The bulk of the fluorescence of lysozyme is located in Trp 62 and Trp 108. By examination of the fluorescence of derivatives in which Trp 62 and/or Trp 108 are specifically oxidized, it has been possible to detect a pH-dependent interaction between tryptophan residues. This interaction is interpreted as energy transfer from Trp 108 to Trp 62.The efficiency of tryptophan fluorescence in proteins varies widely from one protein to another, with quantum yields ranging from about 0.025 to greater than 0.4 (1). In proteins containing more than one tryptophan, each residue can contribute differently to the emission intensity (2). Studies of tryptophan emission in model compounds clearly demonstrate the great sensitivity of the emission intensity to environment (3); fluorescence measurements have been extensively used to obtain information about tryptophan environments in proteins (4). The analysis and interpretation of luminescence data when the protein contains more than one tryptophan are highly uncertain. It is seldom possible to separate unambiguously the contributions of each tryptophan to the total emission. Energy transfer from tyrosine to tryptophan is, in some cases, a complicating factor (5). Evidence for intertryptophanyl energy transfer has been presented (6), although such transfer is commonly ignored.Lysozyme is a very useful substrate for fluorescence studies for three reasons: (i) The tryptophan to tyrosine ratio is high (2: 1), and interference from tyrosine emission and/or tyrosine to tryptophan energy transfer is minimal; (ii) Derivatives can be prepared in which tryptophan (residue 62 or 108) is specifically oxidized (7); and (iii) The crystal structures of the native protein and several protein-saccharide complexes are known (8).In the present work, we evaluate the contributions of individual tryptophans to the emission and provide evidence for intertryptophanyl energy transfer in the fluorescence of lysozyme. MATERIALS AND METHODSThe quantum yields of native, oxindole 62, and oxindole 108 lysozymes were measured relative to tryptophan in H20 with an Aminco-Bowman spectrofluorometer (30°, 0.1 ionic strength). The emission spectra were corrected by comparison with a corrected tryptophan spectrum generously provided by the Turner Instrument Co. The quantum yields of oxindole 62,108 lysozyme were obtained with an instrument of conventional design but greater sensitivity, assembled in this laboratory. Absorbance at 280 nm was in all cases kept below 0.1. Guanidine hydrochloride (guanidine) was ultrapure from Mann; N-bromosuccinimide was from Matheson; lysozyme. was from Worthington. The oxindole-108 derivative was prepared by iodine oxidation to give the oxindolealanine-108, Glu 35 ester, followed by guanidine denaturation and refolding to generate the oxindolealanine-108 protein by hydrolysis of the ester (ref. 7; Imoto and Rupley, unpublished). N-bromosuccinimide oxidation (7) of native or oxindole-108 lysozyme gave the oxindole-62 or oxindole 62,108 derivatives. RESULTS Excitation and emiss...

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Tryptophan lifetimes and relative quantum yields have been determined for a group of small ( I 4 residues) peptides and peptide hormones [luteinizing releasing factor. mellitin, glucagon. glucagon 22-29, glucagon 1-26. glucagon 1-27 (homoserine)]. All of the larger peptides and most of the smaller peptide anions exhibit nonexponential decay. Peptide quenching in the small peptides is more effective when the bonding is at the amino rather than at the carboxyl end of tryptophan. With the exception of tryptophylglycine. quenching by NH; is thought not to involve proton transfer. The results suggest that a decay component of 3-4 ns is expected whenever large peptides and proteins contain a solvent exposed tryptophan.

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