Laser flash photolysis of DNA-intercalated ethidium bromide in the presence of methylviologen

Abstract

Methylviologen quenches the fluorescence of DNA-intercalated ethidium bromide (EB) via electron transfer to yield reduced viologen. The majority of reduced viologen rapidly s) recombines with oxidized EB on the DNA helix; however, a small (ca. 2%) fraction escapes from the helix into bulk solution. Recombination of this fraction then occurs via a second-order process (k = (5.6 * 1.5) X lo9 M-' s-' ). The yield of reduced viologen which escapes the helix increases as the ionic strength of the solution rises. In the absence of methylviologen, excitation leads to small, long-lived absorptions in the visible region to yield oxidized EB.which are attributed to the first excited triplet state of EB. These are quenched by oxygen (k = (3.8 0.6) X lo7 M-' s-I ) IntroductionIn our previous work on the photophysics and photochemistry of DNA intercalated ethidium bromide (EB) we concentrated on the fluorescence quenching of excited EB by transition-metal ions.' V2 In particular we concluded that those ions which quenched EB fluorescence in aqueous solutions were capable of quenching the fluorescence of DNA-intercalated EB with greatly increased efficiency.' In the course of these studies we observed that DNA-intercalated EB fluorescence was also quenched by methylviologen (MV2+) and that reduced viologen was formed on the time scale of the EB fluorescence decay.2 Further, the reduced viologen was stable over hundreds of microseconds in nitrogensaturated solution. This indicates that EB fluorescence is quenched by MVZ+ via electron transfer from excited EB to MV2+ and also that some fraction of the reduced viologen is able to escape fast recombination with oxidized EB. A similar conclusion was reached recently by Fromherz and Rieger who used primarily steady-state methods to observe enhanced yields of electron transfer from EB excited singlet state to MV2+ in the presence of DNA.3In this work we use transient absorption spectroscopy to follow the reactions of the products of this fluorescence quenching. Of particular interest is the environment and quantum yield of the reduced viologen which escapes rapid recombination with oxidized EB. Since to our knowledge no previous work on time-resolved absorption spectroscopy in the EB-DNA system has been reported, with the exception of our own nanosecond study,4 we felt it necessary to investigate this system in addition to the system containing MV2+. This is an extension of our earlier study to longer time scales. We have shown that the singlet excited state

A polarized photobleaching study of DNA reorientation in agarose gels

“…It is helpful to compare the photophysical behavior of the putative triplet state seen in our pFRAP experiments to that of a putative ethidium triplet detected previously (Atherton & Beaumont, 1986, 1987. We first note that the deoxygenated triplet lifetime found here (1.8 ms) agrees well with the literature value.…”

Photophysical Studies on the Binding of Tetrasulfonatophenylporphyrin to Lens Proteins

“…Digitized data from a Biomation 8100 transient recorder were passed to a DEC PDP 11/70 minicomputer for analysis, storage and hard copy. The experimental set-up has been described previously (Atherton and Beaumont, 1987). Figure 2.…”

In vivo AND PHOTOPHYSICAL STUDIES ON PHOTOOXIDATIVE DAMAGE TO LENS PROTEINS AND THEIR PROTECTION BY RADIOPROTECTORS

“…The generated transient species were monitored at right angles to the laser beam in a I cm cell situated in a computer controlled single beam kinetic spectrometer with nanosecond time resolution. The experimental set-up has been described previously (Atherton and Beaumont, 1987). Singlet oxygen generation was monitored by its IR luminescence at 1.27 pM using a germanium photodiode based system (Rogers and Snowden, 1982).…”