Both DNA And Protein Oxidative Damage Mediated By The Zinc-depleted And Normal Superoxide Dismutase

The structural and the metal-binding variations in Cu Zn superoxide dismutase(Cu2Zn2SOD) mutants have been linked to many kinds of neurodegenerative diseases, inparticular, amyotrophic lateral sclerosis, due to their oxidative damage and aggregation.Therefore, we expect to be capable of establishing a relationship among the structural andthe metal-binding variations in Cu2Zn2SOD, Cu2Zn2SOD aggregation and oxidativedamage to DNA and to Cu2Zn2SOD itself by examining both DNA and protein oxidativedamages mediated by the zinc-depleted CunSOD (n = 0, 1, 2, 3, 4) under physiologicalconditions. The results reveal that the zinc-depleted multicopper CunSOD leads to a significantoxidative damage of plasmid DNA in the presence of peroxide hydrogen ormercaptoethanol or both, compared to the apo and holo Cu2Zn2SOD. Free cupric ion onlyshows the weak and even unobservable oxidative activity to DNA under the identicalconditions. The higher concentration of EDTA shows a marked inhibition to the oxidativedamage mediated by the multicopper CunSOD. The free radical quenchers such aspolyethylene glycol (PEG), manitol and dimethyl sulfoxide (DMSO) cannot inhibit thereaction, but can effectively promote the oxidative reaction. The neutral pH values (pH6.8-7.4) facilitate the DNA oxidative damage mediated by CunSOD. The dynamic properties of the oxidative damage of DNA were investigated. Theresults show that the CunSOD-mediated oxidation is a kind of consecutive reaction whichhas two reaction rate constants (k1/k2≈10). In general, k1 values increase as cupric ions inthe CunSOD are added. Two reaction parameters K and Vmax are defined to figure out thereaction of DNA oxidative damage, where K is the least amount of CunSOD in need whenthe rate of the oxidation of DNA is at half of the Vmax, and Vmax is the maximum velocity ofthe oxidative damage of DNA mediated by CunSOD. K? may also measure the interaction IIIbetween CunSOD and DNA. Carbon dioxide can promote CunSOD-mediated oxidation of DNA, but not carbonate.Interestingly, nitrogen can also enhance the oxidation reaction, but not argon. Thepromotion of the DNA damage by nitrogen or carbon dioxide might be involved in abinding to the copper reactive centre, and promote the leaving of hydroxyl radical which isbinding to the centre. The hydroxyl radical as a leaving group, can oxidize diversesubstrates such as DNA. On the other hand, the influence of cupric or zinc ions on the Cu2Zn2SOD aggregationis examined by SDS-PAGE, indicating that the CunSOD dimerization is vulnerable to thecoordination of cupric ion to the His110 residue from two monomer, because the dimer isresistant to sodium dodecyl sulphate (SDS). The SDS-resistant CunSOD dimer dissociatesdue to self-oxidation in the presence of peroxide hydrogen and mercaptoethanol. CunSOD promotes the aggregation of apo, holo and copper- depleted Cu2Zn2SOD, butnot lysozyme and bovine serum albumin, a demonstration that the aggregation is sensitiveto protein structure. Last, the possible binding sites of DNA and CunSOD are discussed, using abioinformatics method.