Interactions of growth inhibitory factor with hydroxyl and superoxide radicals

To show the effects of growth inhibitory factor (Cu₄Zn₃MT-III) involved in the scavenging of reactive oxygen species (ROS), a pulse radiolytic study was employed using N₂O-saturated Cu₄Zn₃MT-III aqueous solutions. It was demonstrated that the oxidizing OH radical efficiently reacted with Cu₄Zn₃MT-III by forming a thiyl radical RS with a second-order constant of 1.46×10¹¹ mol l^–1s^–1, which was determined by competition kinetics against KSCN. The thiyl radical RS reacted rapidly and reversibly with a thiolate in Cu₄Zn₃MT-III to form radical anion RSSR ^– with a constant of 1.65×10⁹ mol lL^–1s^–1 per thiolate, while the constant of the decay of this radical anion was 2.72×10⁵ s^–1, and the equilibrium constant of the formation for RSSR ^– was 6.08×10³ mol^–1 l. These values were close to those of Cd₅Zn₂MT-II. The SOD activity of Cu₄Zn₃MT-III to quench O₂ ^–was assayed by the riboflavine-methionine-nitrobluetetrazolium (NBT) method which catalyzed the dismutation of superoxide (O₂ ^–) at pH 7.8 with an IC₅₀ value of 1.50×10^–6 M for Cu₄Zn₃MT-III and 1.62×10^–6 M for Cd₅Zn₂MT-II. Additionally, the down-regulation of GIF may be a main factor in the decrease of the scavenging ability for the free OH and O₂ ^–radicals, which is possibly associated with the pathogenesis of neurodegenerative disease.