Square Wave Voltammetric and Computational Study of Thyroxine-Thiourea Interaction

The voltammetric behavior of Thyroxine (T4) was studied using square wave voltammetry in phosphate buffer solution at (pH 7.0) as supporting electrolyte. Thyroxine gives two well-defined reduction peaks at Ep1 (-0.359) volt and Ep2 (-1.01) volt versus the reference electrode (Ag/AgCl/Sat.KCl). The calibration curve is linear within a two range of concentration, first is (1.996x10 19.61x10)M with the R equal to (0.999) and (0.9963) for Ep1 and Ep2 respectively, and second (0.996x10 11.857x10) M with the R equal to (0.9819) and (0.9848) for Ep1 and Ep2 respectively. The Gibb`s free energy (∆G), enthalpy (∆H) and entropy (∆S) changes of temperature dependent on (K) were calculated using Van`t Hoff equation for Thyroxine and Thiourea binding. The molecular docking between Thyroxine and Thioureahas been studied, and the results indicates thatthe interaction between T4 and TU was mainly hydrogen bonding and van der Waal`s interaction.


INTRODUCTION
L-Thyroxine (L-T 4 ) (+)-3,5,3`,5`-tetraiodo-L-thyronine (Schem-1) is an important biological compound derived from tyrosine and produced by the thyroid gland (Voet et al., 2002). Also T 4 is the main hormone secreted into the bloodstream by the thyroid gland. It is inactive and most of it is converted to an active form called triiodothyronine (T 3 ) by organs such as the liver and kidneys. Thyroid hormones play vital roles in regulating the body's metabolic rate, heart, digestive functions, muscle control, brain development and maintenance of bones, among many others effects not fully studied. The thyroid hormones T 3 and T 4 are unique in that iodine (as iodide) is an essential component of both (Murray et al., 2009).
However, these methods have some disadvantages such as expensive instrumentation, and time consuming and complicated operations. Electrochemical techniques have also been used for the detection of T 4 . Holak and Shostak (Holak and Shostak,1979) determined T 4 by differential pulse polarography(DPP), with the use of ion-exchange resins to isolate the amino acids from the matrix, and the application of these methods in pharmaceuticals. Jacobsen and Fonahn (Jacobsen and Fonahn, 1980) carried out the most comprehensive study to date on the electrochemical behavior of T 4 by DPP at a dropping mercury electrode (DME); they proposed a possible mechanism of reaction on the electrode surface. Both reports (Holak and Shostak, 1979) (Jacobsen and Fonahn, 1980) showed that the reduction of T 4 involves the substitution of iodine by hydrogen. With the exchange of eight electrons and eight protons. Jacobsen and Fonahn found T 4 to exhibit adsorptive properties at the surface of the DME on analyzing capillarity curves for solutions containing the amino acid. Also Hamdoon used DPP anddifferential pulse stripping voltammetry(DPS) methods for the determination of thyroxine (Hamdoon, 1989).
The glassy carbon electrode modified with Multi-Wall Carbon Nano Tubs(MWCNTs) was reported byK. Wu et al and applied the method to determine T 4 in human serum (Wu et al., 2004).
In the present study, the electrochemical behavior of T 4 anditsinteractionwith thiourea (TU) were studied as related simple compound to the antithyroiddrugs (Schem-2). In addition, the binding constant and thermodynamic parameters were also calculated.

Schem-1 Structure of L-thyroxine
Schem-2Thioureylene drugs are related to thiourea (the thiocarbamide group is essential for their antithyroid activities)

Reagents and Chemicals :
Astock solution (10 -3 M) of L-T 4 was prepared by dissolving T 4 (obtained from Alfa company, Germany) in (0.1 M NaOH in 70% ethanol solution); they were kept in darkness at 4°C , 0.2M K 2 HPO 4 & 0.2M KH 2 PO 4 (obtained from Alfa company, Germany) to prepare 0.1M phosphate buffer solution (PBS) at pH 7.0. The buffer was adjusted to the required pH with the same solutions. Thiourea wasobtained from BDH laboratory reagent, andall solutions were prepared using deionized water and used without further purification.

Apparatus :
All voltammetric measurements were performed using 797-VA Computrace stand (Metrohm AG,CH-9101 Herisav, Switzerland). Reference electrode (RE) was Ag/AgCl/ Sat.KCl and Pt wire was used as auxiliary electrode (AE) and Hanging Mercury Drop Electrode (HMDE) was used as working electrode (WE). pH measurements were performed by using a digital pH meter (HAVANNA) calibrated with standard buffers, for temperature control a HAAKE G water bathwas used.

