme with the N-acetylated unit in subsite C. Assuming this binding mode, we have discussed the contributions in energetic terms from individual subsites of lysozyme towards binding of N-acetylated and de-N-acetylated units.