In this research, the antioxidant property of thymosin alpha-1 -Thα1- peptide was investigated through various antioxidant methods. Thα1 showed 1,1-diphenyl-2-picrylhydrazyl -DPPH- radical scavenging activity -IC50 = 20 µM- and its 2,2-azino-bis -3-ethylbenzothiazoline-6-sulphonic acid- -ABTS- scavenging reached 45.33% at 80 µM -IC50 = 85 µM-. In addition, hydroxyl and superoxide radical scavenging of Thα1 peptide exhibited a concentration-depended manner. The IC50 values of hydroxyl and superoxide radical scavenging were estimated to be 82 µM and 20 µM, respectively. The effect of Thα1 on eliminating superoxide radicals was higher -62.23%- than other antioxidant assays. Moreover, the antioxidant activity of Thα1 peptide was evaluated by measuring cellular reactive oxygen species -ROS-. Results indicated that Thα1 decreased the generation of ROS level in 1321 N1 human neural asterocytoma cells. The inhibitory effect of Thα1 on angiotensin-converting enzyme -ACE- was determined. The kinetic parameters -Km and Vmax- and the inhibition pattern were examined. Based on the Lineweaver-Burk plot, Thα1 displayed a mixed inhibition pattern. The IC50 and Ki values of Thα1 were 0.8 µM and 3.33 µM, respectively. Molecular modeling suggested that Thα1 binds to ACE-domains with higher affinity binding to N-domain with the binding energy of −22.87 kcal/mol. Molecular docking indicated that Thα1 interacted with ACE enzyme -N- and C-domains- due to electrostatic, hydrophobic, and hydrogen forces. Our findings suggested that Thα1 possess a multifunctional peptide with dual antioxidant and ACE-inhibitory properties. Further researches are needed to investigate the antioxidant and anti-hypertensive effect of Thα1 both in vitro and in vivo.

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