CHROMATOGRAPHIC SEPARATION OF ACYLATED EGG WHITE PROTEINS AND BIOPHYSICAL CHARACTERIZATION OF SUCCINYLATED LYSOZYME AND OVALBUMIN

Chemical modification of egg white has been shown to improve its functional properties. In order to fully understand the changes in functionality, it is necessary to separate the proteins and study their corresponding structural changes. Two chromatographic methods are analyzed for their application to native and acylated egg white. A modified DEAE-cellulose ion exchange method was developed which reduced separation time and expense. It was found that increasing net charge caused by acylation with succinic or acetic anhydride changed the ionic state of the proteins, limiting their separation by ion exchange chromatography. A gel chromatography separation using Sephacryl S-200 was developed which separated over 80% of the total succinylated egg white proteins; viz., ovomucin, ovalbumin, conalbumin, ovomacroglobulin and lysozyme. Most proteins of native and acetylated egg white were separated with S-200 gel chromatography, but required rechromatography of the ovalbumin-conalbumin fractions with Sephadex G-100 for complete resolution.; The effect of succinylation on the conformation of ovalbumin and lysozyme was examined in relation to previously observed changes in functionality of succinylated egg white. Parameters measured included hydrodynamic radius, titratable tyrosine, relative exposure of tyrosine and tryptophan, heat stability, relative difference spectra and circular dichroism spectra for native and succinylated lysozyme and ovalbumin. Succinylated ovalbumin had a significantly larger radius, increased exposure of tryptophan and tyrosine and exhibited conformational changes probably affecting secondary and tertiary structure. Succinylated lysozyme's tyrosine residues had pKs shifted to a higher pH and exhibited conformational changes, probably affecting secondary and tertiary structure. The results show the increase in net negative charge accompanying succinylation causes significant conformational change, leading to improved functionality.