Studies of Thyroid Hormones (Propylthiouracil and L-Thyroxine) Interaction with Human Serum Albumin-Spectroscopic Approach

: In this work, UV-absorption, fluorescence spectroscopy and Fourier transform infrared (FTIR) spectroscopy were used to investigate the relationship between Propylthiouracil and L-Thyroxine with human serum albumin. The binding constants of propylthiouracil and L-Thyroxine have been determined of both UV-absorption and fluorescence spectroscopy. The binding Constants values measured at 293k are 1.659×10 3 M -1 for propylthiouracil and 1.013×10 4 M -1 for L-Thyroxine. The constant values of the Stern–Volmer quenching were determined to be 2.144×10 3 L mol -1 for propylthiouracil and 1.937×10 3 L mol -1 L-for Thyroxine. The UV-absorption intensity of HSA-hormones complexes has increased with increasing of propylthiouracil and L-Thyroxine concentration. With the rise in propylthiouracil and L-thyroxine concentrations, the fluorescence results indicate a decrease in HSA-hormone emission intensity. To determine the effects of protein secondary structure and hormone binding mechanisms, we have used FTIR spectroscopy with Fourier self-deconvolution technique and second derivative resolution enhancement, as well as curve-fitting methods for the investigation of the amide I, II, and III regions. The peak positions within the three amide regions (amide I, amide II and amide III) were allocated and any results were explored due to changes in concentration. Due to variations in hormone concentrations, the measurements of the FTIR spectra indicate a difference in the intensity of absorption bands.. FTIR Spectra measurements reflect a difference in the strength of the absorption bands due to changes in hormone concentrations.


Introduction
Propylthiouracil (6-propyl-2-thiouracil) belongs to the class of organic compounds known as pyrimidines.In July 1947, it was used for medicinal use and played an important part in the treatment of hyperthyroidism or Graves' disease [1].The molecular formula of PTU is C H N OS and chemical structure shown in Figure 1.Propylthiouracil is an inhibitor of thyroid hormone synthesis; it functions by inhibiting the thyroperoxidase enzyme, which adds Iodide to the thyroxine hormone precursor thyroglobulin residues of tyrosine [2].The enzyme tertaiodothyronine 5 'deiodinase, which changes thyroxine to triiodothyronine, is also inhibited by propylthiouracil [3].
L-Thyroxine belongs to the Phenylalanine family of organic compounds.It was first made in 1927, and has played a significant role in hypothyroidism care and suppressive therapy to shrink or prevent growth of abnormal thyroid tissue [4].The molecular formula of L-thyroxine is C 15 H 11 I 4 NO 4 and chemical structure shown in Figure 2. L-Thyroxine is synthetic version of the natural hormone thyroxine, naturally formed by the thyroid gland.When the thyroid gland is not producing enough natural thyroxine, L-Thyroxine relieves the symptoms associated with thyroxine deficiency since it increases serum thyroxine concentrations and mimics the action of thyroxine in the body [5].The main protein part of blood plasma is the human serum albumin as shown in Figure 3.It is present in all body fluids, For example its blood concentration is about 40 mg ml (~0.6m mol .It is synthesized and has a half-life of 19 days in the liver [2,6].A single polypeptide chain of 585 amino acids in length consists of human serum albumin, which in a heart-shaped conformation integrates three domains of homology (labeled I, II and III .Every domain is subdivided into smaller subdomains, A and B, each having six and four alpha -helices [7]. Human serum albumin has a high plasma concentration (35-50 g / L human serum) and is used in various essential bio-functions, such as osmotic pressure maintenance and nutritional transfer to cells from the circulation system [8].The osmotic pressure required for the proper distribution of body fluids between the body tissues and intravascular compartments is important for controlling and maintaining [9].
Human serum albumin has capable of binding numerous endogenous and exogenous ligands in the physiological system, counting fatty acids, hormones, pharmaceuticals, metabolites, metal ions, water, various drugs and many other compounds.Such intrinsic properties make human serum albumin a desirable protein material for medicinal use mainly in the delivery of a wide variety of medications or diagnostics.In addition, human serum albumin has become the most widely used biopolymer-based nanocarriers because of its attractive stability, biodegradability, promising distribution of size and easily regulated surface chemistry due to drug delivery and medical diagnosis [6,[10][11][12].
Several high and low affinity ligand binding sites have been differentiated on human serum albumin, the primary of which are responsible for the binding of most protein-associated pharmaceuticals to be known as sites I and II.Site I appears to bind relatively large heterocyclic or dicarboxylic acid compounds [13].Furthermore, this site is large and capable of binding bulky endogenous substances.By differentiating, because binding is more stereo-specific, location II is smaller and less flexible in nature.Within human serum albumin, extra high affinity binding regions are vital for certain drugs and compounds that do not adhere to either position I or II [14].
One of the special feature of human serum albumin 's primary structure is that it has a single tryptophan at amino acid position 214, which is situated within center of subdomain IIA.Since of the location of tryptophan amino acids in Human serum albumin and characteristic fluorescence properties.It provides protein chemists and biochemists with a profitable investigative method.Since the single tryptophan residue, fluorescence quenching tests, molar concentration determination, fluorescence lifetime studies, etc., several spectroscopic and fluorescence studies have been possible [6,11,15].
A spectroscopic technique, including (FT ̵ IR Spectroscopy, fluorescence spectroscopy, ultraviolet visible absorption spectroscopy, also investigates the molecular interactions between human serum albumin and certain compounds.Due to their high sensitivity, precision, rapidity and fast implementation, these methods have been used to investigate the interaction of small molecule substances and proteins. The aim of the present work is to study the interaction between Propylthiouracil and L-Thyroxine and human serum albumin (HSA).The importance of the study comes from the fact that Propylthiouracil and L-Thyroxine applying different supportive data including a binding parameter, quenching, and nature of binding.Therefore, Propylthiouracil and L-Thyroxine and transportation by proteins in the blood plasma becomes an important issue which requires investigations of the interaction mechanisms and determining the binding constant between Propylthiouracil and L-Thyroxine and HSA.In order to attain these objectives, UV-Vis absorption spectroscopy, fluorescence spectroscopy and FTIR spectroscopy were employed to carry out detailed investigation of Propylthiouracil and L-Thyroxine -HSA association.

