Investigating the effect of an arterial hypertension drug on the structural properties of plasma protein
Graphical abstract
Highlights
• Complexation between fibrinogen and propranolol have been characterized. • Propranolol plays two opposite roles on thermal structural stability of fibrinogen, acting as structure stabilizer and destabilizer. • Propranolol changes the conformation of the protein leading to a more compact structure which packs more efficiently onto the interface.
Introduction
Amphiphilic molecules are used widely in both consumer and industrial applications such as food processing, medicines and pharmaceuticals. Because of their peculiar self-assembly behavior, their properties have evolved from being of a purely scientific interest to become a key concept in nano and biotechnology applications, such as drug delivery, sensors, and catalysts. The design of drugs must take into account several attributes, such as the size of the encapsulation system and stability. For applications in drug delivery, previous reports emphasized that the distribution of nanoparticles in tissues and organs is a function of size. In addition, nanoparticles must be sufficiently stable (able to withstand mechanical stress) to increase circulation time in blood and with adequate thermal stability to reduce side effects [1]. Thus, molecular architecture should be involved in the design of self-assembling systems for preparation of nanoparticles [2], [3].
Many pharmacological active compounds are amphiphilic molecules, containing one or more aromatic nuclei. These structures are interesting for their resonance properties and for the cyclic delocalization of the charge [4]. Many of these drugs tend to self-assemble like surfactants; associating to form aggregates, usually with a small aggregation number. Proteins and amphiphilic molecules can interact in different ways in the bulk and at the interfaces, due to the strong amphipatic nature of proteins, resulting in complexes with different surface activity [5], [6], [7], [8]. Protein–amphiphilic molecule interactions have been extensively studied by a variety of experimental methods [9], [10]. In general ionic surfactants are chosen for these studies due to the fact that these kinds of amphiphilic molecules and the proteins have both charged groups and hydrophobic residues. This implies that the properties of the complexes in solution could be very different to those observed for solutions of the individual components. The understanding of molecular recognition in protein–ligand complexes is crucial so as to better comprehend the associated biological function. Non-covalent interactions such as a hydrophobic, electrostatic, van der Waals, and hydrogen bonding govern this ligand-binding process. Elucidating the role of these interactions and the time scales involved provides insights into the mechanism of molecular recognition and the role of binding cooperatively in protein dynamics [11]. It has also a practical importance in the discovery, for instance, of new drugs, and in the understanding of diseases like respiratory distress syndrome (dysfunction attributed to a competition for the air–lung interface between plasma proteins and surfactants).
Recently, we have studied the complexation of different proteins with amphiphilic ligands, including surfactants, lipids and drugs [12], [13], [14], [15]. These experiments have used a combination of different experimental methods (equilibrium dialysis, difference spectroscopy, microcalorimetry, zeta potential, static and dynamic light scattering, circular dichroism) which allowed us to obtain a very complete characterization and deep understanding of the physicochemical phenomena involved in these interactions [12], [16].
Propranolol is a β-adrenergic blocking agent (Scheme 1). It is a racemic compound, with only the 1-isomer having adrenergic blocking activity. It is the most prescribed drug in treating hypertension; it is also applied to manage chronic stable angina. Previous studies on β-adrenoceptor blocking agents have shown that their pharmacological effects arise as a result of modification of the cell membrane [17].
Fibrinogen (FB) is a plasma protein whose major function is related to blood coagulation and this prevents the loss of blood upon vascular injury [18], [19]. This protein is also considered as a major inhibitor of lung surfactants function at the layer lining the alveoli [20], [21]. FB adsorbs rapidly onto a thrombogenic surface and it is often the major protein found on the surface [22], [23], [24]. It is a very large protein (∼340 kDa) and the length of an individual fibrinogen molecule is 45–50 nm [25], [23]. Its native structure has been described as trinodular protein with three hydrophobic domains connected by α-helical coiled-coil domains. FB is composed of two identical subunits, each containing three dissimilar polypeptide chains Aα, Bβ, and γ, which are linked by disulfide bonds. The NH2 terminal portions of the six chains are linked together in the central region of the molecule by 11 disulfide bonds forming a small globular domain, the so-called disulfide knot, in the center [26], [27].
In this study we have investigated the nature of the interactions of propranolol with fibrinogen by using different experimental techniques; differential scanning calorimetry (DSC), dynamic light scattering, circular dichroism (CD), surface tension and atomic force microscopy (AFM). This combination of techniques has offered new insights into the nature of the interaction between propanolol and fibrinogen. Such characterization may provide crucial information towards the design of drugs with optimal performance.
Section snippets
Materials
Bovine plasma fibrinogen, fraction I, type IV, was purchased from Sigma (9001-32-5) and used without purification. Propranolol (1-[Isopropylamino]-3-[1-naphthyloxy]-2-propranol) hydrochloride was obtained from Sigma Chemical Co. (no. P-0884). The buffer solution used was 50 mM glycine plus sodium hydroxide to give a pH of 8.5. Samples were prepared two hours prior to use. All chemical reagents were of analytical grade, and solutions were made using doubly distilled and degassed water.
Differential scanning calorimetry (DSC)
DSC
Differential scanning calorimetry
Fig. 1 shows the DSC thermogram for FB (0.5 g dm−3) in buffer solution (pH 8.5) in the presence of propranolol at different concentrations. Table 1 shows the thermodynamic characteristics of the thermal denaturation obtained for all the systems studied: namely, the melting temperatures (Tm, temperatures at which a maximum occurred in the endothermic peaks), the calorimetric enthalpy (ΔH), and the van’t Hoff enthalpy (ΔHv).
As in the case of pure fibrinogen, we identified no reversible transitions
Conclusions
In this study, we have characterized the interactions and complexation between fibrinogen and propranolol. The results obtained are summarized as follow. First of all, propranolol plays two opposite roles on thermal structural stability of fibrinogen, acting as structure stabilizer and destabilizer, at low and higher molar concentrations respectively. Complexation between fibrinogen and propranolol results in changes in the secondary structure of the protein. This event increase the surface
Acknowledgements
The authors thank the Xunta de Galicia for their financial support (project no. 10PXIB206258PR). J. M. V. thanks MICINN (JCI-2009-03823) and EU (FP7-PEOPLE-PERG07-GA-2010-268315). Research at IFR was supported by the BBSRC core strategic grant. N. H. thanks E.U. for LLP/Erasmus Programme Grant.
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Present address: Biocolloids and Fluid Physics Group, University of Granada, Campus de Fuentenueva, sn. 18071, Granada, Spain.