Binding Interaction Analysis of Lysozyme and Brilliant Blue Using Surface Plasmon Resonance

This study presents a detailed in vitro experimental protocol for analyzing the molecular interaction between lysozyme and the synthetic dye Brilliant Blue FCF (BB). Lysozyme, a natural antimicrobial enzyme found in various secretions, plays a vital role in the innate immune system and oral health. BB, commonly used in food and cosmetic products, is known to interact with biological macromolecules. The binding interaction between these two molecules was investigated using a surface plasmon resonance (SPR) system employing Kretschmann's configuration. The gold SPR sensor surface was functionalized with 11-mercaptoundecanoic acid (MUA). The lysozyme from chicken egg white as a ligand was immobilized onto the functionalized sensor surface and varying concentrations of BB were injected through the SPR channels to evaluate binding kinetics. SPR data were analyzed through multistep software workflow including referencing, baseline correction, alignment, and kinetic fitting. The resulting sensorgrams enabled the determination of key parameters such as association (k_a) and dissociation rate (k_d) constants and equilibrium dissociation constant (K_D). This protocol provides a real-time approach for studying protein-ligand interactions under physiological conditions and contributes valuable insights into the biophysical behavior of lysozyme in the presence of BB.