Materials and Sample Preparations
Human serum albumin, Propylthiouracil (6-propyl-2-thiouracil), and L-Thyroxine were acquired from Sigma Aldrich Company and utilized without advance purification.
Human serum albumin (HAS), its molecular weight is 66.478 kilo Dalton (KDa).Propylthiouracil (6-propyl-2-thiouracile) with a Formula of chemicals (C H N OS) and a weight of molecule is 170.23 g.mol , L-Thyroxine with a Formula of chemicals (C 15 H 11 I 4 NO 4 ) and a weight of molecule is 776.87 g.mol , and Dulbecco's phosphate buffered saline is used to prepare the sample.As spectroscopic cell windows, optical grade silicon windows (NICODOM Ltd) are used.Sigma Aldrich Company bought these windows.
The information were collected utilizing samples within the shape of thin films of HSA, Propylthiouracil mixed with HSA, and L-Thyroxine mixed with HSA.

Preparation of HSA Stock Solution
In phosphate buffer Saline (pH 7.4) HSA was dissolved, with concentration of (80mg/2ml).

Preparation of Propylthiouracil Stock Solution
The molar concentration of stock propylthiouracil solution is 0.01M.The desired concentrations achieved by using the molarity dilution equation (M V = M V ). the following concentrations of propylthiouracil (0.35, 0.3, 0.25, 0.2 and 0.1 mM) were prepared in phosphate buffer solution (pH 7.4) by using the molarity dilution equation.

Preparation of L-Thyroxine Stock Solution
The molar concentration of stock L-Thyroxine solution is 0.01M.The desired concentrations achieved by using the molarity dilution equation (M V = M V ). the following concentrations of L-Thyroxine ( 0.35, 0.3, 0.25, 0.2 and 0.1 mM) were prepared in phosphate buffer solution (pH 7.4) by using the molarity dilution equation.

HSA-Propylthiouracil Solutions
The final concentration of HSA-propylthiouracil solutions was arranged to make the HSA-Hormone complex by mixing equal volumes of HSA and propylthiouracil.The HSA concentration was maintained at 40mg/ml in all samples.The propylthiouracil concentration in the last solution is (0.35, 0.3, 0.25, 0.2 and 0.1 mM) in any case.

HSA-L-Thyroxine Solutions
The final concentration of HSA-L-Thyroxine solutions was arranged to make the HSA-Hormone complex by combining the same amount of HSA and L-Thyroxine.The HSA concentration was maintained at 40mg/ml in all samples.In either case, the L-thyroxine concentration is (0.35, 0.3, 0.25, 0.2 and 0.1 mM) in the final solution.

