Ruth E. Montes Grady Hanrahan Frank A. Gomez Department of Chemistry and Biochemistry, California State University, Los Angeles, CA, USA Received September 15, 2007 Revised January 13, 2008 Accepted January 14, 2008 Research Article Use of chemometric methodology in optimizing conditions for competitive binding partial filling affinity capillary electrophoresis This work expands the knowledge of the use of chemometric response surface methodology (RSM) in optimizing conditions for competitive binding partial filling ACE (PFACE). Specifically, RSM in the form of a Box–Behnken design was implemented in flow-through PFACE (FTPFACE) to effectively predict the significance of injection time, voltage, and neutral ligand (neutral arylsulfonamide) concentration, [L o ], on protein–neutral ligand binding. Statistical analysis results were used to create a model for response surface pre- diction via contour and surface plots at a given maximum response (DRMTR) to reach a targeted K b = 2.50610 6 M 21 . The adequacy of the model was then validated by experi- mental runs at the optimal predicted solution (injection time = 2.3 min, voltage = 11.6 kV, [L o ] = 1.4 mM). The achieved results greatly extend the usefulness of chemometrics in ACE and provide a valuable statistical tool for the study of other receptor–ligand combinations. Keywords: Affinity capillary electrophoresis / Capillary electrophoresis / Chemometrics / Response surface methodology DOI 10.1002/elps.200700693 Electrophoresis 2008, 29, 3325–3332 3325 1 Introduction Molecular recognition refers to the recognition and interac- tion between two or more molecules via noncovalent bond- ing and is at the root of essentially all biological processes and, hence, much of medicine. All cellular functions in the human body depend on protein–protein, protein–small molecule, protein–nucleic acid, or other intermolecular interactions. These biological interactions are essential to life and are involved in all of the enzyme-based reactions involved in cell division, cell death, and cell transformation. Hence, biological interactions are important in the initiation, progression, and effects of all human disease including Par- kinson’s, Alzheimer’s, AIDS–HIV, and cancer [1, 2]. Currently, there are a variety of techniques available to measure affinity parameters between biological species including equilibrium dialysis, RIA, fluorescence quench- ing, ultracentrifugation, NMR, and slab gel electrophoresis. If the amount of bound and free ligand in solution can be distinguished, these techniques can provide reasonable esti- mates of binding constants (K b ). ACE has been shown to be a versatile microanalytical technique to estimate affinity constants, and has emerged as a useful and sensitive method for studying bimolecular noncovalent interactions and for determining binding and dissociation constants of formed complexes. The first reports detailing the use of ACE to measure affinity parameters be- tween biological species were published in the early 1990s [3– 7]. Since these informative studies, a multitude of other inter- actions including protein–ligand, peptide–peptide, protein– peptide, protein–antibody, polymer–peptide, and antibody– antigen have been examined successfully using ACE [8–38]. ACE differentiates between bound and unbound recep- tor (R) as a function of free ligand (L) concentration only when the R–L complexation yields a sizable difference in mass or charge-to-mass ratio. In a typical ACE experiment, a sample of receptor and noninteracting markers are reacted with an increasing concentration of ligand in a running buf- fer, thereby, causing a shift in the migration of the receptor peak. Subsequent analysis of these changes in migration time yields a value for K b [20]. To minimize the amount of sample needed in an ACE assay, partial filling techniques in ACE were developed. In PFACE, the capillary is partially filled with ligand (or receptor) Correspondence: Dr. Frank A. Gomez, Department of Chemistry and Biochemistry, California State University, Los Angeles, CA, USA E-mail: fgomez2@calstatela.edu Fax: 11-323-343-6490 Abbreviations: CAB, carbonic anhydrase B; FTPFACE, flow- through partial filling ACE; HHM, horse heart myoglobin; MO, mesityl oxide; RMTR, relative migration time ratio; RSM, re- sponse surface methodology  2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com