Screening of Protein-Ligand Interactions by Affinity Chromatography Carlos D. Garcı ´a, † Steven C. Holman, † Charles S. Henry, ‡ and W. William Wilson* ,† Department of Chemistry, 118 Hand Chemical Laboratory, Mississippi State University, Mississippi State, Mississippi 39762, and Department of Chemistry, Colorado State University, Fort Collins, Colorado 80523 This paper examines affinity chromatography (AC) as an alternative tool for the determination of protein-ligand interactions for the particular case in which the ligand is the same protein. The methodology is less labor-intensive and more sample-efficient than traditional methods used to measure the second virial coefficient (B 22 ), a parameter commonly used to evaluate protein-protein interactions. The chromato- graphic capacity factor (k′) was studied for lysozyme and equine serum albumin for a wide range of experimental solution conditions such as crystallizing agent concentra- tion, protein concentration and pH. Parallel experiments using AC to determine k′ and static light scattering (SLS) to determine B 22 showed that the two parameters were highly correlated. Two different column volumes (∼1 and ∼0.1 mL) were tested and gave essentially the same values for k′, showing the feasibility of miniaturization. 1. Introduction The determination of protein structure can be ac- complished by X-ray diffraction analysis from protein crystals. Therefore, the growth of diffraction quality crystals is a prerequisite to structure determination (1). However, determining suitable solution conditions for protein crystal growth (PCG) remains an empirical exercise that is usually accomplished by extensive screen- ing experiments (2, 3) whereby the protein is exposed to various crystallizing agents. Many proteins have been crystallized by exploiting the so-called “salting-out” effect, which is induced by the addition of a simple electrolyte as the crystallizing agent. Enhanced association between water and salt ions causes water molecules to be stripped from the protein, thereby increasing hydrophobic attrac- tion between protein molecules (4). For most proteins, this effect is weakly dependent on the cation but is strongly dependent on the anion (5). In addition, there are many other substances (nonelectrolytes) such as mannitol, glycine, dextrans (6) or poly(ethylene glycol)s (1) that can affect protein-protein interactions. Some of them have been used as crystallizing agents. An innovative approach used as a guide for determin- ing suitable PCG conditions is the measurement of the second virial coefficient (B 22 )(7). This parameter is a dilute solution property commonly determined by SLS and gives a quantitative measure of the extent of protein-protein interactions in a given solution (2). A wide variety of proteins have been found to crystallize for slightly to moderately negative values of the B 22 , values generally referred to as being in the “crystalliza- tion slot” (8). The determination of B 22 by light scattering, however, is generally labor-intensive and time-consuming and requires relatively large amounts of protein (mg quantities) (9), making the method unattractive for high throughput studies (10). An alternative approach for measuring protein- protein interactions is the use of AC (6). This form of chromatography utilizes a stationary phase that consists of the protein of interest immobilized on the support material, and the solution to be tested (buffer plus crystallizing agent) is used as the mobile phase. Protein dissolved in the mobile phase or just in the buffer is then injected onto the column, and the retention time is measured and compared to that of a neutral marker. For different solutions (i.e., different crystallizing agents), the injected protein will have different interactions (repulsive/ attractive) with the stationary phase protein, leading to shifts in retention times. Several factors important in determining the chro- matographic retention times include the strength of the protein-protein interaction, the amount of immobilized protein bound to the stationary phase, and the kinetics of protein-protein association and dissociation. In the simplest case, the following equations can be used to describe the interaction between the soluble (S P ) and immobilized protein (I P ) in the column: where k a and k d are the association and dissociation constants, respectively, for the interaction of S P with I P , [S‚‚‚I] is the concentration of the resulting protein- protein complex, [S P ] is the mobile phase concentration of the protein while [I P ] represents the surface concentra- tion of the protein. At equilibrium, the retention of protein solute in the foregoing system is described by (11) * To whom correspondence should be addressed. Fax: 662-325- 1618. E-mail: bilwil@ra.msstate.edu. † Mississippi State University. ‡ Colorado State University. S P + I P { \ } k a k d S‚‚‚I (1) K ) [S‚‚‚I] [S P ][I P ] (2) 575 Biotechnol. Prog. 2003, 19, 575-579 10.1021/bp025725g CCC: $25.00 © 2003 American Chemical Society and American Institute of Chemical Engineers Published on Web 01/30/2003