Triplet state of tryptophan in proteins. 2. Differentiation between tryptophan residues 62 and 108 in lysozyme

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Abstract
Optically detected magnetic resonance (ODMR) was used to characterize the degree of solvent availability of the tryptophan residues in lysozyme that are probably responsible for the observed phosphorescence. The phosphorescence spectra, ODMR zero-field splittings (zfs) and ODMR line widths agree with the X-ray structure and Trp-62 behaves as an exposed residue and Trp-108 is buried. Evidence is presented that ODMR can be used in conjunction with conventional phosphorescence to evaluate the degree of order in the microenvironments of tryptophan in a protein containing several tryptophans. By the specific modification of residues Trp-62 and Trp-108, those portions of the ODMR lines in the native enzyme that are due to those specific residues were identified. Barring major enzyme conformational changes in the vicinity of unmodified tryptophan residues when Trp-62 or Trp-108 are selectively modified, Trp-108 dominates the phosphorescence and the ODMR signals in native lysozyme. The results are discussed in view of previous fluorescence findings.