Partial filling multiple injection affinity capillary electrophoresis (PFMIACE) to estimate binding constants of receptors to ligands

    Authors

    J. Zavaleta; D. B. Chinchilla; A. Ramirez; A. Pao; K. Martinez; S. Nilapwar; J. E. Ladbury; S. Mallik;F. A. Gomez

    Comments

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    Abbreviated Journal Title

    Talanta

    Keywords

    partial filling multiple-injection affinity capillary electrophoresis; vancomycin; teicoplanin; ristocetin; carbonic anhydrase B; binding; constants; scatchard plot; ZONE-ELECTROPHORESIS; GLYCOPEPTIDE ANTIBIOTICS; VANCOMYCIN; PEPTIDES; PROTEIN; RISTOCETIN; DERIVATIZATION; CYCLODEXTRIN; TEICOPLANIN; COMPLEXES; Chemistry, Analytical

    Abstract

    Partial filling multiple injection affinity capillary electrophoresis (PFMIACE) is used to determine binding constants between vancomycin (Van) from Streptomyces orientalis, teicoplanin (Teic) from Actinoplanes teicomyceticus and ristocetin (Rist) from Nocardia lurida to (D)-Ala-(D)-Ala terminus peptides and carbonic anhydrase B (CAB, E.C.4.2.1.1) to arylsulfonamides. Two variations of PFMIACE are described herein. In the first technique, the capillary is partially filled with ligand at increasing concentrations, a non-interacting standard, three or four separate plugs of receptor each separated by small plugs of buffer, a plug containing a second non-interacting standard, and then electrophoresed in buffer. Upon continued electrophoresis, equilibrium is established between the ligand and receptors causing a shift in the migration time of the receptors with respect to the non-interacting standards. This change in migration time is utilized for estimating multiple binding constants (K(b)) for the same interaction. In the second technique, separate plugs of sample containing non-interacting standards, peptide one, buffer, and peptide two, were injected into the capillary column. The capillary is partially filled with a series of buffers containing an antibiotic at increasing concentrations and electrophoresed. Peptides migrate through the column at similar electrophoretic mobilities since their charge-to-mass ratios are approximately the same but remain as distinct zones due to the buffer plug between peptides. Upon electrophoresis, the plug of antibiotic flows into the peptide plugs affecting a shift in the migration time of the peptides with respect to the non-interacting standards occurs due to formation of the of the antibiotic-peptide complex. The shift in the migration time of the peptides upon binding to the antibiotic is used for the Scatchard analysis and measurement of a Kb. The PFMIACE technique expands the functionality and potential of ACE as an analytical tool to examine receptor-ligand interactions. In PFMIACE, a smaller amount of sample is required in the assay compared to both conventional ACE and MIACE. Furthermore, a wide array of data is obtained from a single experiment, thus, expediting the assay of biological species. (c) 2006 Elsevier B.V. All rights reserved.

    Journal Title

    Talanta

    Volume

    71

    Issue/Number

    1

    Publication Date

    1-1-2007

    Document Type

    Article

    Language

    English

    First Page

    192

    Last Page

    201

    WOS Identifier

    WOS:000244059600030

    ISSN

    0039-9140

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