Binding of a highly de-N-acetylated chitosan to Japanese pheasan lysozyme as measured by 1H-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 1H-NMR spectroscopy. The profile of the one-dimensional spectrum of JPL is essentially identical to that of HEWL. Using two-dimensional spectra of JPL and HEWL, several aromatic and aliphatic proton resonances of JPL were assigned by comparison. When a highly de-N-acetylated chitosan (number-average degree of polymerization, about 18; degree of acetylation, 0.04), where the N-acetylated units are predominantly surrounded by de-N-acetylated units (a monoacetylated chitosan), was added to the JPL solution, the NMR signals were clearly affected in Trp28 C5H and Ile98 gammaCH, as in the case of binding to HEWL. 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/mol), 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 significantly 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 monoacetylated chitosan binds to subsites E and F through the left-sided binding mode.
Fukamizo
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Department of Food and Nutrition, Kinki University, Nakamachi, Nara, Japan. fukamizo@nara.kindai.ac.jp
Yamaguchi
T
T
Araki
T
T
Torikata
T
T
Kristiansen
A
A
Vårum
K M
KM
D000465:Algorithms; D000595:Amino Acid Sequence; D000818:Animals; D001717:Birds / Q000378:metabolism*; D002686:Chitin / Q000031:analogs & derivatives / Q000737:chemistry*; D048271:Chitosan; D003640:Dealkylation; D017666:Deuterium Oxide; D004529:Egg White / Q000032:analysis; D004563:Electrochemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009113:Muramidase / Q000737:chemistry*; D011485:Protein Binding; D014364:Tryptophan / Q000737:chemistry
D002686:Chitin; D014364:Tryptophan; D048271:Chitosan; D009113:Muramidase; D017666:Deuterium Oxide