curcumin with human serum albumin: Partial molar volume and partial molar isentropic compressibility studies

Accepted Manuscript Interaction of Methimazole / Curcumin with Human Serum Albumin: Partial Molar Volume and Partial Molar Isentropic Compressibility Studies Sadaf Afrin, Riyaz uddeen PII: DOI: Reference:

S0021-9614(15)00221-9 http://dx.doi.org/10.1016/j.jct.2015.07.001 YJCHT 4298

To appear in:

J. Chem. Thermodynamics

Received Date: Revised Date: Accepted Date:

25 April 2014 12 June 2015 1 July 2015

Please cite this article as: S. Afrin, R. uddeen, Interaction of Methimazole / Curcumin with Human Serum Albumin: Partial Molar Volume and Partial Molar Isentropic Compressibility Studies, J. Chem. Thermodynamics (2015), doi: http://dx.doi.org/10.1016/j.jct.2015.07.001

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1

Interaction of Methimazole / Curcumin with Human Serum Albumin: Partial Molar Volume and Partial Molar Isentropic Compressibility Studies

Sadaf Afrin, Riyazuddeen* Department of Chemistry, Aligarh Muslim University, Aligarh 202002, UP, India E-mail: [email protected] *Corresponding author ABSTRACT  Partial molar volume (
 ) and partial molar isentropic compressibility ( ) values of drugs

methimazole and curcumin in 25 µmol·kg-1 human serum albumin (HSA) solution have been evaluated using density and speed of sound data. The isentropic compressibility values  of curcumin /methimazole + HSA solutions have also been computed. The  values decrease with an increase in the molal concentration of drugs as well as with temperature. The variations of trends  for the studied drugs have been discussed in terms of structure breaking behavior  of drugs. The values of  and   have been interpreted in terms of electrostatic and

hydrophobic interactions operative in the solutions. Keywords: Human serum albumin, Methimazole, Curcumin, Isentropic compressibility, Partial molar volume, Partial molar isentropic compressibility 1. Introduction Human serum albumin (HSA) is the most abundant soluble protein in blood plasma. HSA provides a depot for many compounds, binds some ligands in a strained orientation providing their metabolic modification, renders potential toxins harmless transporting them to disposal sites, accounts for most of the anti-oxidant capacity of human serum, and acts as a NO-carrier [1]. HSA consists of 585 amino acids that form into three structurally similar α-helical domains. These domains are characterized by a common motif of 10 α-helices. Each domain can be divided into sub-domains A and B, which contain six and four α-helical, respectively [2-3]. The domain II and III of HSA contain two primary drug binding sites, known as Sudlow’s site I and site II [4]. Several additional sites were also observed [5-9]. Crystallographic structural analysis of HSA-ligand complexes can reveal the molecular details of drug

2 binding, clarifies the interpretation of drug binding data, and provide a valuable structural template to rationale interaction between drugs and HSA [6]. Study of the interaction between drugs and plasma proteins have been an interesting area of research in chemical biology and pharmacology [10]. Binding of a drug to albumin, results in an increased drug solubility in plasma, decreased toxicity, and protection against oxidation of the bound drug [11]. Drugs distribution is mainly controlled by HSA, because most drugs circulate in plasma and reach the target tissues by binding to HSA [12]. Therefore, drug binding to proteins such as HSA has become an important determinant of pharmacokinetics, e.g. prolonging in vivo half-life, restricting the unbound concentration, and affecting distribution and elimination of the drug [13]. The HSA is the most extensively studied protein, due to its lack of toxicity and immunogenicity make it an ideal candidate for drug delivery [14]. Additionally, HSA is known to accumulate in tumours, being taken up by tumour cells at increased levels compared to normal cells, and serves as carrier conjugate of various anticancer drugs viz, chlorambucil, paclitaxel and doxorubicin [15]. The main role of HSA is to maintain the colloid osmotic pressure in the blood and also plays a key role in the transport and deposition, distribution, and metabolism of several endogenous and exogenous substances such as fatty acids, nutrients, steroids, metal ions, hormones, enzymes, surfactants and a number of therapeutic drugs through the blood plasma to their molecular target [16-17]. Methimazole is an active metabolite of carboxy benzyl and is frequently used in the management of hyperthyroidism in humans [18-20]. It is proven to inhibit the production of new thyroid hormones and thus is effective in the treatment of hyperthyroidism. It is also taken before thyroid surgery or radioactive iodine therapy. The capability of serum albumins to bind aromatic and heterocyclic compounds depends largely on the existence of two major binding regions, namely Sudlow’s site I and site II, [21-22] which are located within specialized cavities in sub domains IIA and IIIA, respectively [23-24]. Curcumin, an antioxidant and anti-tumor agent, is the main constituent of the Indian spice turmeric. It has generated much excitement in the recent years as a potent antioxidant, with promising anti-inflammatory and anticarcinogenic properties [25-27]. There are a few reports on the binding of curcumin to HSA reported mainly by following absorption, fluorescence and circular dichroism methods [28-32]. The present work reports the speed of sound (u) and density (ρ) values of methimazole and curcumin in 25 µmol·kg-1 human serum albumin solutions as functions of drug concentration and temperatures of (298.15, 303.15, 308.15, 313.15, 318.15, 323.15, and 328.15) K. The isentropic compressibility, partial molar volume and partial molar isentropic compressibility values have been computed using the speed of sound and density data. The trends of variation of experimental and computed parameters with the variation in molal concentration of drug and temperature have been discussed in terms of electrostatic and hydrophobic interactions operative in solutions. The curcumin /

