Selected functionality changes of beta-lactoglobulin upon esterification of side-chain carboxyl groups

Area of Science:

• Protein chemistry
• Biochemistry
• Surface science

Background:

• Bovine beta-lactoglobulin (BBL) is a major milk protein.
• Modifying protein functional groups can alter physicochemical properties.
• Understanding these modifications is crucial for food and biomaterial applications.

Purpose of the Study:

• To investigate the impact of esterifying free carboxyl groups on BBL's surface activity.
• To assess changes in protein hydrophobicity and charge.
• To develop a method for quantifying carboxyl group modification in proteins.

Main Methods:

• Esterification of BBL carboxyl groups using methanol, ethanol, and n-butanol.
• Surface and interfacial tension measurements (air/water and oil/water interfaces).
• Fluorescence spectroscopy using 1,8-anilinonaphthalene sulfonate (ANS) as a hydrophobic probe.
• Development of a hydroxamate-ferric ion chelate assay for carboxyl group quantification.

Main Results:

• Esterification reduced negative charge, increasing positive charge on BBL.
• All modified BBL esters exhibited enhanced surface activity compared to native BBL.
• Methyl ester showed the highest surface activity; ethyl and butyl esters showed intermediate activity.
• Hydrophobic probe fluorescence increased with BBL modification, indicating enhanced hydrophobic interactions.
• A strong positive correlation was found between reduced interfacial tension and increased ANS fluorescence.
• The hydroxamic acid reaction provided a method to estimate the extent of carboxyl modification.

Conclusions:

• Esterification of BBL carboxyl groups significantly enhances surface activity and hydrophobic properties.
• The degree of enhancement correlates with the alkyl chain length of the esterifying alcohol.
• The developed hydroxamate-ferric ion assay is a viable method for quantifying protein carboxyl modification.