Proflavine Photosensitization of Double and Single Stranded Dna: Epr Study of the Effect of Various Sulphur‐containing Substances

Abstract

Abstract—The proflavine‐mediated photosensitization of both double‐stranded calf thymus and single‐stranded DNA from bacteriophage φ× 174 was followed in terms of the induction of free radicals in frozen solutions by EPR measurements in the presence of oxygen. The effect of the addition of various sulphur‐containing substances to the proflavine DNA mixtures was studied and quantitatively expressed for definite amounts of proflavine bound to both DNAs. Upon irradiation with visible light, RS radicals were observed in the presence of these sulphur‐containing substances. Some of them caused a decrease in the amount of peroxide radicals normally induced in the photosensitized DNA. This decrease appeared to be linked to the property of the compounds to interact with DNA in such a way that they modify the binding of proflavine to DNA. Cysteine, cysteamine, cystamine and cystine, protect calf thymus DNA against the “proflavine and light” free radical induction by 55, 58, 64 ahd 62% respectively. φ× 174 single‐stranded DNA is also protected by the addition of cysteamine and cystamine, although to a lesser extent than calf thymus DNA (35%). These protection factors were measured under conditions where strong binding of the dye to the DNA was predominant.

In Vitro Toxicity Testing of Environmental Agents

“…As described with other organisms, neither the compounds alone nor light alone inactivate the phage (Spikes, 1977;Carlberg-Bacq et al, 1978). The loss of infectivity is due to interaction of the aza-heterocycles and photons with the free phage particle in solution, and not from effects on the propagation of the phage on infection of the Salmonella host cells.…”

“…Instead, the catalytic role played by photodynamic dyes is believed to involve an initial absorption of a photon to reach a photo-excited state, followed by interaction with dissolved molecular oxygen to generate an activated oxygen species. Several mechanisms have been proposed for this activation (Piette et al, 1978;Foote, 1968). Because we observed the P22 inactivation to vary linearly with light Figure 6 Note: 50 ~L of concentrated cell extracts containing replicating DNA and all the viral proteins but the major capsid protein were incubated with 50 ~L of purified precursor shells (2 x 1011 shells/mL) and the indicated concentration of the indicated acridine derivative (final volume was 100 ~L) for 1 hour at 28° C. The reaction mixtures also contained 0.015 M ATP, 0.02 M Hepes pH 7.4, 0.006M spermidine, 0.02 M MgCI2, and 0.003M 2-mercaptoethanol.…”

“…Singlet oxygen is known to be a transient, highly active molecule which can react with both DNA and proteins (Spikes and Livingston, 1969;Lochman and Micheler, 1973). DNA has generally been assumed to be the target of the lethal event, but the evidence has been indirect (Freifelder and Uretz, 1966;Cramer and Uretz, 1966;Piette et al, 1978). As illustrated here, the results of Bryant (1978) and Bryant and King (in preparation) indicate that, at least for P22, proteins in the phage particle are the target of the lethal damage.…”

“…The map shows the arrangement of the genes coding for the proteins involved in virus structure and assembly (Susskind and Botstein, 1978). and probably proceeds through the generation of a very reactive, short-lived singlet oxygen species (Foote, 1968;Spikes and Livingston, 1969;Piette et al, 1978). This oxidizes nearby groups such as guanidine in DNA or histidine in proteins.…”