The mechanism and kinetics of 2,2-diphenyl-1-picrylhydrazyl (DPPH) reactions in damaging protein and lipid models, and its suitability as a model for oxidative stress damage.

Antioxidant assays based on 2,2-diphenyl-1-picrylhydrazyl (DPPH•) are widely used, yet their relevance to biologically relevant oxidants is often assumed rather than tested. Here, we evaluate DPPH• as a model for peroxyl-type reactivity using quantum-mechanical kinetics (QM-ORSA, M05-2×/6–31 + G(d,p)/SMD) including diffusion and tunneling corrections, together with explicit speciation. Representative substrates include a bis-allylic lipid model, Alanine, Cysteine, Glutathione, Tryptophan, and Guanosine. Compared to HO₂• (water), DPPH• (ethanol) shows significantly higher barriers and rate constants up to several orders of magnitude lower for hydrogen-transfer processes, indicating poor performance as a model for peroxyl-driven oxidation. While DPPH• can exhibit high apparent reactivity under SPLET conditions, these rates depend strongly on medium and speciation. Overall, DPPH• is informative for SPLET-dominated systems but unsuitable as a general model for biologically relevant oxidation. • DPPH fails to reproduce the reactivity and selectivity of biologically relevant oxidants when hydrogen-transfer mechanisms dominate. • QM-ORSA calculations show than f-HAT rate constants for DPPH reactions are several orders of magnitude lower than for peroxyl radicals. • Under SPLET-controlled conditions, DPPH provides a reasonable approximation of antioxidant reactivity systems in polar media. • The study helps to clarify why different radical assays produce inconsistent antioxidant rankings. • Mechanistic insights highlight the need to match assay radicals to biologically relevant oxidative mechanisms.