Dissociation of DNA double strand by hypohalous acids

Abstract

Calf thymus DNA was treated with authentic HOCl, and hypohalous acid-generating systems. This caused a decrease in fluorescence of ethidium-DNA complexes when ethidium bromide was subsequently added to the DNA. The fluorescence continued to decrease up to 30 min after adding HOCl. Loss in fluorescence was proportional to the concentration of HOCl and was complete when a 3-fold excess of HOCl was added to the DNA. No significant decrease in the fluorescence was observed when the chlorination was carried out in the presence of a concentration of monochlorodimedone (MCD) equivalent to that of HOCl. MCD is known to react stoichiometrically with HOCl. The decrease in fluorescence was completely inhibited by H₂O₂, ascorbate and glutathione (GSH). We have estimated the rate constant for the reaction of HOCl with H₂O₂ to be 1–2×10⁵ M^-1s^-1. When compared with authentic HOCl, HOCl-generating systems (Cl^-+H₂O₂+MPO or chloroperoxidase) were found to be inefficient in damaging DNA. This result most likely arises because the rate constant for reaction of HOCl with H₂O₂ is about 1000-fold faster than that for the reaction with DNA. HOBr and HOI generating systems also had a limited ability to damage DNA. We conclude that good chlorine acceptors and antioxidants protect DNA from hypohalous acid-induced oxidative damage.