Energetics of binding and protein unfolding upon amphiphilic drug complexation with a globular protein in different aqueous media

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Abstract

The interactions and complexation process of the structurally related amphiphilic phenothiazines promazine and triflupromazine hydrochlorides with horse myoglobin in aqueous buffered solutions of pH 2.5, 5.5 and 9.0 have been examined by ζ-potential, isothermal titration calorimetry (ITC), UV–vis spectroscopy and dynamic light-scattering techniques with the aim of analyzing the effect of hydrophobic and electrostatic forces, the alteration of protein conformation and the effect of substituents in the drug molecular structure on the binding mechanism and structure of the resulting complexes. The energetics and stoichiometry of the binding process was derived from ITC. The enthalpies of binding obtained are small and exothermic, and the Gibbs energies of binding are dominated by large increases in entropy consistent with hydrophobic interactions. Binding isotherms were obtained from microcalorimetric data by using a theoretical model based on the Langmuir isotherm. ζ-Potential data showed a reversal in the sign of the protein charge at pH 9.0 as a consequence of drug binding. Gibbs energies of drug binding per mole of drug were also derived from ζ-potential data. On the other hand, binding of the phenothiazines causes a conformational transition on protein structure which was followed as a function of drug concentration by using UV–vis spectroscopy. These data were analyzed to obtain the Gibbs energy of the transition in water (ΔGw°) and in a hydrophobic environment (ΔGhc°). Finally, the population distribution of the different species in solution and their size was analyzed through dynamic light scattering. The existence of an aggregation process of drug/protein complexes, mainly at pH 2.5, was observed. We think this is a consequence of the already expanded structure of the protein at this pH and the subsequent binding of drug molecules to the protein.

Introduction

Proteins are complex macromolecules that can adopt a large number of slightly different conformations within their native state, called conformational substrates. Even small structural differences between these substrates can lead to drastic changes in functional parameters. In addition, the marginal stability of the native conformation is a delicate balance of various interactions in proteins (van der Waals, electrostatic, hydrogen bonds, hydrophobic and disulfide bridges) [1], which is affected by pH, temperature or addition of small molecules such as substrates, coenzymes, inhibitors and activators that bind especially to the native state and alters this fragile equilibrium.

In this regard, many drugs, particularly those with local anesthetic, tranquillizer, antidepressant and antibiotic actions, exert their activity by interaction with biological membranes. These drugs have also to be carried to their sites of action by means of protein carriers (as human serum albumin) at which they bind with different affinities. Accumulation of drug molecules at certain sites in the body causing a localized high concentration, adverse drug reactions [2], [3], and ligand-induced protein structure conformational changes [4], [5] are major problems complicating drug medical therapy. Therefore, studies which combine the study of the conformational changes in proteins through variations of external parameters such as pH, temperature, salinity, etc, and the analysis of the binding mechanism between proteins and amphiphilic drugs and the structure of the resulting complexes are of particular interest. These works enable to elucidate how ligand affinity is regulated and how the protein conformation is altered upon complexation [1], which are of crucial importance in a vast range of important biochemical phenomena, as for example, the reversible binding of oxygen by myoglobin and the noncovalent association of serum albumin with fatty acids and other compounds containing nonpolar groups [6], [7].

Thus, in the present work, we analyze the complexation process of two structurally related phenothiazine drugs, promazine and triflupromazine hydrochlorides (see Fig. 1) to the oxygen transport protein myoglobin in aqueous buffered solutions of pH 2.5, 5.5 and 9.0. Myoglobin is a monomeric heme protein found mainly in muscle tissue where it serves as an intracellular storage site for oxygen [8]. It has been usually used as a model protein to check binding mechanisms and reveal structural changes in its native conformation due to its peculiar structure and function. In recent reports, the self-aggregation process and the physico-chemical properties of these two drugs have been extensively studied [9], [10], [11], [12]. We tried to elucidate and quantify the forces involved in the drug-binding process, the subsequent alteration of protein conformation and the effect of the presence of substituents in the drug molecular structure on the binding mechanism. By comparison with the results obtained in a previous work [13], the role of the hydrophobic moiety structure of the drug on the binding affinity can be also derived. To do this, isothermal titration calorimetry measurements (ITC) were performed to determine the type and magnitude of the energies involved in the complexation process of the two-phenothiazine drugs to myoglobin. The extent of drug adsorption was calculated using the theoretical model of Ueda and Yamanaka [14]. The electrophoretic mobility of the myoglobin/phenothiazine complexes was measured, providing information on the adsorbed layer, the ζ-potential of the complexes and the adsorption energies. UV–vis data were used to follow the conformational changes of the myoglobin structure upon binding, and to calculate its free energy of unfolding. Finally, dynamic light-scattering measurements were performed in order to characterize the complexes size distributions.

Section snippets

Materials

Horse skeletal muscle myoglobin (100684-32-0; 0.30% iron content), promazine (N,N-dimethyl-3-(10H-phenothiazin-10-yl)propan-1-amine HCl) and triflupromazine (N,N-dimethyl-3-[2-(trifluoromethyl)-10H-phenothiazin-10-yl]-propan-1-amine HCl) hydrochlorides were obtained from Sigma Chemical Co. and used after a further purification by liquid chromatography using a Superdex 75 column equilibrated with 0.01 M phosphate. Experiments were carried out using double distilled, deionized and degassed water.

Myoglobin structure

The tertiary structure of myoglobin is typical of a water-soluble globular protein. Its secondary structure is unusual since it contains a very high proportion (75%) of α-helical secondary structure [8]. A myoglobin polypeptide is comprised of 8 separate right-handed α-helices, designated A–H, connected by short non-helical regions. Each myoglobin molecule contains one heme prosthetic group inserted into a hydrophobic cleft in the protein. Each heme residue contains one central coordinately

Conclusions

The present study shows that the structurally related phenothiazines promazine and triflupromazine hydrochlorides bind to horse myoglobin in different aqueous media. ITC demonstrates that hydrophobic interactions between the phenothiazine and the protein play a predominant role in this process, although the existence of electrostatic interactions is also noted, even at acidic pH due to the heterogeneous charge distribution on the biopolymer. This agrees with the small exothermic values of

Acknowledgements

Authors thank MEC by research project MAT2004-02756 and Xunta de Galicia for financial support.

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    Permanent address: Department of Chemistry, Quaid-i-Azam University, Islamabad 45320, Pakistan.

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