Optimizing Purification and Quantitative Detection of Avidin and Lysozyme

Hen egg white is one of the major raw materials used in food industry especially in foaming and gelling. Egg white consists of many different proteins that vary greatly in molecular weights and pI (isoelectric point) values. Two of its minor proteins, avidin and lysozyme have important commercial applications. In this research project, we tested the effect of pH and heat denaturation at 65o on the purification and recovery of avidin and lysozyme proteins from egg white. Four different pH conditions were chosen; these included ascorbate buffers at pH values of 4 and 5 and phosphate buffers at pH values of 6 and 7. Following heat denaturation and dialysis, the minor proteins were purified by cation exchange chromatography. We also optimized a Western Blot method to quantitate differences in the recovery of the egg white proteins. In this method we were successfully detected small amounts of both proteins using IR detection. It was found that an ascorbate buffer with an initial pH of 5.0 gave the best recovery of both avidin and lysozyme proteins in the eluate 1 fraction. The pH values of 5 and 6 were the best for the removal of a major contaminant, ovotransferrin. These findings allow new insights into the future improvements in the purification of lysozyme and avidin, and have also provided methods that can be used in the laboratory project for Biochemistry 360 students