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.

FIGURE 1:

High-pressure fluorescence cell.

FIGURE 2:

High-pressure absorbance and enzyme assay cell.

FIGURE 3:

Absorption spectra of oxygen, 1500 psi, 10.7-cm light path.

FIGURE 4:

Oxygen quenching of N-acetyl-l-tryptophanamide (엯) and riboflavine (Δ) in 0.1 m sodium phosphate, pH 7.0.

FIGURE 5:

Arrhenius plot of the oxygen bimolecular quenching constant of tryptophan.

FIGURE 6:

Oxygen quenching of tryptophan as observed by fluorescence yield and lifetime. Solvent is 0.1 m sodium phosphate, PH 7.0.

FIGURE 7:

Oxygen quenching of perylene in dodecane.

FIGURE 8:

Comparison of quenching by oxygen (this paper) and iodide (Lehrer, 1971) of charge copolymers. The same conditions as described for tryptophan (Table II) were used, except 360-nm emission for the glutamate-tryptophan copolymer. The solvent was 0.06 m Tris-HCl, pH 7.54.

FIGURE 9:

Oxygen quenching of ethidium bromide when free in solution (엯) and when intercalated into double helical DNA (Δ). The solvent was 0.01 m Tris-HCl-0.001 m EDTA–1 m NaCl, pH 7.8; free dye. 480 ± 2 nm excitation, 585 ± 3 nm emission; ethidium bromide–DNA complex, 505 ± 2 nm excitation, 582 ± 3 nm emission, [DNA] = 3.6 × 10⁻⁴ m, [ethidium bromide] = 0.24 × 10⁻¹ m.

FIGURE 10:

Oxygen quenching of ANS–bovine serum albumin, $\overline{n}=0.5$ (a) and $\overline{n}=9$ (b) as observed by fluorescence yield (엯) and lifetime (Δ). See legend to Table V for experimental details. Lifetimes were measured by the modulation method, 14.2 MHz.