Comparative Analysis of the Binding Characteristics of Fluorescein and Rhodamine B with Bovine Serum Albumin

Abstract Bovine serum albumin (BSA) serves as a key model for investigating protein–ligand interactions and is crucial for understanding drug pharmacokinetics and developing biomedical probes. This study presents a comprehensive comparative analysis of the binding of fluorescein and rhodamine B to BSA, employing UV–vis absorption, fluorescence, molecular docking, and 1000 ns molecular dynamics (MD) simulations. The results revealed competitive binding, with rhodamine B exhibiting a greater affinity (ΔGMMGBSA = −48.17 kcal/mol) than fluorescein (−23.16 kcal/mol), which was supported by fluorescence quenching and binding analyses. Molecular docking confirmed Sudlow’s site I as the primary binding site, whereas MD simulations highlighted stable complexes driven by van der Waals and electrostatic forces, particularly for rhodamine B. Unexpectedly, the superior affinity of rhodamine B under physiological pH deviates from lipophilic trends. These findings enhance the molecular understanding of dye–protein interactions, providing a robust basis for designing advanced fluorescent probes, drug conjugates, and diagnostic platforms.