This work presents the first known use of response surface methodology (RSM) in electrophoretically mediated microanalysis. This concept is demonstrated by examining the optimization of reaction conditions for the conversion of nicotinamide adenine dinucleotide to nicotinamide adenine dinucleotide, reduced form by glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) in the conversion of glucose-6-phosphate to 6-phosphogluconate. Experimental factors including voltage, enzyme concentration, and mixing time of reaction at the applied voltage were selected at three levels and tested in a Box-Behnken response surface design. Upon migration in a capillary under CE conditions, plugs of substrate and enzyme are injected separately in buffer and allowed to react at variable conditions. Extent of reaction and product ratios were subsequently determined by CE. The model predicted results are shown to be in good agreement (7.1% discrepancy difference) with experimental data. The use of chemometric RSM provides a direct relationship between electrophoretic conditions and product distribution of microscale reactions using CE, thereby offering a new and versatile approach to optimizing enzymatic experimental conditions.

An ACE predictive investigation of protein-ligand binding using a highly effective chemometric response surface design technique is presented. Here, K(d) was estimated using one noninteracting standard which relates to changes in the electrophoretic mobility of carbonic anhydrase B (CAB, EC 4.2.1.1) on complexation with the ligand 4-carboxybenzenesulfonamide (CBSA) present in the electrophoresis buffer. Experimental factors including injection time, capillary length, and applied voltage were selected and tested at three levels in a Box-Behnken design. Statistical analysis results were used to create a mathematical model for response surface prediction via contour and surface plots at a given target response (K(d) = 1.19x10(-6) M). As expected, there were a number of predicted solutions that reached our target response based on the significance of each factor at appropriate levels. The adequacy of the model was validated by experimental runs with the predicted model solution (capillary length = 47 cm, voltage = 11 kV, injection time = 0.01 min) presented in detail as an example.

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

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