Quenching of the fluorescence of various fluorophores by molecular oxygen has been studied in aqueous and nonaqueous solutions equilibrated with oxygen pressures up to 100 atm. Temperature dependence of quenching, agreement with the Stern-Volmer equation, and fluorescence lifetime measurements indicate that essentially all the observed quenching is dynamic and close to the diffusion-controlled limits. Studies of charged polyamino acids containing tryptophan show that oxygen quenching, in contrast to I-, is completely insensitive to charge effects. Ethidium bromide, when M olecular oxygen is known to be an efficient quencher of the fluorescence of aromatic hydrocarbons (Berlman, 1965;Ware, 1962). The studies so far published show quenching by oxygen to be a diffusion-controlled process in which virtually every collision with the excited fluorophore is effective in quenching. Although a good deal of work has been done in nonaqueous solutions, very little work has been done in aqueous solutions. This discrimination is due to the low solubility and diffusion coefficient of oxygen in water as compared to organic solvents, and the concomitant low levels of quenching that are observed in solutions equilibrated with air, or even with pure oxygen at atmospheric pressure.Previous work in aqueous systems used pyrenebutyric acid (Vaughan and Weber, 1970)' Due to the long fluorescent lifetime of pyrenebutyric acid (100-200 nsec) it was possible to quench a significant fraction of its fluorescence using dissolved oxygen at 1 atm pressure. It was shown that when pyrenebutyric acid was bound to bovine serum albumin and other proteins the amount of quenching was greatly reduced. reported the use of oxygen to quench photo-and radioluminescence of aromatic amino acids, proteins, and nucleic acids in solution and as dry powders. The use of polychromatic excitation (240-293 nm) comprising wavelengths of strong 0 2 absorption, the smaller range of 0 2 pressures (16 atrn), the small number of proteins examined, and the absence of fluorescence lifetime measurements preclude comparison of his results with the present studies. intercalated into double helical DNA, is quenched with 1/30th of the efficiency of the free dye in solution. Three dyes bound to bovine serum albumin were also found to be relatively protected from the free diffusion of oxygen. Quenching of intrinsic or bound fluorophores by molecular oxygen is therefore an appropriate method to determine the accessibility to oxygen of regions of the macromolecule surrounding the fluorophore and indirectly the structural fluctuations in the macromolecule that permit its diffusion to the fluorophore.The protein structure prevented the diffusion of oxygen to the fluorophore.Typical fluorescence probes have lifetimes of 10-20 nsec. Native tryptophan fluorescence of proteins displays lifetimes ranging from 2 to 6 nsec. Upon equilibration with oxygen at a pressure of 1 atm the fluorescence of a dye solution with a 2nsec lifetime would decrease by only 3 %; in a system where there is so...

Environmentally sensitive fluorescent probes involve two groups, an electron donor and an electron acceptor, attached to an aromatic ring system, and maximal effects may be expected when these groups are as far apart as feasible. The syntheisis, characterization, and spectroscopic properties of 6-propionyl-2-(dimethylamino)naphthalene (PRODAN), a compound that fulfills these conditions, are described. The maximum of emission is at 401 nm in cyclohexane solution and at 531 nm in water solution, indicating an increase of dipole moment of approximately 20 D units on excitation to the lowest singlet state. The effect of temperature upon the spectral distribution and the bandwidth of fluorescence of PRODAN in 1:1 complexes with albumin shows the existence of a dynamic relaxation process of the protein surroundings within th 2-4 ns of the fluorescence lifetime.

Quenching of the tryptophan fluorescence of native proteins was studied using oxygen concentrations up to 0.13 M, corresponding to equilibration with oxygen at a pressure of 1500 psi. Measurement of absorption spectra and enzymic activities of protein solutions under these conditions reveal no significant perturbation of the protein structure. The oxygen quenching constant (k + *) for a variety of proteins indicates that the apparent oxygen diffusion rate through the protein matrix is 20-50% of its diffusion rate in water. No tryptophan residues appear to be excluded from quenching, and no correlation of the fluorescence emission maxima with k + * was found, indicating that the rapid oxygen diffusion is present in all regions of the protein, even those normally considered inaccessible to solvent. Energy transfer among tryptophans was excluded as a possible mechanism for the rapid quenching by studies using 305-nm excitation, where energy transfer is known to fail. The dynamic character of the observed quenching was proven by the proportional decrease of the fluorescence lifetimes and yields measured under the same conditions. We conclude that proteins, in general, undergo rapid structural fluctuations on the nanosecond time scale which permit diffusion of oxygen.The previous paper (Lakowicz and Weber, 1973) described the methodology and presented experimental data for the quenching of small molecules, and some linear biopolymers, by oxygen. Here we examine the quenching of the fluorescence of proteins by oxygen. X-Ray determined structures and solvent perturbation studies of many proteins have shown that tryptophan residues are often situated in the interior of the protein matrix and appear inaccessible to solvent. It must be pointed out, however, that both of these techniques yield information about the average conformation and solvation of the amino acid residues, but no information about the existence of the structural fluctuations which may occur. Since quenching of fluorescence by oxygen depends on the collisional rate between oxygen and fluorophore, we expected oxygen quenching of tryptophan fluorescence in proteins to yield information on the dynamics of those structural changes in the nanosecond time scale that would allow diffusion of oxygen through the protein matrix. Such local fluctuations must be indispensable for the effective quenching of a fluorophore shown by X-ray structural studies to be out of direct contact with water.

We shall not attempt here to enumerate the results or review in a systematic way the significant literature dealing with the use of high pressure in studies of proteins and other molecules of biological interest. Two recent reviews on this subject, one by MOrild (1981) and another by Heremans (1982), and a further article by Jaenicke (1981) on enzymes under extreme environmental conditions contain expositions and references that would render redundatn such a task. Rather we concentrate here on the examination of othe conceptual framework employed in the interpretation of high pressure experiments and in the critical discussion of our knowledge of selected areas of present interest and likely future significance.

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