Quenching of protein fluorescence by oxygen. Detection of structural fluctuations in proteins on the nanosecond time scale.

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 (*) 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 * 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.