3 methimazole-HSA molecular interaction and their temperature dependence play an important role in the understanding of drug action. Such results can be helpful in predicting the absorption and transport of drugs across the biological membranes. Therefore, it may be interesting to investigate their properties at different drug concentration and temperature for understanding the mechanism of drug action.

Methimazole

Curcumin FIGURE 1. Structures of the drugs

2. Experimental 2.1 Chemicals Human serum albumin (fatty acid free and lyophilized), curcumin and methimazole were purchased from Sigma-Aldrich and used without further purification. All other reagents were of analytical grade. 2.2 Equipment and procedure The concentration of HSA was determined spectrophotometrically using Є1%1cm of 5.3 M-1cm-1 (HSA) at 280 nm by using UV-Visible Perkin Elmer Lambda 25 spectrophotometer. A stock solution of HSA (25 µmol·kg-1) in 0.02 mol·kg-1 sodium phosphate buffer (pH 7.4) was also prepared at room temperature. 50 ml stock solution of 0.02 mol kg-1 sodium phosphate buffer (pH=7.4) was prepared by mixing 15.50 ml of 0.02 mol kg-1 solution of Na2HPO4 and 4.50 ml of 0.02 mol kg-1 solution of NaH2PO4. Drug solutions were prepared by dissolving 5-10 mg of a drug in a known amount of HSA solution at (298.15±0.1K). All the solutions were kept in the dark and used soon after mixing the components. The density and speed of sound values of the solutions were measured at temperatures (298.15, 303.15, 308.15, 313.15, 308.15 and 323.15) K with an oscillating-tube digital density and sound velocity meter (DSA 5000M, Anton Paar, Austria). The temperature of water around the densimeter cell was controlled to within ±0.01 K. Before each series of measurements, the instrument was calibrated at (298.15, 303.15, 308.15, 313.15, 318.15 and 323.15) K with the triply distilled water and dry air. The uncertainties in

4 density and speed of sound measurements were within ±5×10-2 kg·m-3 and ±0.5 m·s-1, respectively. The reproducibility of the density and speed of sound values were found to be within ±1×103 kg·m-3 and ±0.1 m·s-1, respectively. The uncertainties in the concentrations of sodium phosphate buffer solution, HSA solution (in buffer solution) and drugs solutions (in HSA + buffer solution) were found to be within ±1×10-3 mol·kg-1, ±1×10-7 mol·kg-1 and ±1×10-4 mol·kg-1, respectively. Basic data of the studied pure compounds are summarized in Table 1, including the name of the compounds, their CAS number, molar mass, source, the purification methods, water content and purity. 3. Result and Discussion The density and speed of sound data of curcumin/methimazole-HSA solutions at different concentration of drug in 25 µmol·kg-1 HSA solution at temperatures (298.15, 303.15, 308.15, 313.15, 318.15 and 323.15) K have been listed in Tables 2 and 3, respectively. 3.1 Partial molar volumes The apparent molar volumes, VΦ, of drugs in 25 µmol·kg-1 HSA solution have been evaluated using the expression,
 = ⁄ − ( −  )/ 

