Ion-Exchange Membranes for Stable Derivatization of Protein Crystals

Ion-exchange membranes were applied in this work to diffuse ions and heavy atoms inside protein crystals in order to gently perform their derivatization. The ion-exchange process rate for three different ions, bromide (Br^-), platinum (Pt⁺ through PtCl₄ ^2-), and mercury (Hg²⁺), was evaluated, allowing to determine the concentration of these ions in the crystal solution over time and to evaluate their effect on the crystals. Nafion and Neosepta AXE01, cation and anion exchange-membranes, respectively, were used for transport of cations and anions to hen egg white lysozyme (HEWL) crystals, selected as model protein. X-ray diffraction analysis of the crystals confirmed the attainment of the derivatives and allowed the ab initio building of the bromide derivative model. Derivatization experiments were also conducted by the conventional method, directly soaking the crystals in the heavy atom solution. It was possible to conclude that the controlled diffusion, regulated by the membrane, increases the crystal's stability, avoiding handling procedures (in situ derivatization) and maintaining a safer environment near the crystals without disturbing the vapor diffusion equilibrium.