The synergistic effect of ascorbic acid (AsA) and vitamin K_5 in the inactivation of phage J1 was previously reported. This paper reports the mechanism of the synergistic effect. Bubbling air or oxygen through the reaction mixture containing phage J1, AsA and K_5, and addition of hydrogen peroxide or metal ions into the reaction mixture enhanced inactivation of phage by combination of AsA and K_5 In contrast, nitrogen bubbling and addition of reducing agents, chelating agents or general radical scavengers inhibited the inactivation of phage. Spectrophotometric study showed that K_5 increased the rate of oxidation of AsA. The results indicate involvement of oxygen radicals in the inactivation reaction. The experiments using specific radical scavengers, superoxide dismutase or catalase showed that among oxygen radicals hydroxyl radical would be mainly responsible for the inactivating effect. Taking all of the results into consideration, K_5 could enhance the inactivating effect of AsA by increasing the rate of generation of hydroxyl radical during the oxidation of AsA by molecular oxygen. In addition, induction of single-strand scission in phage DNA by combination of AsA and K_5 has been shown by agarose gel electrophoresis.