(1)

where bB is the molality (mol·kg−1) of the solution; M is the relative molar mass of solute (kg·mol−1); and ρo, ρ are the densities (kg·m−3) of the solvent and solution, respectively. The apparent molar volume, VΦ, values of the methimazole and curcumin in HSA solution are listed in Table 4. The apparent molar volumes of drugs have been fitted to the following equation by the least-square method,
 =
 +  

(2)

where
 is the apparent molar volume at infinite dilution, which is also referred to as the partial molar volume of the solute, and Sv is the volumetric pair-wise interaction coefficient. The
 and Sv values of the drugs under investigation are presented in the Table 5. The partial molar volumes increase with increasing temperatures. This trend is also indicative of the existence of electrostatic interactions between the HSA and the drug molecule. The increase may be due to the release of some amount of water molecules from the hydration shell of the drug to the bulk with raising temperature [33]. The characteristic of the co-sphere depends upon the drug structure, size, shape and hydrophobicity of the drug. At the highest drug concentration, an association process of protein complexes is detected, possibly resulting in the clustering of drug molecules around the hydrophobic amino acid residues of the unfolded

5 polypeptide chain, which aggregates. The initial binding of drug monomers seems to take place via electrostatic interactions to the ionic sites of the protein molecules owing to opposite signs of the net electric charges macromolecule and drug. Once the specific binding sites are saturated, an additional hydrophobic adsorption is probably produced onto the hydrophobic cavities of the protein molecules with a certain expansion of complex structure, due to hydrophobic interactions, the main force behind complexation. The higher  values of curcumin have been ascribed to an expansion of the protein structure induced by drug binding to primary binding sites, which contributes to the increase in the number of available binding sites, allowing a major space in the interior of the protein to accommodate curcumin molecules. This behavior results in an increase of the complex size, as shown by dynamic light scattering experiment, and even leading to the possible formation of clusters of drug along the HSA molecule [34]. As the drug concentration increases, the size of the complex also increases which is assigned to a further expansion of the protein structure induced by drug binding as a result of protein destabilization. According to the simplistic continuous model [35] the expression for the partial molar volume of a solute at infinite dilution is given as follows:  =
 +
 +   

(3)

where VC is the volume of the cavity in the solvent enclosing a non interacting solute; VI is the interaction volume that represents a change in volume accompanying the switching on of solute–solvent interactions;   is the coefficient of isothermal compressibility of the solvent. The   RT term, which originates from the availability of the entire volume of the solution to the solute, describes the volume effect related to the kinetic contribution to the pressure of a solute molecule due to its translational degrees of freedom. The cavity volume, VC, consists of the van der Waals volume of the solute and the thermal volume, VT. With increasing temperature, waters solvating non-polar groups become highly oriented in an attempt to maximize their mutual hydrogen bonds within a restricted configurational space. Therefore, the solution becomes compressible and hence increases the volume. Drugs used in the solution are bulkier than water and can form numerous hydrogen bonding and electrostatic interactions with its neighboring solvent and co-solvent molecules. Consequently, despite its being engaged in solute-solvent interactions, methimazole and curcumin can still develop numerous interactions with protein molecules in the bulk. The creation of a cavity is by definition a positive contribution to the partial molar volume of a solute, whereas the attractive intermolecular solute-solvent interactions cause a negative contribution by shrinking the cavity. The results are also viewed in terms of the geometrical fit of the drug molecules in an ordered solvent. As the temperature of the solution is increased, cavities are produced in the ordered solvent environment,

6 resulting in the better fit of the complex structured solutes in the solvent. With increasing temperature the contribution from the drug-solvent binding is weakened and the partial molar volume of the drug compounds increases significantly with temperature. 3.2 Isentropic compressibilities The isentropic compressibility [36] is computed by the Newton Laplace expression:

 = 1⁄ 

(4)

The isentropic compressibility values of HSA-drug solutions as functions of molal concentration of the drug and temperature have been listed in Table 6. The isentropic compressibility values decrease with an increase in concentration of drugs. The isentropic compressibility values of HSA-curcumin solution is slightly higher than HSAMethimazole values which may be explained by the view presented by Masterton [37] and Hepler [38]. Masterton and Hepler have indicated that the aromatic ring causes little influence on water structure, presumably due to the counterbalancing effects arising from its hydrophobic and structure breaking character. The structure-breaking effect is due to a poor fit of the planar ring into the tetrahedral structure of water. However, the effect of substituents and the negative charge on the curcumin ion may disturb this apparent balance such that a weaker hydration of the anion can be expected as a result of the diffuse charge and the potential for hydrogen bond formation between water and phenolic OH group. 3.3 Partial molar isentropic compressibilities The apparent molar isentropic compressibilities,  , [39] have been calculated using the relation,

