While the phosphorescence of. aromatic chromophores. in solution is normally quenched through diffusion of dissolved oxygen and other solvent-mediated processes, the phosphorescence of some proteins in solution is observed at room temperature. The tryptophan phosphorescence arises from residues which are hindered from interaction with oxygen by the folding of the polypeptide chains. Measurements of the phosphorescence lifetime of horse liver alcohol dehydrogenase (alcohol: NAD+ oxidoreductase, EC 1.1.1.1) as a function of oxygen concentration indicatethat internal tryptophan residues are periodically exposed to oxygen. This permits the calculation of rate constants for conformational oscillations in the enzyme. The present article illustrates the feasibility of employing phosphorescence in the stuedy of proteins in solution in general-and the utility of such experiments in probing the dynamic aspects of protein structure.The properties of the triplet state of aromatic chromophores may be profitably exploited in the study of biomolecular structure and dynamics. Phosphorescence measurements have been used in biochemical systems both to detect the proximity of aromatic groups (1-3) and to monitor long-range interactions (4, 5) in small molecule-biopolymer complexes. In addition tryptophan and tyrosine (6), and in favorable cases individual tryptophan residues (7), can be resolved in protein phosphorescence spectra.Despite the molecular information which can be derived from Rigid media have been employed due to the normally efficient quenching of the triplet state in fluid solution by dissolved oxygen and other solvent-quenching processes. In that aromatic amino acids residues are frequently buried within the globular structure of proteins, it was anticipated that their triplet states would be less susceptible to quenching, the degree of protection afforded a particular aromatic residue being a function of the location of the residue within the protein structure and the flexibility of the protein conformation.Our observations of protein phosphorescence with increasinig temperature reveal that the folding of the polypeptide chains in protein molecules does hinder quenching of the triplet states of internal tryptophan residues. Phosphorescence from proteins is observed in fluid solution at temperatures where it cannot be observed from free chromophores, and in the present article preliminary data on the room temperature phosphorescence of horse liver alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) and Escherichia coli alkaline phosphatase (orthophosphoric-monoester phosphohydrolase, EC 3.1.3.1) are presented. The oxygen dependence -of the phosphorescence lifetimes observed at room temperature provides a measure of the kinetics of conformational flexibility in proteins. The triplet lifetimes are also found to be sensitive to the binding of coenzymes and the presence of protein stabilizing and destabilizing agents.We feel the present study not only indicates the usefulness of phosphorescence in p...

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Abstract. The emission spectra of polar aromatic molecules in rigid, polar solution are shown to depend on the exciting wavelength. Occurrence of the phenomenon depends on both the excited-state lifetime of the chromophore and the degree of rigidity of the medium. The results are interpreted in terms of a model which stresses the contribution of micro-environmental heterogeneity to electronic absorption and emission spectra.Shifts in chromophore electronic absorption and emission spectra as a function of the polarity and polarizability of the solvent are well known and have been interpreted by Bayliss and McRae in terms of permanent and induced dipolar interactions between a molecule in its ground and excited states and the solvent.' The interactions have been qualitatively treated as occurring between the chromophore and a general molecular environment. For aromatic amino acid residues in proteins2-4 and for aromatic hydrocarbons trapped in polycrystalline matrices' a considerable body of evidence exists that indicates that chromophores of the same species can have different electronic energies as a consequence of the existence of several distinct local environments. Even in a homogeneous phase, solute molecules can be expected to occupy a variety of solvation sites at any given time, generating an array of electronic transition energies whose summation comprises the absorption or emission spectrum of the sample as a whole. Such variation in solute-solvent local interactions is thought to be a significant source of the broadening of the absorption and emission spectra of a chromophore in a condensed phase.In the present article we provide evidence for the contribution of microenvironmental heterogeneity to solution spectra through our observation of an exciting-wavelength dependence for fluorescence and phosphorescence spectra of aromatic molecules in dilute rigid solution. In exploring the consequences of this environmental heterogeneity we show that it provides a rationalization for the failure of fluorescence concentration depolarization at the red edge of the solute absorption band7-9-the so-called "Weber red edge effect." 10 The Model. No attempt is made in this paper to present a quantitative model for the contribution of environmental heterogeneity to electronic spectra. We only emphasize three qualitative assumptions: (1) that electronic energies of chromophores in solution are a function of geometry-dependent solute-solvent interactions, (2) that at any instant in time there is an ensemble of interac-1116

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