2.8. Metal Chelating Activity

The metal chelating activity was determined as described by Arise et al. [33] but modified as follows. The synthesized peptides and L-glutathione (GSH) were each diluted with distilled water to obtain 1 mg/mL assay concentration. Then 1 mL of the sample was pipetted into an Eppendorf tube, followed by 25 µL of 2 mM FeCl₂, 50 µL of 5 mM ferrozine [3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-4,4-disulfonic acid sodium salt] solution and 925 µL of distilled water. The solution was vortexed and allowed to stand at room temperature for 10 min. Thereafter, a 200 µL aliquot of the reaction mixture was pipetted into a flat bottom 96-well plate. Distilled water was used as the experiment blank, and the percentage metal chelating activity was calculated as follows:

where Ab and As, are absorbance of the blank and sample respectively.