Decay of Fluorescence Emission Anisotropy of the Ethidium Bromide-DNA Complex Evidence for an Internal Motion in DNA

Abstract

Abstract. Evidence for an internal oscillatory Brownian motion in the DNA helix is obtained from the measurement of the decay of the fluorescence emission anisotropy of the ethidium bromide-DNA complex. The amplitude of the oscillation is found to be equal to 350 and the relaxation time equal to 28 nanoseconds.The dye ethidium bromide has been found to bind specifically to doublestranded nucleic acid helix. The interesting characteristics of this binding are:(1) The fluorescence quantum efficiency of the dye increases considerably on binding to double-stranded nucleic acids." 2 (2) Binding occurs by intercalation.2' Then ethidium bromide is rigidly bound to the double-stranded helix and the direction of the absorption and emission transition moments which are in the plane of the dye have a known direction with reference to the helix axis.2 Then, ethidium bromide is an ideal candidate as a fluorescence label for fluorescence depolarization measurements since the ethidium bromide lifetime of fluorescence is relatively large.4It was first observed5 that the polarization fluorescence coefficient for ethidium bromide bound to DNA was dependent on the viscosity of the solution. In other words, in aqueous solvent a depolarization of fluorescence was observed. Since the DNA is a large molecule, the extent of its rotation during the time of the ethidium bromide excited state can be assumed negligible. Then, two hypotheses had to be postulated, either the DNA was not rigid and was able to undergo some kind of internal motion, or ethidium bromide was not really rigidly bound as predicted by the intercalation model.6 In these conditions it was of interest to further study this problem by measuring the emission anisotropy during the fluorescence decay. This method has been already used with macromolecules bearing covalently bound fluorescent chromophores. Detailed information on the Brownian motion and the internal deformation of these macromolecules have been obtained.7'0 Methods. Let Ill(t), I1(t), be, respectively, the decay of fluorescence intensity of the horizontal and perpendicular components when fluorescence excitation is made by an infinitely short flash of natural light. The time of the flash is taken as the origin.

Rapid Segmental and Subdomain Motions of DNA Polymerase β

“…23); in particular, emission anisotropy has a long history of revealing local motions (24,25). In addition, timeresolved methods have the advantage of quantifying both the angular extent and the rate of these motions (24,26,27). In favorable cases, the fast motion of a segment on a loose "hinge" can be interpreted both in terms of the size of the independent segment and the angular range of facile hinging.…”

“…The measurement of segmental flexibility has a long history (see Ref. 23); in particular, emission anisotropy has a long history of revealing local motions (24,25). In addition, timeresolved methods have the advantage of quantifying both the angular extent and the rate of these motions (24,26,27).…”

A Study on the Motion Of Protenis in Excitable Membrane Fragment by Nanosecond Fluorescence Polarization Spectroscopy

“…I n particular, fluorescence spectroscopy appears to be especially convenient and versatile since it provides deep insight on the structure, interaction and motion of macromolecules both dispersed in solutions or integrated in a biological membrane A n important improvement of this last technique comes from the utilisation of nanosecond light pulses. By the measurement of the polarization of the fluorescent light emitted as an explicit function of time in the nanosecond range [6,[14][15][16][17]22,23], the estimation of the rotational motion of molecules labelled by convenient fluorophores becomes possible.We have carried out a nanosecond fluorescence polarization study of membranes prepared from the electric organ of the eel Electrophorus electricus. These membrane fragments derive from the innervated, and thus excitable surfaces of the electroplaxes, the elementary units of the electric organ.…”

Theory for Spin−Lattice Relaxation of Spin Probes on Weakly Deformable DNA

“…1 In 1970, the timeresolved decay in the fluorescence polarization anisotropy (FPA) from ethidium intercalated between base-pairs revealed that DNA in solution is a flexible polymer that undergoes both aboutaxis twisting and bending. 2 To explain these data, Barkley and Zimm developed a continuous elastic model of internal Brownian twisting and bending motions of the double helix. 3 Concurrently, Allison and Schurr generated a theory for the twisting motion contribution to the FPA decay in which DNA is represented by a series of identical rigid rods connected by Hookean torsion springs.…”