Thin Film Preparations
Silicon windows (NICODOM Ltd) are used as windows for spectroscopic cells.The 100% line of a NICODOM silicon window indicates that the silicon bands do not show add-up absorption in the mid-IR region and can be efficiently subtracted.A silicon widow was dispersed with 20µl of each HSA-propylthiouracil or L-Thyroxine sample and the dissolvable was evaporated using an incubator, to get a clear thin film on the silicon window.At a room temperature of 25°C, both solutions were arranged at the same time.

Fourier Transform Infrared Spectroscopy (FT-IR)
The FT-IR measurements were acquired with a liquid KBr beam splitter and a nitrogen-cooled MCT detector on a Bruker IFS 66 / S spectrophotometer.During the measurements, the spectrometer was persistently washed with dry air.Information was analyzed using the software Bruker Uncertainties 66 / S and the origin program.
Within the wave number range of 400-4000 cm -1 , the absorption spectrum was obtained.To broaden the signal to noise ratio, a spectrum of 126 scans was taken as an average.Baseline correction, normalization, Fourier self-deconvolution and measurements of peak areas were performed by Optic Consumer (OPUS) software for all the spectra.The second derivative of the continuum was used to determine the peak positions [16,17].
The HSA, HSA-propylthiouracil or L-thyroxine complex infrared spectra were obtained within the 1200-1800 cm region.By subtracting the buffer solution absorption spectrum from the protein solution spectrum, the FT-IR spectrum of free HSA was procured.The differential spectra (protein and propylthiouracil or L-thyroxine-protein solution) were produced using the featureless locale of the 1800-2200 cm -1 protein solution as an inside standard solution for the net interaction effect [6,12,18].

UV-VIS Spectrophotometer (NanoDrop ND-1000)
Use the NanoDrop ND-1000 spectrophotometer, the absorption spectrum of HSA with propylthiouracil or L-Thyroxine was obtained.It is used to calculate, with high precision, the sample absorption spectrum is in the range of 220-750 nm.

Fluorospectrometer (NanoDrop 3300)
NanoDrop ND-3300 Fluoro-spectrophotometer at 25°C was used for the fluorescence measurements.One of the three solid-state LEDs produces the excitation source.The excitation source options include: 365 nm UV LED with greatest excitation, 470 nm Blue LED with excitation, and excitation from 500 to 650 nm white LED.

Results and Discussion
UV absorption spectrums are discussed and analyzed in the first section.The second section deals with the effects of Fluorescence spectroscopy.Spectroscopy charts and information analysis of Fourier transform infrared (FT-IR) are given in the last section.

UV-Absorption Spectroscopy and Binding Constant (K)
One of the successful techniques used to investigate the interaction between hormones and HSA was UV-absorption spectroscopy.The absorption spectra of different concentration Human Serum Albumin-Spectroscopic Approach of propylthiouracil and L-Thyroxine with fixed amount of HSA are shown in Figure 4 and Figure 5. On the other hand, the excitation was done at 210 nm, while the absorption was recorded at 280 and 277 nm.The figures show that the UV intensity of HSA increases as the concentration of propylthiouracil and L-Thyroxine increases, and the absorption peaks of this solutions showed moderate shifts.
The spectrum of pure hormones propylthiouracil and L-thyroxine showed little or no absorption of UV.These results support the assumption that the peak shifts between the free HSA solution and hormones are due to the interaction between hormones and HSA complexes.The absorption data on the following equation were treated utilizing linear double reciprocal plots [19].
Where A in the absence of ligand corresponds to the initial absorption of protein at 280 nm, A Q is the final absorption of the ligand protein, and A is the recorded absorption at different propylthiouracil or L-Thyroxine concentration (L . The double reciprocal plot of 1/( A 8 A ) vs. 1/L is Linear for HSA with different concentrations of propylthiouracil or L-Thyroxine as shown in the Figure 6

