Impact of esterification on the folding and the susceptibility to peptic proteolysis of beta-lactoglobulin.

beta-Lactoglobulin was esterified and the differences between unmodified and ethylated beta-lactoglobulin were studied by microcalorimetry, circular dichroism and limited proteolysis. Microcalorimetric studies and circular dichroic spectra in aromatic regions revealed changes of esterified beta-lactoglobulin tertiary structure compared with native beta-lactoglobulin conformation in aqueous media. These changes are characteristic of molten globule state. While beta-lactoglobulin is resistant to peptic hydrolysis in aqueous and physiological conditions, a study of peptic action on esterified (ethylated, approximately 40% of the carboxyl groups substituted) beta-lactoglobulin in aqueous conditions showed that it is hydrolysed rapidly by this enzyme. The main part of the obtained peptic peptides has been purified and identified. Their analysis shows that 22 new sites of pepsin cleavage are induced by esterification of beta-lactoglobulin. Fourteen cleavage sites are pepsin specific and their unveiling is due to imposed tertiary structure changes. Eight of the observed new cleavage targets are entirely atypical containing either one or two distal dicarboxylic acid moieties. Apparently, the ethylation of beta- and/or gamma-carboxylates removing charges and grafting hydrophobic ethyl groups adapts substituted dicarboxylic amino-acid side chains for the recognition by pepsin.