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We have shown earlier that restraint-cold stress-induced gastric ulceration in rats is caused by metal ion dependent generation of hydroxyl radical (OH^̇) and oxidative inactivation of the gastric peroxidase (GPO), an important H₂O₂ scavenging enzyme. To study the mechanism of the oxidative damage of GPO, the purified enzyme was exposed to an OH^̇ generating system containing Cu²⁺, ascorbate, and H₂O₂. Kinetic studies indicate that the enzyme is inactivated in a time-dependent process showing saturation with respect to Cu²⁺ concentration. The enzyme specifically requires Cu²⁺ and is not inactivated by the same concentration of Fe²⁺, Mn²⁺, or Zn²⁺. Sensitivity to catalase indicates the critical role of H₂O₂ in the inactivation. Inactivation is insensitive to superoxide dismutase, suggesting no role of superoxide. The rate of inactivation is not increased in D₂O excluding the involvement of singlet oxygen in …