Chemical Equilibrium Of SOD1 Binding To DNA

Copper-zinc superoxide dismutase(SOD1)has been considered as a key antioxidant enzyme since its discovery,catalyzing O2·-disproportionation of O2 and H2O2,making intracellular ROS in homeostasis.Although this enzyme can regulate the key intracellular ROS signaling network,except for its anti-oxidation function,its interacting with DNA to regulate gene expression has not attracted attention.According to our research before,we have put forward a hypothesis:SOD1 may be a gene expression regulatory protein that senses intracellular reactive oxygen species levels.We expressesWT-SOD1,mutant A4V and H46R-H48Q and studies the combination of SOD1 and DNA under different redox conditions.Thus,it was confirmed that SOD1 is a metal regulatory protein.In this paper,the binding of wild-type SODl and two other mutants in physiological environment,redox,and copper chelating agents.The results are as follows:1.Construct recombinant plasmids of WT-SOD1,A4V and H46R-H48Q and introduce them into E.coli for next expression and purification,we optimize the induction conditions and extract a large amount of highly active proteins;2.The binding of WT-SOD1 and 5’-ATGGAATGGAAT-3’ DNA double strands(High12)can reach 108M-1.The oxidative environment weakens the binding of DNA to WT-SOD1,and the binding of WT-SOD1 to DNA becomes static in the reducing environment.In combination,after active copper ions in SOD1 has been chelated,the protein cannot combine with DNA;3.The binding of the mutant A4V and High12 DNA sequences was stronger than that of WT-SOD1,while that of H46R-H48Q is weaker.The oxidative environment promoted the binding of A4V to DNA.The binding of A4V or H46R-H48Q to DNA in the reducing environment were also electrostatic interaction.After active copper ions in mutants have been chelated,the proteins cannot combine with DNA;It shows that the deletion of metal ions caused by the mutation of the natural copper ion binding site doesn’t bind SOD1 to DNA.The redox environment weakens the binding of WT-SOD1 and H46R-H48Q to DNA but enhances the binding of A4V to DNA.An additional copper ion chelator inhibits the binding of SOD1 protein to DNA.