Use of chemometric methodology in optimizing conditions for competitive binding partial filling affinity capillary electrophoresis

Abstract

Use of chemometric methodology in optimizing conditions for competitive binding partial filling affinity capillary electrophoresisThis 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) 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 between biological species were published in the early 1990s [3][4][5][6][7]. Since these informative studies, a multitude of other interactions including protein-ligand, peptide-peptide, proteinpeptide, protein-antibody, polymer-peptide, and antibodyantigen have been examined successfully using ACE .ACE differentiates between bound and unbound receptor (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 buffer, 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, flowthrough partial filling ACE; HHM, horse heart myoglobin; MO, mesityl oxide; RMTR, relative migration time ratio; RSM, response surface methodology

Use of CE for the determination of binding constants

“…Competitive binding methods with a second substrate or ligand have been reported 20, 22, 62, 80–82 for systems that have small or no system‐response shifts for the substrate and complex.…”

Chemometrics applications in biotech processes: A review

“…57,58 Response surface methodology (Box Behnken design) has been used to model the effect of injection time, voltage, and ligand concentration on proteinneutral ligand binding. The model was subsequently validated by a series of experimental runs.…”

Affinity in Electrophoresis

“…In its lifetime a substantial number of papers and reviews dealing with affinity interactions in electrophoretic techniques have been published in Electrophoresis (Table 1). More than 125 papers 18, 38, 46, 47, 49–53, 69–71, 93, 101, 110, 118, 129–238 with the word affinity in the title and dealing with some aspect of affinity electrophoresis have been published in the journal since 1989, the year it started being indexed in PubMed. Prior to that, i.e.…”