title: Studies of chemical modifications of proteins by carbon 13 neuclear magnetic resonance spectroscopy. Reaction of hen egg white lysozyme with iodine.
abstract: The reaction of iodine with aromatic residues of hen egg white lysozyme is examined by means of natural abundance 13C nuclear magnetic resonance spectroscopy. In the unfractionated product of the reaction at PH 5.5 (with I2/lysozyme molar ratios of 0.5, 1.0, and 2.5), the only detectably modified aromatic residues are Trp-108 and either Tyr-20 or Tyr-23 (probably the latter). The rates of reaction at the two sites are similar. The extents of modification (at each site) are approximately 25%, 50%, and approximately greater than 80% for I2/lysozyme molar ratios of 0.5, 1.0, and 2.5, respectively. At pH 4.5, the rates of reaction of both residues are about one-third or less of the rates at pH 5.5. When the reaction is carried out at pH 8.5 (with an I2/lysozyme molar ratio of 1.0), only the tyrosine residue is modified. Resonances observed in the spectra of the modified protein mixtures (but not in the spectrum of intact lysozyme) indicate that the modified Trp-108 residue is not oxindolealanine, but either delta1-hydroxytryptophan or an ester thereof. This result is consistent with previous evidence which indicates that the modified tryptophan is the Glu-35 ester of delta1-hydroxytryptophan-108 (Imoto, T., and Rupley, J.A. (1973) J. Mol. Biol. 80, 657-667; Beddell, C. R., Blake, C. C. F., and Oatley, S. J. (1975) J. Mol. Biol. 97, 643-654). The spectra also indicate that the modified tyrosine residue is predominantly monoiodinated. The spectra of modified protein samples subjected to denaturation with 6M guanidinium chloride for 24 h at 37 degrees (and the renatured) indicate that residue 108 is converted to about equal amounts of the two diastereoisomers of oxindolealanine. However, incubation in 6M guanidinium chloride for 2 h at 25 degrees does not cause measurable hydrolysis of the Glu-35 ester of delta1-hydroxytryptophan-108.
mesh_terms: D000818:Animals; D002247:Carbon Isotopes; D002645:Chickens; D004529:Egg White; D005583:Fourier Analysis; D007455:Iodine; D007467:Iodoproteins; D009682:Magnetic Resonance Spectroscopy; D009113:Muramidase; D011485:Protein Binding; D011487:Protein Conformation
chemical_list: D002247:Carbon Isotopes; D007467:Iodoproteins; D007455:Iodine; D009113:Muramidase