In Vitro Study of Acriflavine Interaction with Horseradish Peroxidase C

Acriflavine (3,6-diaminoacridine) is an anticeptic drug developed in 1912. Previous research has focused on investigation of the intercalating features of acriflavine, but little is known about its interaction with proteins. Drug-receptor interaction is of major interest in clinical science. The aim of the present study was to evaluate the ability of acriflavine to induce alterations in conformation and function of peroxidase, a critical enzyme in cell survival. Horseradish peroxidase C (HRPC) activity was determined by measuring H2O2-dependent oxidation of o-dianisidine at 460 nm using an extinction coefficient of 11.3 Mm-1 cm-1. Apparent Km and Vmax values were then calculated. The electronic absorption spectra were recorded for 300-700 nm. Both Kd and ΔG were calculated from changes in the absorbance of 403 nm. Intrinsic fluorescence was detected for the 297 nm excitation and the emission was recorded for 300-700 nm wavelengths. The Stern-Volmer constant and Hill coefficients were then obtained. All measurements were performed in 0.1 M citrate buffer, pH 4.0. Results indicated that acriflavine either stimulated or inhibited HRPC activity depending on concentration and pre-incubation time. The Drug-receptor complex formation occurred via binding of four molecules of acriflavine in two different binding sites on HRPC, and the heme environment became more polar. Finally, acriflavine quenched the only tryptophan residue of HRPC. The alterations in HRPC conformation identified in the present study, suggest that drugs that induce apoptosis could alter critical cell protein conformation and function.