# Elucidation of the Synergistic Interaction Between Bilirubin and Casein Protein: An Integrated Spectroscopy and Computational Approach

## TL;DR

This study explores how bilirubin interacts with casein protein using a combination of experiments and computational methods.

## Contribution

The study reveals the synergistic interaction and structural dynamics between bilirubin and casein through integrated spectroscopy and computational analysis.

## Key findings

- Bilirubin undergoes conformational changes when encapsulated in casein micelles.
- Binding of bilirubin to casein is spontaneous and driven by entropy gains.
- TDDFT calculations show red shifts in bilirubin's absorption spectra due to the protein environment.

## Abstract

Herein, we unveil the interaction between bilirubin (BIL),
a liver
metabolite, and a milk protein, casein (CAS), through an integrated
experimental–computational approach. Encapsulation of BIL within
CAS protein micelles was characterized by using UV–vis absorption,
steady-state fluorescence, and circular dichroism (CD) spectroscopy.
CD analysis revealed conformational modulation of BIL upon encapsulation,
accompanied by Förster resonance energy transfer (FRET) from
CAS’s tryptophans to BIL. 1H NMR measurements determined
specific binding interactions of BIL functional groups involved in
micellar interactions, correlating photophysical and electronic properties.
The binding affinity of BIL in CAS micelles was found to be on the
order of 104 M–1 with a spontaneous binding
process (−24.56 kJ/mol) driven by entropy gains (467.17 J/mol).
TDDFT calculations unveiled red shifts in BIL’s absorption
spectra caused by the protein environment. This integrated experimental–computational
study provides novel insights into synergetic interactions and structural
dynamics between BIL and CAS, shedding light on the influence of milk
proteins on bilirubin’s behavior.