Quest for the binding mode of malachite green with humic acid

•Spectroscopic methods combination with molecular modeling were used.•MG located in the hydrophobic cavity of HA.•The π–π stacking and hydrophobic forces played a key role in the binding.•HA binding caused coplanar conformation changes of MG.

The association of malachite green (MG) with humic acid (HA) was investigated by using fluorescence, UV–vis spectroscopy and molecular Modelling method. The fluorescence spectral results indicated that the binding between MG and HA occurred by mainly hydrophobic and electrostatic forces with association constants of KA (298 K) = 6.24 × 105 L/mol and KA (310 K) = 10.20 × 105 L/mol. There were more than one binding sites on HA to bind with MG. The binding sites of MG with HA primarily located at the aromatic rings of HA. MG could enter into the hydrophobic cavities of HA to quench the fluorescence of HA. On the contrary, HA binding caused MG to a coplanar conformation with more extended π bond distribution by π–π stacking interactions. The experiment and calculation data both showed that the hydrophobic binding cavities in HA played a key role in its binding with MG.

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