Pitfalls of using 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay to assess the radical scavenging activity of peptides: Its susceptibility to interference and low reactivity towards peptides.

DPPH assay is widely used to evaluate the radical scavenging activities of peptides. Effects of pH and buffers on the stability of DPPH• and its reduced product (DPPHH) in the ethanol-buffer solution were investigated in this study and the reactivity of DPPH• towards several dipeptides was compared to that of 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS•) and the peroxyl radicals in oxygen radical absorbance capacity (ORAC) assay. Results showed that the deprotonation of DPPHH under basic condition could interfere with the spectrophotometric measurement at 515-525nm. It was suggested that the reaction mixture be maintained at a final pH range of 5.0-6.5 in 1:1 ethanol-acetate/citrate buffer medium when evaluating the activities of peptides. Additionally, among tested dipeptides, only Cys-containing dipeptides displayed DPPH• scavenging activity with 0.14-0.28μmol TE (Trolox equivalent)/μmol, while Tyr/Trp-containing dipeptides with high reactivity towards ABTS• and peroxyl radicals were inert to DPPH• with TE values less than 0.02.