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Hydroxyl radicals (OH .) are one of the most active reactive oxidants recognized for their deleterious effects to cause protein oxidative damage. Thymoquinone, a monoterpene molecule abundantly present in black cumin and known for its pharmacological activities, but its activity against the OH . -induced protein oxidative damage has never been explored. This study determined the therapeutic potential of thymoquinone against OH . -induced oxidative human hemoglobin damage. Novel data demonstrated that thymoquinone provides structural protection of hemoglobin against oxidative damage. Treatment of hemoglobin with OH . induces hypochromicity at 280 and 405 nm, whereas thymoquinone reversed these hypochromic effects. In addition, OH . cause significant reduction in tryptophan fluorescence, however thymoquinone also reversed these damaging effects. Thymoquinone also reduces OH . -induced hydrophobicity and also reduces OH . -induced carbonylation. Moreover, it also inhibits thermal stabilization of OH . -hemoglobin complex. SDS-PAGE of unmodified hemoglobin showed four bands, which disappeared upon OH . treatment and these changes were also retained by thymoquinone. In conclusion, this is the first study that shows the therapeutic potential of thymoquinone against OH . -induced oxidative damage in human hemoglobin. • Oxidative by-product hydroxyl radicals caused extensive damage of hemoglobin. • Thymoquinone stabilizes hemoglobin structure against ROS. • Thymoquinone reverses ROS-induced tryptophan fluorescence. • Thymoquinone reduces ROS-induced hydrophobicity. • Thymoquinone also reduces ROS-induced carbonylation. [ABSTRACT FROM AUTHOR]