Fluorescence Spectroscopy
Fluorescence spectroscopy has long been one of the most useful biophysical procedures accessible to researchers considering the structure and work of biological molecules, especially proteins.
Protein comprises three fluorescent aromatic amino acids: tryptophan, tyrosine and phenylalanine.Tryptophan, which is the dominant intrinsic fluorophore, is normally present in proteins at around% mol.If it is ionized or near to an amino group, a carboxyl group, or a tryptophan residue, tyrosine fluorescence is almost completely quenched [25].In most studies, phenylalanine is not excited since phenylalanine's quantum yield in proteins is low, so emissions from phenylalanine residues in proteins are rarely observed [26].
So far we see in Figures 8 and 9 the fluorescence emission spectra of HSA at different concentration of propylthiouracil or L-Thyroxine showed that HSA had a strong fluorescence emission band at wavelength 394 nm.Moreover it can be seen that, increasing the concentration of propylthiouracil or L-Thyroxine from (0.1mM to 0.35mM) in the system with HSA causes a decrease in fluorescence intensity without changing the shape of the peaks.These results indicated that there was an interaction between propylthiouracil or L-Thyroxine and HSA.Dynamic quenching results from collisional between the fluorophore and the quencher and static quenching resulting from the formation of a dynamic ground state between the fluorophore and the quencher [19,27].

Determination of Stern-Volmer
The Stern-Volmer equation explains collisional quenching of fluorescence: Where K `a is the Stern-Volmer constant, and τ is the mean fluorescence life time of the fluorophore in the absence of quencher, and L is the quencher concentration.In addition to F and F, respectively, the intensity of fluorescence in the absence and presence of a quencher.
The K `a value indicates the importance of the accessibility of Fluorophores to the quencher.Although the meaning of K ^ gives an indication of the nature of the quencher's diffusion within the medium [28].
As shown in Figures 10 and 11, linear curves have been plotted according to the Stern-Volmer equation for propylthiouracil-HSA and L-Thyroxine-HSA complexes, respectively.From the curve slope obtained in Figures 10 and  11, the quenching constant K `a was obtained.The K `a values for propylthiouracil-HSA or L-Thyroxine-HSA complexes equal to (2.144 × 10 T L mol , 1.937 × 10 T L mol ), respectively.
The Stern-Volmer quenching constant can be represented as K `a = K ^t that we can obtain the value of K ^ using the fluorescence life time of 10 e s for HSA.we calculated the K ^ values for propylthiouracil-HSA and L-Thyroxine-HSA complexes equal to (2. 144 × 10 L mol S , 1.937 × 10 L mol S ) , respectively.There are larger than the maximum dynamic quenching constant for different quenchers with biopolymer ( 2 × 10 L mol S ).It could indicate that the quenching is not initiated by a dynamic collision, but by the formation of a complex that results in static quenching dominance [19,23].

Determination of Binding Constant (k) by
Fluorescence Spectroscopy The modified Stern-Volmer could be used when static quenching is dominant [29].
Where K is the binding constant of Propylthiouracil or L-Thyroxine with HSA, and can be obtained by plotting 1/(F -F) vs. 1/L of HSA hormones system as shown in Figure 12 and Figure 13.The obtained values of K for Propylthiouracil or L-Thyroxine -HSA complexes are ( 1.756 × 10 T M , 1.017 × 10 U M ), respectively.That agrees well for the value results provided via UV spectroscopy and confirms the successful function of static quenching.
We can see L-Thyroxine has higher binding constant of (1.013-1.017× 10 U M ) than propylthiouracil (1.659 -1.756 × 10 U M ).For the two molecules, the contrast is based on molecular structure variation.The binding constant of L-Thyroxine arises from the electrostatic interaction of the anionic phenolate group of thyroxine with cationic lysyl єamino groups in HAS [17,30].
Propylthiouracil is sulfhydryl compound, Sulfhydryl (-SH) is a functional group that may perform a role in the control of binding processes for several ligands.In secondary, tertiary or quaternary protein structures, disulfide (-S-S-) bonds bind the required amino acids together for functional purposes [6,31].Propyl group is also needed for the binding of propylthiouracil to HSA.This binding is strengthened by hydrophobic interaction between the propyl group in propylthiouracil and a polar substituents on HSA.

