Effect of resveratrol and tiron on the inactivation of glyceraldehyde-3- phosphate dehydrogenase induced by superoxide anion radical.

Protein damage mediated by oxidation has been associated with aging and age-related diseases, in particular neurodegenerative diseases. The protein that is known to be one of the major targets of oxidative stress is glyceraldehyde- 3-phosphate dehydrogenase. GAPDH is believed to play a key role in certain neurodegenerative disorders, such as Alzheimer's, Parkinson's, and Huntington's diseases. Several recent studies have suggested that a wide range of variety of polyphenols including resveratrol may have neuroprotective effects. Here, we present data that clearly indicate the prooxidative properties of resveratrol and tiron in the inactivation of GAPDH induced by the superoxide anion generated via xanthine oxidase mediated oxidation of xanthine. Generated in the studied system tiron and resveratrol radicals are much more efficient in the inactivation of GAPDH than the superoxide anion alone. The analysis of CD spectra of protein exposed to the tiron and resveratrol radicals revealed little effect on the secondary structure of GAPDH. In both cases reduction of α-helical structure was followed by the increase in ß-sheet conformation. Thus, the most probable mechanism of inactivation of GAPDH in the studied system is oxidation of cysteine residues in the catalytic center of the enzyme. Finally, molecular modeling of the resveratrol - GAPDH and tiron - GAPDH complexes showed potential binding sites for those antioxidants with binding affinity -45 kcal/mol and -48 kcal/mol respectively.

Can melatonin delay oxidative damage of human erythrocytes during prolonged incubation?

PURPOSE: Melatonin (MEL) is an effective antioxidant in numerous experimental models, both in vitro and in vivo. However, it should be stressed that there are also papers reporting limited antioxidative activity of MEL or even giving evidence for its pro-oxidative properties. In the present paper we investigated the influence of MEL on the oxidative damage of human erythrocytes during prolonged incubation. MATERIAL/METHODS: Human erythrocytes suspended in phosphate-buffered saline (PBS), pH 7.4 were incubated at 37ºC either in absence or presence of melatonin at concentration range 0.02 mM-3 mM for up to 96 hrs. The influence of MEL on erythrocyte damage was assessed on the basis of the intensity of intracellular oxidation processes (the oxidation of HbO2, GSH, fluorescent label DCFH2) as well as damage to the plasma membrane (lipid peroxidation, the potassium leakage) and the kinetics of hemolysis. RESULTS: The prolonged incubation of erythrocytes induced a progressive destruction of erythrocytes. Melatonin prevented lipid peroxidation and hemolysis whereas the oxidation of HbO2 and DCFH2 was enhanced by melatonin at concentrations higher than 0.6 mM. In the case of erythrocytes incubated with 3 mM of MEL, the hemolysis rate constant (0.0498±0.0039 H%•h⁻¹) was 50% lower than that of the control while the HbO2 oxidation rate constants were about 1.4 and 1.5 times higher for 1.5 and 3 mM of MEL, respectively. Melatonin had no influence on the oxidation of GSH and the potassium leakage. CONCLUSIONS: Probably, MEL can stabilize the erythrocyte membrane due to interaction with lipids, thus prolonging the existence of cells. On the contrary, in the presence of MEL the accelerated oxidation of HbO2 and generally, increased oxidative stress was observed in erythrocytes. Pro- and antioxidative properties of melatonin depend on the type of cells, redox state, as well as experimental conditions.