ENHANCEMENT OF OXIDATIVE DNA-DEGRADATION BY HISTIDINE - THE ROLE OF STEREOCHEMICAL PARAMETERS

The nicking of supercoiled DNA by H2O2 and ferrous iron has been studied in a variety of environmental conditions. The replicative form of phage fd DNA (fd RF DNA) was used for investigating the phenomenon. The rate of nicking was measured in 10 mM NaCl. The addition of 1 mM Tris-HCl buffer (pH 7.5) slowed down the rate of nicking, the addition of 0.1 mM histidine enhanced it. The simultaneous presence of 1 mM Tris-HCl buffer and of 0.1 mM histidine further enhanced the rate of nicking of fd RF DNA. Increasing the concentration of NaCl dramatically reduced the rate of the reaction. The degradation of fd RF DNA was determined as a function of the concentration of histidine (0-5 mM): the rate increases with concentration, reaches a maximum and then decreases. In the presence of histidine, increasing the concentration of Tris leads to a similar phenomenon. In the absence of histidine, Tris always quenches the degradation of DNA. Electron spin resonance measurements failed to detect an enhancement of the signal characteristic for the hydroxyl radical when histidine was added to the solution containing hydrogen peroxide and ferrous iron. When the nicking of DNA is achieved via the process of auto-oxidation of ferrous iron (i.e., in the absence of added H2O2), histidine only reduces the rate of reaction in a dose-dependent manner, in the explored range of concentrations. In the presence of H2O2 and ferrous iron, histidine enhances the rate of nicking of double-stranded DNA in its supercoiled as well as in its relaxed state, but fails to modify the rate of nicking of fd DNA when it is in its vegetative, single-stranded form.