Interaction of normelinonine F and related N-methyl-β-carbolines derivatives with bovine serum albumin. Spectroscopic profiles, multivariate analysis and theoretical calculations.

β-carbolines (βCs) represent a large family of bioactive alkaloids, including norharmane and normelinonine F, known for their diverse pharmacological activities. The effects of these alkaloids may depend, among other factors, on their delivery, accumulation in different subcellular compartments, and interactions with biomacromolecules such as serum albumins. In this study, we investigated the pH dependence of the interactions between bovine serum albumin (BSA) and four βCs (norharmane, normelinonine F, and their corresponding N(9)-methyl derivatives) using UV-vis and fluorescence spectroscopy, combined with multivariate analysis and molecular docking. This selected set of N-methyl derivatives provides valuable insights into molecular-level binding interactions, clarifying aspects observed in previous studies. The results reveal a distinct spectroscopic interaction pattern for quaternary βCs compared to derivatives with a free N(2)-pyridinic nitrogen. Specifically, normelinonine F and N(9)-methyl-normelinonine F exhibited weak interactions, likely with external sites (site 3, subdomain IB) and/or the protein surface. In contrast, for N(2)-unsubstituted derivatives such as norharmane and N(9)-methyl-norharmane, stronger interactions and internalization into less polar and/or hydrophobic BSA sites predominate across the investigated pH-range (4 < pH < 9), likely site 2 (subdomain IIIA). However, the interaction of the corresponding cationic species of norharmane and N(9)-methyl-norharmane with BSA remains unclear due to low interaction levels and similar UV-vis absorption and emission spectra between free and BSA-bound species.