Quantification of Binary Diffusion in Protein Crystals Aleksandar Cvetkovic, ² Cristian Picioreanu, ² Adrie J. J. Straathof,* ,² Rajamani Krishna, ‡ and Luuk A. M. van der Wielen ² Department of Biotechnology, Delft UniVersity of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands, and Van ‘t Hoff Institute for Molecular Sciences, UniVersity of Amsterdam, Nieuwe Achtergracht 166, 1018 WV Amsterdam, The Netherlands ReceiVed: January 17, 2005; In Final Form: April 1, 2005 The use of confocal laser scanning microscopy for visualization and quantification of binary diffusion within anisotropic porous material is described here for the first time. The dynamics of adsorption profiles of dianionic fluorescein, zwitterionic rhodamine B, and their mixture in the cationic native orthorhombic lysozyme crystal were subsequently analyzed. All data could be described by a classical pore diffusion model. There was no change in the adsorption characteristics, but diffusion decreased with the introduction of a second solute in the solution. It was found that diffusion is determined by the combination of steric and electrostatic interactions, while adsorption is dependent on electrostatic and hydrophobic interactions. Thus, it was established that the outcome of binary transport depends on the solute, protein, and crystal characteristics. Introduction A rapid surge in the number of resolved protein crystal structures has occurred because of enhanced ability for the determination of optimal crystallization conditions by atomized screening techniques, 1 theoretical predictions on the basis of the second virial coefficient, 2 or protein engineering 1 and easier characterization of protein crystallization. 1 Protein nanocrystals have been produced and characterized, 3 large scale crystalliza- tion processes for the production of uniform protein crystals with sharp cutoff crystal sizes have been described and developed, 4 and crystals made of monoclonal antibodies have successfully been applied in enantioselective separations. 5 These noteworthy advances provide support for the applications of protein crystals, in separation processes such as chromatog- raphy, 6-8 in enzymatic production processes, 9-13 in medical formulations for pharmaceutical delivery, 14-16 in biosen- sors, 17,18 and in detergents. 9 The combination of the crystals’ open structure (a high porosity and pore surface area) with the wide variety of molecular topologies and with the proteins’ ability of regio- and stereoselective recognition makes protein crystals a novel class of nanoporous materials 6 of vital interest for many industrial fields. Despite the progress made in improving the characteristics of protein crystals, comprehension of solute transport phenomena in the crystal pores is still modest. In the case of single-solute uptake, significant progress has been achieved. Solute diffusion in protein crystals was found to be anisotropic 19,20 and dependent on steric interactions in the pores. 20 Electrostatic and hydrophobic characteristics of both the solute and the crystals dictated solute adsorption by protein crystals. 21,22 Real processes, involving multicomponent transport and adsorption, have not been investigated; therefore, they may or may not be realistically represented by a combination of single-solute data. The true challenge in dealing with actual processes is a quantitative comprehension of multicomponent phenomena, which will be the subject of this paper. Confocal laser scanning microscopy (CLSM), being the only available experimental technique capable of simultaneous spatial and temporal monitoring in situ, has already been applied to study single-solute diffusion in isotropic 23-29 and anisotropic materials. 19 Furthermore, the same technique has been used for visualization of the binary uptake of labeled proteins by isotropic spherical materials. However, there are no measurements of such processes in anisotropic materials. Therefore, the methods for qualitative 19 and quantitative 20 studies of fluorescein diffusion in native lysozyme crystals of different structures will be adopted in this paper to enable the quantitative visualization of the in situ binary diffusion in anisotropic porous material. Quantitative analysis of the binary diffusion of fluorescein and rhodamine B in native orthorhombic lysozyme crystals was performed. Orthorhombic lysozyme, being the most anisotropic of all lysozyme structures, is chosen as the model crystal. The properties of lysozyme and the morphologies of its crystals are well-known, and the crystals can be obtained easily and reproducibly. Fluorescein and rhodamine B are suitable solutes for this study because of their fluorescence, their similarity in structure, and their difference in charge. Materials and Methods Materials. Chicken egg-white lysozyme was obtained from Sigma (Product No. L-6876; 95% purity; M ) 14 307 g mol -1 ) and was used without further purification. Orthorhombic structures of lysozyme crystals were prepared according to the procedure described by Cvetkovic et al. 19 The disodium salt of fluorescein (Sigma, Product No. F-6377) and the hydrochloride of rhodamine B (Fluka, Product No. 83690) were used without further purification. Preparation of the Solutions. The mother liquor of crystal- lization was filtered using Schleicher & Schuell (Germany) syringe filters with a cutoff value of 200 nm, and a concentrated solution of sodium fluorescein or/and rhodamine B was added until the desired concentration was reached. Solutions of * Author to whom correspondence should be addressed. Phone: +31- 15-2782330. Fax: +31-15-2782355. E-mail: a.j.j.straathof@tnw.tudelft.nl. ² Delft University of Technology. ‡ University of Amsterdam. 10561 J. Phys. Chem. B 2005, 109, 10561-10566 10.1021/jp050289c CCC: $30.25 © 2005 American Chemical Society Published on Web 05/05/2005