Inactivation of bacteriophage øX174 by mitomycin C in the presence of sodium hydrosulfite and cupric ions

Abstract

Bacteriophage øX174 was inactivated by mitomycin C reduced with sodium hydrosulfite in the presence of cupric ions (Cu 2+). 99% of the phage particles lost their plaque-forming abilities when incubated with 1.5 · 10 −4 M mitomycin C, 5.7 · 10 −4 M sodium hydrosulfite and 1.0 · 10 −4 M CuCl 2 for 120 min at 37°C in 0.05 M Tris-HCl buffer (pH 8.1). Sodium borohydride and thiol-reducing agents such as L-cysteine, 2-mercaptoethanol or dithiothreitol could not serve as a substitute for sodium hydrosulfite and other transition metal ions such as Fe 2+, Fe 3+, Mn 2+, Co 2+ and Zn 2+ were of no effect. Inactivated phage sedimented at 114S just as intact phage, but phage DNA was degraded. Strand-scission was observed when øX174 single-stranded DNA was directly reacted with mitomycin C reduced with sodium hydrosulfite in the presence of CuCl 2. Phage inactivation was inhibited by catalase, EDTA and several scavengers such as cysteamine, 2-aminoethylisothiuronium bromide HBr (AET), 4,5-dihydroxy-1,3-benzenedisulfonic acid (Tiron) or 1,4-diazabicyclo[2,2,2]octane (DABCO). These results suggest that free oxygen radicals and mitomycin C semiquinone radical generated during autoxidation of reduced mitomycin C in the presence of cupric ions cause the degradation of øX174 DNA.