CRYSTAL GROWTH & DESIGN
Polymorphism of Urate Oxidase in PEG Solutions Denis Vivares,‡ Ste´phane Veesler, Jean-Pierre Astier, and Franc¸ oise Bonnete´* 1
Centre de Recherche en Matie` re Condense´ e et Nanosciences, CRMCN - CNRS, Campus de Luminy, Case 913, F-13288 Marseille Cedex 09, France
2006 VOL. 6, NO. 1 287-292
ReceiVed January 19, 2005; ReVised Manuscript ReceiVed July 12, 2005
ABSTRACT: In a previous paper, we have shown that three different crystal habits exist in the urate oxidase/PEG phase diagram (Vivares, D.; Bonnete´, F. J. Phys. Chem. B 2004, 108, 6498-6507). In the present paper, we show with a temperature-controlled device that these different crystal habits correspond to two polymorphs (namely, S/N and P). Furthermore, we point out how an accurate control of the crystallization parameters and a deep understanding of the phase diagram enable us to select one or the other polymorph. Finally, in a narrow part of the phase diagram, we observe an unusual nucleation and growth of one polymorph by epitaxy. 1. Introduction Intensive theoretical and experimental studies have been done in the last two decades to better understand and predict protein crystallization, a bottleneck in the determination of protein threedimensional structure by X-ray diffraction.1-5 Few of them have focused on the polymorphism of protein crystals, although most proteins exhibit several crystal polymorphs, usually induced by modifying the crystallization conditions such as pH, temperature, pressure, or crystallizing agent concentration.6-12 This polymorphic variation in proteic crystals arises from the large number of contacts between proteins in the crystal lattice and from the weakness of some of these contacts.13 Different polymorphs reflect different arrangements of protein molecules and therefore different protein-protein contacts.13-17 The study of crystal polymorphism can therefore reveal which proteinprotein contacts in given experimental conditions are required to induce protein crystallization. Mapping the different polymorphs helps to yield crystals of better quality.16,18 Moreover, different polymorphs have different solid-state properties such as their dissolution rate or their solubility, which implies different bioavailability if used as drugs. The delivery of proteic drugs as crystals presents several advantages such as high concentration in proteins and low viscosity of the crystalline suspension,19 although today only crystalline suspension of insulin has been approved for therapeutic use. This would mean an accurate control of the polymorphism of the protein crystals. Here we study the modification of both crystal habits and polymorphism of urate oxidase (uox) from Aspergillus flaVus in the presence of polyethylene glycol (PEG). This enzyme, whose three-dimensional molecular structure was first solved at 2.05 Å resolution20 in complex with a stabilizing inhibitor, 8-azaxanthine, is used as a drug to reduce toxic uric acid accumulation during chemotherapy. Recently, we correlated uox crystallization in PEG solutions with protein-protein interactions21 and studied the effect of metastable phases on its crystallization.22 Different crystal habits were found depending upon the PEG and protein concentrations. In the present work, we characterize the polymorphic variety of these crystal habits and determine the various crystal nucleation and growth pathways * Corresponding author. E-mail:
[email protected]; phone: + 33 (0) 6 62 92 28 39; fax: + 33 (0) 4 91 41 89 16. ‡ Present address: Laboratoire de Bioe ´ nerge´tique Cellulaire, De´partement d’Ecophysiologie Ve´ge´tale et de Microbiologie, Baˆt 156, CEA-Cadarache, 13108 Saint-Paul-Lez-Durance, France. 1 Laboratory associated with the Universities Aix-Marseille II and III.
of polymorphs depending upon the position in the uox/PEG phase diagram. 2. Materials and Methods 2.1. Solutions. The natural extracted uox from A. flaVus, complexed with its inhibitor 8-azaxanthine, was supplied by Sanofi-Aventis and purified in 50 mM Tris buffer at pH 8.5 by gel filtration chromatography on Superdex S200PG with an A ¨ KTA basic system and concentrated by ultrafiltration on an Amicon cell.21,22 The concentration of the uox stock solution was about 20 mg/mL. A 4 M stock solution of sodium chloride (Sigma-Aldrich) was prepared by dilution of the appropriate amount of salt in 50 mM Tris buffer at pH 8.5. A solution of 40% w/v of PEG 8000 Da in 50 mM Tris buffer at pH 8.5 was prepared from a 50% w/v solution (Hampton research). All the experiments were performed with 100 mM sodium chloride. 2.2. Crystallization Setup. The crystallization trials were observed with an inverted optical microscope (Nikon TE 200) and recorded via a digital video camera. The initial crystallization experiments performed to observe the different crystal habits were performed in an airconditioned room (set to 20 °C) with the Microbatch technique.22 Experiments to differentiate the polymorphs were carried out in a Peltier controlled-temperature device using small glass cells (
