Abstract
The interaction between human serum albumin (HSA) and the acetylcholinesterase inhibitor donepezil, has been studied by means of capillary electrophoresis frontal analysis (CE/FA) and circular dichroism. CE/FA enabled rapid and direct estimation of the quantity of free donepezil present at equilibrium with a physiological level of serum albumin (600 μmol L−1). Application of Scatchard analysis enabled estimation of the binding parameters of HSA towards donepezil, such as association constant and number of binding sites on one protein molecule. Furthermore, due to enantioseparation ability shown by HSA on donepezil in CE mode, displacement experiments were carried out using ketoprofen and warfarin as coadditives to the HSA based running buffer. The addition of these compounds reduced the enantioresolution of donepezil by HSA only when used at high concentration. These data were confirmed and corroborated by circular dichroism (CD) experiments. Using CD, bilirubin was also applied as a ligand specific to site III of HSA. The observed behaviour suggested that donepezil could be considered a ligand with independent binding to sites I and II; although site III is not the highest affinity site, indirect interaction (i.e. cooperative binding) can be assumed.
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Acknowledgements
This work was supported by a grant from MIUR (Rome, Italy, Cofin. 2002). The authors thank Professor P. Biscarini (Facoltà di Chimica Industriale, Università di Bologna, Italy) for the availability of the Jasco J-810 spectropolarimeter.
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Gotti, R., Bertucci, C., Andrisano, V. et al. Study of donepezil binding to serum albumin by capillary electrophoresis and circular dichroism. Anal Bioanal Chem 377, 875–879 (2003). https://doi.org/10.1007/s00216-003-2171-z
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DOI: https://doi.org/10.1007/s00216-003-2171-z