Optical properties of lysozyme. pH and saccharide binding difference spectra

• Published in: The Journal of biological chemistry 1975-10, Vol.250 (20), p.8275-8282
• Main Authors: Imoto, T, Andrews, L J, Banerjee, S K, Shrake, A, Forster, L S, Rupley, J A
• Format: Article
• Language: English
• Subjects: Acetylglucosamine; Animals; Binding Sites; Chickens; Egg White; Glucosamine - analogs & derivatives; Hydrogen-Ion Concentration; Muramidase - metabolism; Oligosaccharides; Protein Binding; Protein Conformation; Spectrophotometry, Ultraviolet; Turkeys
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)40847-8

Difference spectra associated with changes in pH and with binding of saccharides have been recorded for hen egg white (HEW) lysozyme, turkey egg white (TEW) lysozyme, and for the derivatives of the hen protein in which Tre-62 or Trp-108 had been oxidized specifically to oxindolealanine to give the Oxa-62 or Oxa-108-proteins. Identical pH difference spectra were obtained for HEW, TEW, and Oxa-62-lysozymes. Oxidation of Trp-108 is reflected in both the high and low pH (pH 7 versus 5 and pH 2 versus 5) difference spectra. The magnitude of the low pH difference spectrum is enhanced by binding of saccharide for HEW and Oxa-62-lysozymes but not for TEW lysozyme. The shapes and magnitudes of saccharide binding difference spectra are affected by oxidation of residues 62 or 108. These results can be interpreted in terms of the perturbations responsible for the lysozyme difference spectra. The pH 7 versus 5 difference spectrum results from perturbation by Glu-35 of Trp-108 and another tryptophan, probably Trp-63. Perturbation of Trp-108 and one or more other tryptophan residues by several carboxylate groups is responsible for the low pH difference spectra of the unliganded HEW and TEW lysozyme molecules. Perturbation of Trp-108 makes a principal contribution to the saccharide-binding difference spectrum. Perturbation of the Oxa-108 chromophore by ionization of Glu-35 or by saccharide binding produces absorbance changes in the 250 to 265 nm region.