Dynamics of Free Radical Formation from the Reaction of Peroxides with Haemproteins as Studied by Stopped-Flow Chemiluminescence

Abstract

Metal ions such as iron and copper are implicated in the generation of free radicals in vivo, but the dynamics of free radical production in the decomposition of hydrogen peroxide and hydroperoxides by iron complexes have not been shown unequivocally due to their rapid rates. In the present study, we have devised a stopped-flow chemiluminescence system and succeeded in elucidating the time course of the free radical formation in the decomposition of hydrogen peroxide and organic hydroperoxides by haemproteins. The rate of free radical formation was very much dependent on the metal complexes and peroxides. Methemo-globin and cytochrome c decomposed hydrogen peroxide slower than microperoxidase but generated free radicals continuously. Methyl linoleate hydroperoxide was decomposed more rapidly than hydrogen peroxide. The chemiluminescence intensity was directly proportional to the amount of hydroperoxide, and the chemiluminescence progress curve reflected the time course of free radical flux. These results show that this stopped-flow chemiluminescence system is suitable to follow the radical flux from the decomposition of peroxides by metal ions.

Key Words:

• free radical
• haemprotein
• active oxygens
• chemiluminescence
• oxidative damage
• stopped-flow analysis