Ferric Reducing Ability of Peptides

FRAP assay adapted to microscale with minor modifications [27 (link)] was used to measure the ferric reducing ability of peptides. Acetate buffer (0.3 M, pH 3.6), 2,4,6-Tris(2-pyridyl)-s-triazine (TPTZ, 10 mM prepared in 40 mM HCl), and ferric solutions (20 mM) were freshly mixed at a ratio of 10:1:1 and place in a 96-well microplate. Then, 198 µL of FRAP reagent and 2 µL of the peptide sample was mixed and incubated at 37 ºC in the dark for 30 min. The absorbance was measured at 593 nm using a microplate reader (Bioteck, Epoch/2 microplate reader, Winooski, VT, USA). The difference between the absorbance of the test sample and the blank reading was calculated and expressed as µM of FeSO₄/g of the peptide. FeSO₄ was used as standard.