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...