Multiple-Injection Affinity Capillary Electrophoresis

Please address correspondence to Frank A. Gomez at [email protected]. nteractions between biological species are essential to life and are involved directly in many of the enzyme-based reactions involved in cell division, cell death, and cell transformations. These biological interactions are important in the initiation, progression, and harmful effects of human disease including Parkinson’s, Alzheimer’s, AIDS-HIV, and cancer. Advances in molecular biological tools have yielded a huge array of biological interactions focusing foremost on receptor–ligand interactions. Similarly, combinatorial chemical techniques have generated millions of potential drugs and drug precursors. Combined, these two areas of research have made the development of new analytical techniques an important area of research. During the last decade, affinity capillary electrophoresis (ACE) has emerged as a useful and sensitive technique for studying bimolecular noncovalent interactions and for determining binding and dissociation constants of formed complexes. In 1992, the first reports documenting the use of ACE to study receptor–ligand interactions were published (1–5). Since then, myriad interactions including protein–ligand (6–17), peptide–metal (18,19), peptide–peptide (20–30), protein–peptide (36), protein–antibody (37), polymer–peptide (38), antibody–antigen (39), enzyme–drug (40), and polymer–cyclodextrin (41) have been examined successfully using ACE. For example, Varenne and colleagues used ACE to examine the binding of fucoidan, an anticoagulant polysaccharide of marine origin, to antithrombin (6). Kaddis and colleagues recently determined binding constants for the activator fructose-6-phosphate (F6P) and substrate ATP to the recombinant wild-type (WT) Rhodobacter Sphaeroides adenosine 5’diphosphate-(ADP)-glucose pyrophosphorylase (ADPGlc PPase) using ACE (7). Finally, Lewis and colleagues described the screening of antimicrobial targets using ACE (30). In ACE, the mobility of a receptor (or ligand) changes upon binding to a ligand (or receptor) that is present in the electrophoresis buffer. The change in migration time can be correlated to a binding constant via Scatchard analysis or other form of analysis. In general, there are three different types of ACE. In the first type, affinity ligand and receptor are premixed and an aliquot of the solution analyzed by CE. This form of ACE has been used successfully in the analysis of DNA–protein, DNA–DNA, and DNA–small molecule Multiple-injection affinity capillary electrophoresis (MIACE is a versatile analytical technique to probe bimolecular noncovalent interactions and to estimate binding constants between receptors and ligands. In this article, we demonstrate the use of MIACE and several variations to MIACE to determine binding constants between the glycopeptide antibiotics vancomycin, ristocetin, and teicoplanin from Streptomyces orientalis, Nocardia lurida, and Actinoplanes teichomyceticus, respectively, and D-Ala-D-Ala terminus peptides.