Computational study:
The Molecular Operating Environment MOE version (2009) software developed by (Chemical Computing Group, Montreal, Canada) was used for the graphical illustrations and molecular interaction study.
Molecular mechanics and quantum chemical calculations were performed to study the geometries and electronic structures. The 3D structures were drawn and used as the starting point for energy minimization. The energy minimizations were performed until the gradient was below (Minimum RMS Gradient 0.0001 Kcal/mol/Aº). Initial geometry optimization of molecule was carried out using molecular mechanics by the force field method (MMFF94x).

RESULT AND DISCUSSION Electrochemical behavior of L-T 4 :
Preliminary measurements of T 4 using SWV and the three-electrode system with HMDE as working electrode in PBS at pH 7.0 as supporting electrolyte give two well-defined peaks at (-0.359 and -1.01) Vversus Ag/AgCl/Sat.KCl. As shown in Fig. (1) using optimum instrument conditions.

Optimum Condition for T 4 using SWV :
A set of S.W. experiments were carried out using a solution containing (4.98×10 -6 )M T 4 in PBS pH 7.0.
The optimum conditions were obtained by changing the operating condition continuously and the best results obtained either givethe highest peak current or the best shape voltammogram, as showed in Fig. (1). The results obtained are shown in (Table 1)

Effect of L-thyroxine concentration :
The calibration curve of T 4 was constructed using SWV under the optimum conditions (Table 1) and potential between ( -1.4 --0.1)V.The S.W.Voltammograms were recorded for the sequence additions of (10 -4 ) M and (10 -3 ) M respectively as stock solution of T 4 in (10 ml) PBS(pH 7.0). Fig. (2) shows the result of these measurements, and the peak current plotted against the T 4 concentration is shown in Fig. (3).

Voltammetric study of T 4 -TU Interaction:
To study the interaction betweenThyroxine andThiourea,a successive amount of Thiourea (1×10 -4 Mas a stock solution) was added to voltammatric cell containing (9.9×10 -6 M)(L-Thyroxine) in phosphate buffer solution at (pH 7.0) at different temperatures (288,293,295,303) ºKand the voltammogram was recorded for each addition. The peak current was measured at Ep 1 = (-0.365 V) because it is more sensitive than Ep 2 , which belongs to the reduction peak of L-T 4 ; denoted as Ipº Fig (4 A). Gradually. In each addition, the voltammogram was recorded Fig (4 B) for second addition of Thiourea and the peak currents wave was measured at corresponding Ep and denoted as Ip.It is very clear from Fig. (4), the peak current Ip decreased gradually with the sequence addition of Thiourea until it reached constant value (saturation) . Where K is apparent binding constant, Ipº and Ip, the peak current of the free (T 4 ) and the complex (T 4 -TU), respectively. Then the plot of ln (1/[Conc.Thiourea (M)]) versus ln (Ip/(Ipº-Ip)) giveslinear relation with intercept of -ln (K) Equation (1) .  The result shows that the value of K was decreased with increasing temperature.   (Table 3), it can be seen that the negativevalue of ∆G reveals that the interaction process is spontaneous, the negativevalue of ∆H indicates that the interaction is exothermic andthe negativevalue of ∆S indicates that the interaction is order.

Calculation of Thermodynamic Parameters
From thermodynamics parameters(∆H<0, ∆S<0), it is clear that the van der Waal`s and hydrogen bonding is the main force in the interaction (Zhao, 2010).

Molecular Docking:
To predict the structure of molecular complex between two or more molecules (Ferreira et al., 2015), the molecular docking technique was performed as the best orientation and conformation of complex, as shown in Fig. (7).   Fig.(7a,7b, 7c) shows that Thyroxine interacts with Thiourea by H-bonding and electrostatic forces.
It can be seenfrom Fig. (7a, 7b) that the oxygen of carboxylic group of T 4 was very closed with hydrogen of TU with distance 1.51 and 1.53 °A as hydrogen bonding between them (as shown in Fig.(7a) and 7b with white dashed line) and which wasaccepted with thermodynamic result about Ep 1 (∆H<0 and ∆S<0).
On the other hand, the phenolic ring (π electrone) of thyroxine, also interacted with nitrogen's of TU ring, makes a cation-pi interaction with the phenolic ring between the Nitrogen and the ring π electrons (as shown in Fig.(7c) with a yellow dashed line) suggest electrostatic forces. The result of molecular docking between T4 and TU shown in (Table 4).

CONCULSION
In this paper, the interaction of thyroxine hormone with thiourea has been studied by electrochemical method. The experimental results indicates that thiourea can interact with thyroxine through hydrogen bond and van der Waals force. The binding constant (K) between thyroxine and thiourea was determined to be (16.67x10 6 -3.40x10 6 ) at temperature range (288-303)ºK, thermodynamic parameters also were calculated. The molecular docking also has been studied between thyroxine and thiourea.