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

Free carboxyl groups of bovine beta-lactoglobulin were esterified with methanol, ethanol, and n-butanol. The modified proteins showed increased positive charge as the number of ionizable carboxyl groups was reduced. The methyl, ethyl, and butyl esters of beta-lactoglobulin showed enhanced surface activity, determined with surface and interfacial tension measurements at an air/water and oil/water interface, respectively. The methyl ester showed the largest enhancement in surface activity relative to the native protein. The ethyl and butyl esters were less effective in lowering the surface and interfacial tension but were more surface active than the native protein. The hydrophobic probe, 1,8-anilinonaphthalene sulfonate, showed enhanced fluorescence in the presence of native and modified beta-lactoglobulin. The largest enhancement in fluorescence of the hydrophobic probe was noted in the presence of the methyl ester of beta-lactoglobulin. The ethyl ester also effectively enhanced the fluorescence of the probe. The slopes of the curves of the fluorescent response for the native and modified proteins were graphically related to the interfacial tension at an oil/water interface for the corresponding protein. A large positive correlation was noted between the effectiveness of the proteins in reducing the interfacial tension and the enhancement in fluorescence of ANS with hydrophobic binding by the proteins. A general method to quantify carboxyl groups of proteins, employing the formation of a colored hydroxamate-ferric ion chelate, was also developed. Modification of the reaction for proteins in general, and carboxyl-modified proteins in particular, appears to have potential. Use of the hydroxamic acid reaction made it possible to estimate the apparent extent of carboxyl modification of beta-lactoglobulin through esterification with methanol, ethanol, and n-butanol.