Fourier Transform Infrared (FTIR) Spectroscopy
FTIR spectroscopy is a sensitive and useful technique which may be relevant to protein structure investigations and studies.It is useful non-destructive, requires less planning for samples, and can be used in a wide range of conditions.It provides data on the protein content in the secondary structure.This could arise from the amide band that comes from the vibrations of the groups around the protein peptide.The attachment between peptides involves changes in hydrogen bonding.When drugs and globular proteins such as HSA are associated, this may alter the amide modes' vibrational frequency [23,32].
The FTIR was used in this research study to investigate changes in the HSA secondary structure after the binding of propylthiouracil and L-Thyroxine to HSA.Infrared protein spectral data is commonly interpreted in terms of structural unit vibrations.These repeated units give rise to nine characteristic bands of infrared absorption, called A, B, and I-VII amide.This is a product of the groups vibrating around the protein peptide.
Protein structure analysis by FTIR spectroscopy is mainly performed using the amide Ι, ΙΙ and ΙΙΙ band.With a significant contribution from C=O stretching vibration and a small contribution from C-N stretching vibration, Amide I absorption is directly connected to the backbone conformation.The absorption of amide Ι occurs in 1600 − 1700 cm .The amide ΙΙ absorption results both from N − H bending vibration and C − N stretching vibration.The absorption of this band occurs in 1480 − 1600 cm .Amide ΙΙΙ absorption occurs in 1220 − 1330 cm .This band originates from the C − N stretching vibration as well as N − H in-plane bending vibration, with weak contribution from C − C stretching and C = O in-plane bending vibration [19].
The peak positions of HSA with different propylthiouracil and L-Thyroxine concentrations for amide Ι, ΙΙ and ΙΙΙ regions are listed in Table 1, and Table 2, respectively.For HSA-propylthiouracil system, the amide Ι bands of HSA infrared spectrum shifted as listed in table 1: 1613 to 1609 cm , 1627 to 1625 cm , 1641 to 1639 cm , 1658 to 1656 cm , 1675 to 1674 cm , 1691 to 1689 cm after interaction with propylthiouracil.In amide ΙΙ region of the peak positions have shifted in the following order: 1514 to 1513 cm , 1530 to 1528 cm , 1548 to 1546 cm , 1564 to 1565 cm , 1582 to 1580 cm , 1598 to 1593 cm .In amide ΙΙΙ region of the peak position have shifted in the following order: 1224 to 1225 cm , 1247 to 1242 cm , 1264 to 1266 cm , 1293 to 1299 cm , 1308 to 1307 cm .
For HSA-L-Thyroxine interaction, the amide Ι bands of HSA infrared spectrum shifted as listed in table 2: 1613 to 1609 cm , 1627 to 1625 cm , 1641 to 1639 cm , 1658 to 1656 cm , 1675 to 1674 cm , 1691 to 1689 cm after interaction with propylthiouracil.In amide ΙΙ region of the peak positions have shifted in the following order: 1514 to 1513 cm , 1530 to 1528 cm , 1548 to 1546 cm , 1564 to 1565 cm , 1582 to 1580 cm , 1598 to 1593 cm .In amide ΙΙΙ region of the peak position have shifted in the following order: 1224 to 1225 cm , 1247 to 1242 cm , 1264 to 1266 cm , 1293 to 1299 cm , 1308 to 1307 cm .
Changes in peak positions and peak shapes have explained that the secondary structures of HSA have been influenced by the interaction of propylthiouracil or L-thyroxine with HSA.
The difference spectra for [ ( HSA+ Propylthiouracil or L-Thyroxine)-(protein)] were collected in able to monitor the differences in intensity of these vibrations.The results are display in Figures 16,17,18 and 19).For HSA-Propylthiouracil interaction, Figure 16 shows FTIR spectra (top two curves and difference spectra of HSA and its complexes with different propylthiouracil concentrations in the amide Ι and amide ΙΙ regions. In the amide Ι region, a strong negative feature appears at 1654 cm , In amide ΙΙ a strong negative feature appears at 1545 cm .for amide ΙΙΙ two negative features appear at 1308 cm and 1243 cm as shown in Figure 17.These features were detected at a low concentration of Propylthiouracil (0.2mM).
For HSA-L-Thyroxine interaction, Figure 18 shows FTIR spectra (top two curves and difference spectra of HSA and its complexes with different L-Thyroxine concentrations in the amide Ι and amide ΙΙ regions.
In the amide Ι region, a strong negative feature appears at 1656 cm , In amide ΙΙ a strong negative feature appears at 1545 cm .for amide ΙΙΙ two negative features appear at 1318 cm and 1242 cm as shown in Figure 19.These features were observed at low L-Thyroxine concentration (0.3mM .
The C-O stretching vibration in propylthiouracil has main contribution at 1627 cm and at 1 626 cm .The FTIR bands in amide ΙΙΙ of propylthiouracil assigned to C-N stretching mode of vibrations [33].
In dehydrated films, in order to obtain quantitative data of the protein secondary structure for free HSA, HSA-Propylthiouracil, HSA-L-Thyroxine complexes are calculated from the amide I, II and III band shape.In the range of (1700-1600 cm ), ( 1600-1480 cm ), and (1330-1220 cm ) , baseline correction was performed to obtain amide bands I, II and III.For the purpose of increasing spectral resolution and thus estimating the numbers, location and each component band's area, to such three ranges, Fourier self-deconvolution and second derivative were then applied respectively.To bring the best Lorentzian-shaped curves to match the original HSA spectrum, the curve-fitting method has been done by origin.
In keeping with the frequency of its maximum, Amide I, II and III regions were allocated to secondary structures after Fourier self-deconvolution was applied.
The decrease of the α-helical percentage with the increase of propylthiouracil or L-thyroxine concentration is evident and this trend is consistent for three Amide regions.For the β-sheet the relative percentage increased with the increase of propylthiouracil or L-thyroxine concentration.

