The direct time-resolved fluorescence anisotropy of the single tryptophan residue in the polypeptide hormone adrenocorticotropin-(1-24) (ACTH) and the fluorescence decay kinetics of this residue (Trp-9) are reported. Two rotational correlation times are observed. One, occurring on the subnanosecond time scale, reflects the rotation of the indole ring, and the other, which extends into the nanosecond range, is dominated by the complex motions of the polypeptide chain. The fluorescence lifetimes of the single tryptophan in glucagon (Trp-25) and the 23-26 glucagon peptide were also measured. In all cases the fluorescence kinetics were satisfied by a double-exponential decay law. The fluorescence lifetimes of several tryptophan and indole derivatives and two tryptophan dipeptides were examined in order to interpret the kinetics. In close agreement with the findings of Szabo and Rayner [Szabo, A. G., & Rayner, D. M. (1980) J. Am. Chem. Soc. 102, 554-563], the tryptophan zwitterion exhibits emission wavelength dependent double-exponential decay kinetics. At 320 nm tau 1 = 3.2 ns and tau 2 = 0.8 ns, with alpha 1 = 0.7 and alpha 2 = 0.3. Above 380 nm only the 3.2-ns component is observed. By contrast the neutral derivative N-acetyltryptophanamide has a single exponential decay of 3.0 ns. The multiexponential decay kinetics of the polypeptides are discussed in terms of flexibility of the polypeptide chain and neighboring side-chain interactions.

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We have used optically detected magnetic resonance (ODMR) to characterize the degree of solvent availability of the tryptophan residues in lysozyme that are likely to be responsible for the observed phosphorescence. From the phosphorescence spectra, ODMR zero-field splittings (zfs), and ODMR line widths, we concur with the X-ray structure [Blake, C. C., Mair, G. A., North, A. C. T., Phillips, D. C., & Sarma, V. R. (1967) Proc. R. Soc. London, ser. B 167, 365-377] that Trp-62 behaves as an exposed residue and Trp-108 is buried. In addition, we present evidence 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, we have identified those portions of the ODMR lines in the native enzyme that are due to those specific residues. Barring major enzyme conformational changes in the vicinity of unmodified tryptophan residues when Trp-62 or Trp-108 are selectively modified, we find that Trp-108 dominates both the phosphorescence and the ODMR signals in native lysozyme. The results are discussed in view of previous fluorescence findings.

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