Elsevier

Journal of Luminescence

Volume 176, August 2016, Pages 149-158
Journal of Luminescence

Full length article
Binding of carvedilol to serum albumins investigated by multi-spectroscopic and molecular modeling methods

https://doi.org/10.1016/j.jlumin.2016.02.001Get rights and content

Highlights

  • The quenching mechanism between Carvedilol and HSA /BSA is a static process.

  • Hydrogen bonds and Van der Waals contacts were stabilized the Carvedilol albumin complexes.

  • Molecular modeling simulations confirmed the fluorescence spectroscopy and FRET analysis.

  • According to the binding mechanism differences between HSA and BSA, the results of BSA experiments could not be applied for HSA binding.

Abstract

Carvedilol (CAR) binding to human and bovine serum albumins (HSA and BSA) was studied using fluorescence, UV–vis absorption and Fourier transform infrared spectroscopy (FTIR) and molecular docking techniques at different temperatures (288, 298 and 308 K) under physiologic pH. Results obtained from fluorescence data indicated that values of binding sites (n), effective quenching constants (Ka) and binding constants (Kb) decreased under higher temperature and that the quenching mechanism was static. The thermodynamic parameters including enthalpy (ΔH), entropy (ΔS) and Gibb׳s free energyG) changes were calculated by the van׳t Hoff equation and these data showed that hydrogen bonds and Van der Waals contacts were the main binding force in HSA–CAR and BSA–CAR systems. Binding distance (r) between HSA–CAR and BSA–CAR were calculated by the Fӧrster (fluorescence resonance energy transfer (FRET)) method. FTIR absorption studies showed that the secondary structure was changed according to the interaction of HSA/BSA and CAR. Results determined by molecular docking were in agreement with thermodynamic and FRET data and confirmed that the binding mechanism of Carvedilol to HSA and BSA is different.

Introduction

Biological activity is affected by ligand protein binding extensively [1]. Mechanism of drug–protein binding information (e.g. serum albumin binding) is needed to investigate particular pharmacokinetic (distribution, excretion, metabolism, and interaction with target tissues) and pharmacodynamic (therapeutic effect) properties of drugs [2], [3].

Human serum albumin (HSA) and bovine serum albumin (BSA) are two of the most extensively studied serum albumins. They are homologous proteins (76%) with three structural domains (I–III). Each domain composed of two sub-domains (A and B). HSA (MW= 66,500  Da), contains 585 amino acids and one tryptophan (Trp214) in sub-domain (IIA), while BSA (MW=65,000 Da) consists of 582 amino acids with two tryptophan (Trp 134 and Trp 213). Trp 134 is located on the surface of the molecule and Trp 213 is located in the hydrophobic pocket (Fig. 1) [4], [5], [6].

Several techniques have been developed which can be categorized to separative techniques (e.g. high- performance affinity chromatography [7]; frontal analysis capillary electrophoresis [8], hummel and dreyer [9], [10], flow injection-capillary electrophoresis frontal analysis [11]) and non separative techniques (e.g. circular dichroism spectroscopy, isothermal titration calorimetry, zeta-potential and fluorescence polarization spectroscopy [12]; fluorescence spectroscopy [13], [14], [15], [16], [17]; synchronous fluorescence spectroscopy [18]; resonance light-scattering (RLS) technique [19]; atomic force microscopy [20] and electrochemical methods [21], [22]). Other techniques used to study albumin drug binding by quantification of non-bound drug concentration include equilibrium dialysis [23], [24], [25] and ultrafiltration [26], [27]. In addition molecular modeling [28], [29] and chemometrics methods [30] have been used to study binding characteristics of some molecules.

Spectroscopic methods (spectrofluorimetry, UV–vis absorption and Fourier transform infrared (FTIR) spectroscopy) are often applied because of their high sensitivity and fastness. Fluorescence spectroscopy has exceptional sensitivity, selectivity and convenience and its mechanism is supported by a strong theoretical foundation that provides a popular and comprehensive method to study the binding mechanism [1], [2], [31].

Albumin has intrinsic fluorescence due to the presence of tryptophan, tyrosine and phenylalanine residues [32]. The intrinsic fluorescence of HSA is mostly attributed to tryptophan because phenylalanine has very low quantum yield and the fluorescence of tyrosine is almost totally quenched if it is ionized or located near an amino/carboxy group or a tryptophan residue. Binding constant, number of binding site, binding location, binding mechanism and binding distance [13] can be obtained by flourescence studies.

Albumin binding of β adrenergic blocking drugs i.e. Metoprolol [33] and propranolol [34] has been studied recently. Carvedilol (1-(9H-carbazol-4-yloxy)-3-[2-(2-methoxyphenoxy) ethylamino] propan-2-ol) (Fig. 2) is a nonselective β adrenergic blocking agent [35]. Carvedilol is used in the treatment of mild to moderate hypertension and congestive heart failure (CHF) and is often used in combination with other drugs. [35], [36], [37]. Carvedilol is a weak base (pKa value is approximately 7.8) [38] with poor aqueous solubility (10 mg/ml to 25 °C) [39]. It is known that more than 95% of the drug is bound to plasma proteins [36] while the mechanism of its binding to HSA or BSA has not been studied to date.

BSA binding studies׳ results are applied to predict HSA binding properties, while Akdogan et al., were studied the water-tuned structural differences between HSA and BSA and showed that conformational adaptability and flexibility dominate in the HSA solution structure while BSA seems to lack these properties [40]. In this paper Carvedilol binding to BSA and HSA was studied using the spectroscopic methods in the present study and the results are discussed based on the applicability of BSA binding data to predict HSA binding. The detailed mechanism of carvedilol binding to HSA/BSA was further investigated by the application of molecular docking methods.

Section snippets

Reagents

HSA/BSA were purchased from Sigma Aldrich. Stock solutions of HSA/BSA (0. 5% w/v) were freshly prepared in 0.05 M phosphate buffer (pH 6.5, 7.0, 7.4 and 7.6) daily. pH of the phosphate buffer were adjusted to the desired pH by NaOH (1.0 M).

CAR was purchased from Sobhan pharmaceutical Co. (Rasht, Iran). Stock solution of CAR (4.96×10−4 M) was prepared in methanol and stored at 4 oC. The working solution of CAR was prepared by dilution of the stock solution with phosphate buffer, in which a 3 mL

UV spectroscopy studies

UV absorption spectra of HSA and BSA in absence and presence of CAR at room temperature are shown in Fig. 3. The maximum absorption wavelength of HSA/BSA was observed at around 279 nm (which was mainly caused by the transition ππ* of aromatic amino acid residues). Absorbance enhancement according to increasing of CAR concentration indicates formation of HSA–CAR and BSA–CAR complexes. Slight red-shift (1–2 nm) was observed at the maximum absorption wavelength following the addition of CAR to

Conclusion

Interaction between CAR and HSA/BSA was investigated using Fluorescence, FTIR and UV–vis absorption spectroscopy and molecular docking techniques at physiological conditions. To overcome the fluorescence spectra overlapping of carvedilol and albumin, emissions of complexes (CAR–BSA and CAR–HSA) were studied in a non-maximum wavelength (i.e. 320 nm).

Results for spectroscopic and molecular docking studies suggest that CAR could bind to HSA/BSA (subdomain IB, IIA and IIIA) using hydrogen bonding

Acknowledgment

The authors would like to acknowledge Drug design laboratory of pharmacy faculty (Tabriz University of medical sciences) for providing hardware and software of molecular docking studies.

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