Using Capillary Electrophoresis To Study the Electrostatic Interactions Involved in the Association of d -Ala- d -Ala with Vancomycin

This work examines the electrostatic interactions involved in the recognition of d -Ala- d -Ala ( d A d A ) by vancomycin ( Van ) by using capillary electrophoresis (CE) and affinity capillary electrophoresis (ACE). Acetylation of the N-terminal amine of Van decreases its affinity for Di-Ac- l -Lys- d -Ala- d -Ala ( Ac 2 K d A d A ) by a factor of 11 at pH 7.1 (from 4.3 μM to 48 μM). Succinylation of the N-terminus of Van introduces a pendant negative charge that further decreases its affinity for Ac 2 K d A d A about 2-fold at pH 7.1. The association of Ac- d -Ala- d -Ala ( Ac d A d A ) with Van shifts the p K a of the N-terminal amine of Van by 1.7 units from 7.1 to 8.8, and thus changes its net charge in the range of values of pH between 6 and 10. The electrostatic interaction between the −CO 2 - group of the d A d A moiety and the −NH 2 + CH 3 group of Van contributes approximately 5.9 kJ/mol to the free energy of binding of these species. In addition to establishing or confirming these thermodynamic parameters, this paper illustrates the use of CE as a physical-organic tool in examining electrostatic interactions in biomolecular recognition.