Temperature and pH Dependence of the Binding of Oligosaccharides to Lysozyme

Association of lysozyme with the following ligands was measured at 0.1 ionic strength, using absorbance and fluorescence procedures: (a) the β(1 → 4)-linked trimer of N-acetylglucosamine, at pH 0 to 9 and 8–60°; (b) the dimer, at pH 0 to 9 and 30° and at 9–60° and pH 5.3; (c) N-acetylglucosamine, at 9–60° and pH 5.3. The complex-free protein difference spectrum varied with temperature and pH. Analysis of the association data indicated that trimer binding perturbs the pK of three ionizations, glutamic 35, 6.1 to 6.4; aspartic 101, 4.3 to 3.4; aspartic 66, 1.9 to 1.5. Since aspartic 66 does not interact directly with the saccharide, its pK shift presumably reflects the change in conformation that has been seen in crystallographic analysis (Blake, C. C. F., Mair, G. A., North, A. C. T., Phillips, D. C., and Sharma, V. R. (1967) Proc. Roy. Soc. London B Biol. Sci. 167, 365; Blake, C. C. F., Johnson, L. N., Mair, G. A., North, A. C. T., Phillips, D. C., and Sharma, V. R. (1967) Proc. Roy Soc. London B Biol. Sci. 167, 378) and involves this carboxyl. Aspartic 66 is buried as a carboxylate that is not part of an ion pair. The enthalpy of association increases by 5.9 kcal between pH 5 and 2 and is compensated by a nearly equal increase in entropy. This behavior may be related to desolvation of aspartic 101 in complex formation. The apparent average enthalpy of hydrogen-bond formation in the trimer complex is -1.5 kcal.