Kinetic behavior of the reaction between hydroxyl radical and the SV40 minichromosome

Abstract

Aqueous solutions containing the minichromosomal form of the virus SV40 and the radical scavenger DMSO were subjected to gamma-irradiation, and the resulting formation of single strand breaks (SSB) was quantified. Under the irradiation conditions, most SSBs were produced as a consequence of hydroxyl radical ((•)OH) reactions. By controlling the competition between DMSO and the viral DNA substrate for (•)OH, we are able to estimate the rate coefficient for the reaction of (•)OH with the SV40 minichromosome. The results cannot be described adequately by homogeneous competition kinetics, but it is possible to describe the rate coefficient for the reaction as a function of the scavenging capacity of the solution. The experimentally determined rate coefficient lies in the range 1×10(9) - 2×10(9) L mol(-1) s(-1) at 10(7) s(-1), and increases with increasing scavenging capacity.

Figure 1

Loss of supercoiled SV40 with increasing radiation dose. An aerobic solution containing the SV40 minichromosome (30 µg ml⁻¹), DMSO (5×10⁻³ mol dm⁻³), was irradiated at 2.43×10⁻¹ Gy s⁻¹ to the doses indicated. The fraction of intact SV40 remaining after each dose was determined by using agarose gel electrophoresis (see Materials and Methods). The data are fitted with a least mean square straight line of the form y = ce^−mx. From the value of the slope m, under these conditions the D₀ is 171 Gy and the SSB yield is 5.16×10⁻⁵ µmol J⁻¹ or 1.72×10⁻⁹ Gy⁻¹ Da⁻¹.

Figure 2

Effect of SV40 concentration on the SSB yield. The SV40 concentration is expressed here as the molarity of nucleotide residues. This is a double reciprocal plot of the data from Figure 2 according to competition kinetics (see Discussion). The data are fitted with a least mean square straight line of the form y = mx+c. Values for the slope m and the intercept c are respectively 1.83 MJ dm⁻³ and −2.17×10³ J µmol⁻¹.

Figure 3

Effect of ^•OH scavenging capacity (σ(^•OH)) on the rate coefficient for the reaction of ^•OH with DNA in the form of the SV40 minichromosome (closed symbols). The data are fitted with a least mean square straight line of the form y = cx^m (solid line). Also plotted in this figure are straight lines of the same type fitted to previously published data for plasmid DNA (long dash) and single stranded DNA (short dash). The values of the slopes m are 0.38 (solid line), 0.35 (long dash), and 0.20 (short dash).