HETEROGENEITY DETERMINATION AND PURIFICATION OF COMMERCIAL HEN EGG-WHITE LYSOZYME

Hen egg-white lysozyme (HEWL) is widely used as a model protein, altho ugh its purity has not been adequately characterized by modern biochem ical techniques. We have identified and quantified the protein heterog eneities in three commercial HEWL preparations by sodium dodecyl sulfa te polyacrylamide gel electrophoresis with enhanced silver staining, r eversed-phase fast protein liquid chromatography (FPLC) and immunoblot ting with comparison to authentic protein standards. Depending on the source, the contaminating proteins totalled 1-6% (w/w) and consisted o f ovotransferrin, ovalbumin, HEWL dimers, and polypeptides with approx imate M(r) of 39 and 18 kDa. Furthermore, we have obtained gram quanti ties of electrophoretically homogeneous [> 99.9% (w/w)] HEWL by single -step semi-preparative scale cation-exchange FPLC with a yield of abou t 50%. Parallel studies of crystal growth kinetics, salt repartitionin g and crystal perfection with this highly purified material showed fou rfold increases in the growth-step velocities and significant enhancem ent in the structural homogeneity of HEWL crystals.