2.5. Determination of Copper Chelating Activity

The copper chelating activity was determined according to Kubglomsong et al. [9]. Briefly, 4 mM of chromogenic reagent, i.e., pyrocatechol violet was prepared in 50 mM sodium acetate buffer (pH 6.0). Then, 10 µL of peptide sample (10 mg/mL), 280 µL sodium acetate buffer, 10 µL CuSO₄∙5H₂O and lastly 6 µL pyrocatechol violet were added to a 96-well plate. The sample solution was mixed well and the absorbance was measured at 632 nm using a spectrophotometer (Spectramax M5, Molecular Devices, San Jose, CA, USA). Ethylenediaminetetraacetic acid (EDTA) was used as the positive control. The copper chelating activity is calculated as follows:

where A_control denotes the absorbance of the system containing sodium acetate buffer, pyrocatechol violet and CuSO₄∙5H₂O whereas A_sample denotes the absorbance of the system containing peptide, sodium acetate buffer, pyrocatechol violet and CuSO₄∙5H₂O.