Human serum albumin unfolding pathway upon drug binding: A thermodynamic and spectroscopic description

Mohammad Arif Cheema, Pablo Taboada*, Silvia Barbosa, Josué Juárez, Manuel Gutiérrez-Pichel, Mohammad Siddiq, Víctor Mosquera

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

44 Scopus citations

Abstract

The interest on phenothiazine drugs has been increased during last years due to their proved utility in the treatment of several diseases and biomolecular processes. In the present work, the binding of the amphiphilic phenothiazines promazine and thioridazine hydrochlorides to the carrier protein human serum albumin (HSA) has been examined by ζ-potential, isothermal titration calorimetry (ITC), fluorescence and circular dichorism (CD) spectroscopies, and dynamic light scattering (DLS) at physiological pH with the aim of analyzing the role of the different interactions in the drug complexation process with this protein. The ζ-potential results were used to check the existence of complexation. This is confirmed by a progressive screening of the protein charge up to a reversal point as a consequence of drug binding. On the other hand, binding causes alterations on the tertiary and secondary structures of the protein, which were observed by fluorescence and CD spectroscopies, involving a two-step, three-state transition. The thermodynamics of the binding process was derived from ITC results. The binding enthalpies were negative, which reveal the existence of electrostatic interactions between protein and drug molecules. In addition, increases in entropy are consistent with the predominance of hydrophobic interactions. Two different classes of binding sites were detected, viz. Binding to the first class of binding sites is dominated by an enthalpic contribution due to electrostatic interactions whereas binding to a second class of binding sites is dominated by hydrophobic bonding. In the light of these results, protein conformational change resembles the acid-induced denaturation of HSA with accumulation of an intermediate state. Binding isotherms were derived from microcalorimetric results by using a theoretical model based on the Langmuir isotherm. On the other hand, the population distribution of the different species in solution and their sizes were determined through dynamic light scattering (DLS). Aggregation of drug/protein complexes was found as a result of a possible expansion of protein structure induced at high drug concentrations. In addition, the presence of free drug aggregates at concentrations below the drug critical micelle concentration was also detected.

Original languageEnglish
Pages (from-to)439-447
Number of pages9
JournalJournal of Chemical Thermodynamics
Volume41
Issue number4
DOIs
StatePublished - Apr 2009
Externally publishedYes

Bibliographical note

Funding Information:
Authors thank MEC by Research Project MAT2007-61604 and Xunta de Galicia for financial support.

Keywords

  • Binding
  • Cluster
  • Complexation
  • Human serum albumin
  • Phenothiazine
  • Unfolding

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