Showing papers in "Biochemistry in 1973"

Quenching of fluorescence by oxygen. A probe for structural fluctuations in macromolecules.

Abstract: 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 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.

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

Abstract: Quenching of the tryptophan fluorescence of na- tive proteins was studied using oxygen concentrations up to 0.13 M, corresponding to equilibration with oxygen at a pres- sure of 1500 psi. Measurement of absorption spectra and en- zymic 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 indi- cates that the apparent oxygen diffusion rate through the pro- tein matrix is 20-50z of its diffusion rate in water. No tryp- tophan residues appear to be excluded from quenching, and no correlation of the fluorescence emission maxima with k+* T he previous paper (Lakowicz and Weber, 1973) de- scribed the methodology and presented experimental data for the quenching of small molecules, and some linear biopoly- mers, by oxygen. Here we examine the quenching of the fluo- rescence 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 ex- istence of the structural fluctuations which may occur. Since quenching of fluorescence by oxygen depends on the colli- sional 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 fluoro- phore shown by X-ray structural studies to be out of direct contact with water. The quenching of the fluorescence of fluorophore residues forming part of a protein, or in general of a compact macro- molecule, presents many complexities that are absent in the case of an isolated fluorophore in solution. These complexities can best be examined by reference to the modified Stern- Volmer equation discussed in the previous paper