Protection of Biomolecules by Antioxidants - Mechanisms and Applications

Abstract: Reactive oxygen species (ROS) consisting of various oxygen-based free radicals as well as other reactive non-radical species produced in O2-related metabolism or through other processes are involved in the oxidation of such vital biomolecules as DNA, proteins and lipids. The types of oxidation represented here are known to cause many different diseases and disorders in human beings, such as cancer, Alzheimer's disease and ageing. In addition, oxidation by ROS causes deterioration of food and is sometimes involved in the deactivation of enzymes. A particularly important source of ROS is believed to be the reaction of hydrogen peroxide with transition metal ions, mostly iron and copper, resulting in the formation of the highly deleterious hydroxyl radical, which is able to basically oxidize any organic molecule present. Several antioxidative defence mechanisms have evolved, serving to keep the biomolecules intact and prevent them from being damaged by ROS. The action of small-molecule antioxidants having the function of reacting with free radicals so as to prevent more vital molecules from being oxidized are an important part of this defence. The thesis is based on papers dealing with research on naturally occurring small-molecule antioxidants and the oxidation of DNA and proteins. In the case of DNA, a study was performed of the mechanisms involved, in which reaction intermediates and the formation of adducts were examined, using the iron-mediated Fenton reaction to induce oxidation of the nucleoside dG. Several oxidation products were identified and quantified by use of various reaction conditions. It was found that 8-oxodG, frequently used as a marker for oxidative DNA-damage, was highly susceptible to secondary oxidation. The measured level of this adduct being highly dependent upon both the reaction time and Fenton reagent concentrations involved. Two antioxidants, the iron-chelating antioxidant catechin and the strong radical-scavenger ascorbic acid, were evaluated by use of the same reaction system. It was concluded that catechin was more effective. Again, problems regarding 8-oxodG were observed, its being clearly shown that more reliable results could be obtained when a marker of more than one type was used to evaluate the effects of the antioxidants. In another study the effects of antioxidants were evaluated using a cellular test developed based on the Ames-tester strain Salmonella typhimurium TA102. Oxidation was induced by use of either hydrogen peroxide or the organic peroxide tert-butyl hydroperoxide (tBHP). Similar to the study just referred to, chelating antioxidants, in this case phenolic quercetin and caffeic acid, were the antioxidants found to be most effective. In the protein oxidation study carried out, use was made of the commercially interesting enzyme chloroperoxidase (CPO) from Caldariomyces fumago. This enzyme is able to catalyze various stereoselective oxygen-insertion reactions useful in the synthesis of drugs, for example, a clean source of hydrogen peroxide being used as the external oxidant. The main limitation of CPO is its poor stability, caused by its oxidative inactivation by hydrogen peroxide during the catalytic cycle. It was found that the inactivation was correlated with the oxidation of a key amino acid, a cysteine residue acting as the axial ligand to the prosthetic heme group of the enzyme, along with the disappearance of the heme. Furthermore, the addition of antioxidants significantly improved its operational stability, leading in the best case to 10 times as many turnovers of CPO being observed prior to its inactivation. Again it was found that a chelating antioxidant, caffeic acid in this case, was the antioxidant inhibiting unwanted oxidation the most. One thing these rather differing oxidation studies had in common was that the chelating antioxidants were clearly superior to the antioxidants that only possessed a radical scavenging mechanism. The results indicate two things in particular, first that the Fenton reaction is highly relevant in vivo and is thus a good way of producing radicals in in vitro assays, and secondly that the chelating mechanism is an important antioxidative mechanism, one which involves the action of small-molecule antioxidants both in vitro and in vivo.