Probing the Binding of Tetracyclines With Bovine Serum Albumin Using Computational and Spectroscopic Methods

Tetracycline (TC) and its degradation products, 4-epitetracycline (ETC), anhydrotetracycline (ATC), and 4-epianhydrotetracycline (EATC), are known to contribute to environmental toxicity, yet their molecular interactions with bovine serum albumin (BSA), a model for human serum albumin, remain insufficiently understood. This study explores the binding characteristics of TC and its degradates with BSA using fluorescence spectroscopy and molecular docking. Experimental results revealed fluorescence quenching of BSA upon binding with all four compounds, with ATC and EATC causing the highest quenching. The Stern-Volmer analysis confirmed static quenching and ranked binding affinities as ATC > EATC > ETC ≈ TC, with K_SV values of 9.54 × 10⁵, 6.36 × 10⁵, 1.47 × 10⁵, and 1.79 × 10⁵ M^-1, respectively. Spectral shifts indicated conformational changes in BSA upon ligand binding. Docking simulations supported the experimental findings, showing stronger binding of ATC and EATC, attributed to their planar structures, which favor hydrogen bonding and hydrophobic interactions. TC and ETC, due to internal hydrogen bonding and curled conformations, formed weaker interactions. This is the first study to report strong binding of EATC to BSA, highlighting its potential biological relevance and underscoring the need for further in vivo or in vitro validation of its toxicological impact.