EFFECTS OF PURIFICATION ON THE CRYSTALLIZATION OF LYSOZYME

We have additionally purified a commercial lysozyme preparation by cat ion exchange chromatography, followed by recrystallization. This mater ial is 99.96% pure with respect to macromolecular impurities. At basic pH, the purified lysozyme gave only tetragonal crystals at 20 degrees C. Protein used directly from the bottle, prepared by dialysis agains t distilled water, or which did not bind to the cation exchange column had considerably altered crystallization behavior. Lysozyme which did not bind to the cation exchange column was subsequently purified by s ize exclusion chromatography. This material gave predominately bundles of rod-shaped crystals with some small tetragonal crystals at lower p Hs. The origin of the bundled rod habit was postulated to be a thermal ly dependent tetragonal <----> orthorhombic change in the protein stru cture. This was subsequently ruled out on the basis of crystallization behavior and growth rate experiments. This suggests that heterogeneou s forms of lysozyme may be responsible. These results demonstrate thre e classes of impurities: (1) small molecules, which may be removed by dialysis; (2) macromolecules, which are removable by chromatographic t echniques; and (3) heterogeneous forms of the protein, which can be re moved in this case by cation exchange chromatography. Of these, hetero geneous forms of the lysozyme apparently have the greatest affect on i ts crystallization behavior.