Exploring the Structural Importance of the C3=C4 Double Bond in Plant Alkaloids Harmine and Harmaline on Their Binding Interactions with Hemoglobin

[Image: see text] Harmine and harmaline are two structurally similar heterocyclic β-carboline plant alkaloids with various therapeutic properties, having a slight structural difference in the C3=C4 double bond. In the present study, we have reported the nature of the interaction between hemoglobin (Hb) with harmine and harmaline by employing several multispectroscopic, calorimetric, and molecular docking approaches. Fluorescence spectroscopic studies have shown stronger interaction of harmine with Hb compared to that of almost structurally similar harmaline. Steady-state anisotropy experiments further show that the motional restriction of harmine in the presence of Hb is substantially higher than that of the harmaline–Hb complex. Circular dichroism (CD) study demonstrates no conformational change of Hb in the presence of both alkaloids, but CD study in 1-cm cuvette path length also demonstrates stronger affinity of harmine toward Hb compared to harmaline. From the thermal melting study, it has been found that both harmine and harmaline slightly affect the stability of Hb. From isothermal titration calorimetry (ITC), we have found that the binding process is exothermic and enthalpy driven. Molecular docking studies indicated that both harmine and harmaline prefer identical binding sites in Hb. This study helps us to understand that slight structural differences in harmine and harmaline can alter the interaction properties significantly, and this key information may help in the drug discovery processes.