Encapsulation of testosterone and its aliphatic and aromatic dimers by milk beta-lactoglobulin

• Encapsulation of testosterone and its dimers by milk beta-lactoglobulin was studied.
• Steroid-binding sites were located on beta-lactoglobulin.
• Testosterone showed stronger affinity toward protein complexation than its dimers.
• Steroids alter protein morphology causing increase in protein diameter.
• Milk beta-lactoglobulin can be used as steroid carrier.

The encapsulation of testosterone and it aliphatic dimer (alip) and aromatic dimer (arom) with milk β-lactoglobulin (β-LG) was studied in aqueous solution at pH 7.4. Multiple spectroscopic methods, transmission electron microscopy (TEM) and molecular modeling were used to characterize testosterone-β-LG binding and protein aggregation process. Spectroscopic analysis showed that steroids bind β-LG via hydrophobic and H-bonding interactions with overall binding constants Ktest--β-LG = 5.6 (±0.6) × 104 M−1, Ktest-dimeralip-β-LG = 4.8 (±0.5) × 103 M−1 and Ktest-dimer-arom-β-LG = 2.9 (±0.4) × 104 M−1. The binding affinity was testosterone > testosterone dimer-aromatic > testosterone dimer-aliphatic. Transmission electron microscopy showed major changes in protein morphology as testosterone–protein complexation occurred with increase in the diameter of the protein aggregate indicating encapsulation of steroids by β-LG. Modeling showed the presence of H-bonding stabilized testosterone-β-LG complexes with the free binding energy of −9.82 Kcal/mol indicating that the interaction process is spontaneous at room temperature.