Figure 6 .
Figure 6.The plot of 1/(X 8 X vs. 1/7 for HSA with different concentrations of propylthiouracil. Quenching Constants (Y Z[ ) and the Quenching Rate Constant of Biomolecule (Y \ )Fluorescence quenching includes any type that reduces the fluorescence intensity of a sample.Different mechanisms, generally divided into dynamic quenching and static quenching, may cause fluorescence quenching.

Figure 16 .Figure 17 .
Figure 16.FTIR spectra (top two curves and difference spectra of HSA and its complexes with different propylthiouracil concentrations in the region of 1800-1500 (% .

Figure 18 .
Figure 18.FTIR spectra (top two curves and difference spectra of HSA and its complexes with different L-Thyroxine concentrations in the region of 1800-1500 (% .

Figure 19 .
Figure 19.FTIR spectra (top two curves) and difference spectra of HSA and its complexes with different L-Thyroxine concentrations in the region of 1330-1220 (% .

Figure 20 .
Figure 20.Second-derivative resolution and curve-fitted area of amide I (1700-1612(% ) and secondary structure assessment of the free human serum albumin.

Figure 21 .Figure 22 .
Figure 21.Second-derivative resolution and curve-fitted area of amide I region (1700-1612(% ) and secondary structure assessment of the 0.25mM concentration of HSA-Propylthiouracil complexes.

Figure 23 .
Figure 23.Second-derivative resolution and curve-fitted area of amide I region (1600-1480 (% ) and secondary structure assessment of the 0.25mM concentration of HSA-Propylthiouracil complexes.

Figure 24 .
Figure 24.Second-derivative resolution and curve-fitted area of amide I (1330-1220(% ) and secondary structure assessment of the free human serum albumin.

Figure 25 .
Figure 25.Second-derivative resolution and curve-fitted area of amide I region (1330-1220 (% ) and secondary structure assessment of the 0.25mM concentration of HSA-Propylthiouracil complexes.

Figure 26 .
Figure 26.Second-derivative resolution and curve-fitted area of amide II (1600-1480(% ) and secondary structure assessment of the free human serum albumin.

Figure 27 .
Figure 27.Second-derivative resolution and curve-fitted area of amide II region (1600-1480 (% ) and secondary structure assessment of the 0.25mM concentration of HSA-Propylthiouracil complexes.

Figure 28 .
Figure 28.Second-derivative resolution and curve-fitted area of amide III (1330-1220(% ) and secondary structure assessment of the free human serum albumin.

Figure 29 .
Figure 29.Second-derivative resolution and curve-fitted area of amide III region (1330-1220 (% ) and secondary structure assessment of the 0.25mM concentration of HSA-Propylthiouracil complexes.
O 10 T M 2.144 O 10 T L mol 1.659 O 10 T M L-Thyroxine 1.017 O 10 U M 1.937 O 10 T L mol 1.013 O 10 U M

Table 1 .
Band assignments in the absorbance spectra of HSA with different Propylthiouracil concentrations for amide p, pp and ppp regions.

Table 2 .
Band assignments in the absorbance spectra of HSA with different L-Thyroxine concentrations for amide p, pp and ppp regions.

Table 3 .
Secondary structure determination for amide I, II and III regions in HSA and its propylthiouracil complexes.

Table 4
Secondary structure determination for amide I, II and III regions in HSA and its L-Thyroxine complexes.

Table 5 .
Propylthiouracil and L-Thyroxine with HSA comparison.