Interaction of Proflavine and Acriflavine with Acetylcholinesterase
Authors: Wermuth Bendicht , Brodbeck Urs 
Published: March 2005

Purified acetylcholinesterase from toluene‐treated organs of the electric eel (form B) was inhibited in a noncompetitive manner by the acridine derivatives proflavine and acriflavine. With proflavine secondary plots of slopes and intercepts revealed hyperbolic inhibition patterns. The secondary plots of acriflavine showed a biphasic inhibition pattern with two different slopes (slope1 > slope2). A similar biphasic inhibition pattern was observed in the Dixon plot. From the two slopes two apparent Ki‐values were obtained. Between 0 and 1 μM acriflavine (low concentrations of acriflavine) the apparent inhibition constant was 0.6 μM, at acriflavine concentrations greater than 1 μM (high concentrations of acriflavine) the apparent inhibition constant was 2.2 μM. The apparent Km values for acetylthiocholine were 52 μM and 99 μM respectively. The relative V were 0.37 IU and 0.26 IU respectively. At high concentrations of acriflavine the double‐reciprocal plot of substrate concentration versus enzyme activity became hyperbolic and the Hill coefficient for acetylthiocholine was 0.6. However, in presence of 2 mM hexamethonium it increased to 0.9.

Double inhibitor studies were carried out with acriflavine and atropine, d‐(+)tubocurarine, hexamethonium, decamethonium, nicotine or tensilone. At a constant concentration of acriflavine, atropine, d‐(+)tubocurarine, hexamethonium and to a lesser extent decamethonium decreased the inhibition by acriflavine. Nicotine and tensilone showed no such effect. Yonetani‐Theorell plots with atropine, d‐(+)tubocurarine, hexamethonium or decamethonium as constant inhibitors and acriflavine as variable inhibitor revealed a decrease in slopes2 at acriflavine concentrations above 1 μM. At acriflavine concentrations below 1 μM slopes1 did not significantly differ from each other for the pairs of inhibitors investigated. From the Yonetani‐Theorell plots α‐values for acriflavine (fixed inhibitor) and nicotine and tensilone (variable inhibitors) were determined. For slopes1 the values ranged between 10 and 100 depending on substrate concentration, for slope2 the values were 1–10 at 0.1 mM substrate and reached infinity at 5 mM substrate.

Addition of proflavine and acriflavine to acetylcholinesterase produced a bathochromic and hypochromic shift in the visible absorption spectrum. A maximum difference in the spectra was obtained at 465 nm for proflavine and at 472 nm for acriflavine. Spectral titration of acetylcholinesterase with proflavine revealed normal binding with proflavine concentrations up to 22 μM. At higher concentrations the change in absorbance was significantly greater than expected for a simple stoichiometry of one mole of dye bound per mole of active site. The Hill coefficients were 1.03 and 2.25 respectively. The binding of acriflavine followed a theoretical binding curve up to a concentration of 10 μM. At higher concentrations the change in absorbance was again greater. For the acriflavine binding the Hill coefficients were 1.05 and 1.75 respectively. Addition of a second ligand decreased the minimum and maximum of the difference spectrum, indicating a displacement of acriflavine by the second ligand. Atropine and hexamethonium were more effective in displacing acriflavine than nicotine and tensilone.

From these data it nas concluded that acetylcholinesterase exists in two catalytically different states. At high concentrations of acriflavine an apparent activation of the enzyme was observed in presence of atropine, d‐(+)tubocurarine, hexamethonium and substrate. At low concentrations of acriflavine the enzyme was insensitive to these compounds.