 = ( −  )⁄  + 


(5)

where bB is the molality of the solution (mol·kg−1); ρo is the density (kg·m−3) of the HSA solution; and κs (=1/(ρu2) and κo= 1/(ρouo2) are the isentropic compressibilities (m2·N−1) of the solution and solvent (HSAbuffer solution), respectively. The values of  listed in Table 7 have been fitted by the least-squares method with the following equation,   =  +  

(6)

7 

where  is the apparent molar isentropic compressibility at infinite dilution, which is also referred to as the partial molar isentropic compressibility, and is a measure of solute–solvent interactions. The 

experimental slope Sk represents solute–solute interactions in the solutions. The  and Sk values are listed in Table 8. Gekko and Noguchi [40] proposed a method to estimate hydration term and found that the compressibility is a function of the hydrophobicity of proteins. A property directly related to cavity and hydration is the hydrophobicity of proteins. The cavity would be mainly generated by imperfect packing of hydrophobic amino acid residues localized in the interior of the protein molecules, and the nonpolar surface would cause the decrease in hydration demonstrating that the more hydrophobic a protein is the more compressible it is. The partial molar compressibilities of HSA + methimazole and HSA + curcumin solutions are 5.067 and 16.287 bar-1.m3.mol-1, respectively at 298.15 K which represent that drug and HSA interactions are highly hydrophobic. Drug induced release of individual water molecules may contribute favorably or unfavorably to the net binding energetics depending on the location of the hydration site of protein [41]. The higher partial molar compressibility of HSA + curcumin solution is higher than that of the HSA + methimazole solution which may be due to their molecular structure causing an enhanced activity, which is directly related to their structure conformation and binding affinity. This is a result of a change in the environment of tryptophan and tyrosine residues upon drug binding, indicating a severe change in the region where these residues are located, pointing out a certain denaturation of the protein molecules. 4. Conclusion The density and speed of sound data have been used to evaluate the partial molar volume, isentropic compressibility and partial molar isentropic compressibility values to study the binding of methimazole and curcumin to human serum albumin. The obtained results used to draw certain conclusions about the nature of the molecular interactions involved in the binding and subsequent complex formation between HSA and methimazole/curcumin. The drug complexation involves a certain neutralization of the net protein charge and the protein conformational change. These results suggest that the interaction of the drug with HSA undergoes a significant change in its conformation. The hydrophobic interactions between the methimazole/curcumin and the protein play the predominant role in the complexation process, although the existence of electrostatic interactions is also noted. These studies are done to understand about the extent and strength of the interactions between plasma proteins and drugs which are required to determine the optimal dose of administration of these compounds and to avoid

8 irreversible structural alterations in protein molecules, which can lead to a loss of their biological activity and to side reactions complicating medical therapy. Acknowledgment One of the authors (SA) acknowledges to CSIR, New Delhi, India, for providing the financial assistance in form of Research Associateship (RA).

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10 TABLE 1 Compounds used in this study with their CAS number, molar mass, source, purification method, water content and mass fraction purity. Compound

CAS number

Human Serum Albumin

Molar mass

Source

Purification Method

Water content

Mass fraction Purity

70024-90-7 67 kDa

SigmaAldrich

without further <5.0 ppm purification (Karl Fisher)

≥0.99

Methimazole

60-56-0

114.17 g/mol

SigmaAldrich

without further N.D. purification

≥0.99

Curcumin

458-37-7

368.38 g/mol

SigmaAldrich

without further N.D. purification

≥0.94

N.D.: Not determined. TABLE 2 Densities, ρ·10-3/kg.m-3 as a function of the drug concentration at different temperatures and at pressure p = 0.1 MPa. bB/10−3 T/K mol.kg-1 298.15 303.15 308.15 313.15 318.15 323.15

(i) Methimazole in 25 µmol·kg-1 HSA 0.0

0.99972

0.99830

0.99666

0.99483

0.99280

0.99059

1.0

0.99974

0.99832

0.99669

0.99485

0.99283

0.99062

5.0

0.99976

0.99836

0.99673

0.99489

0.99285

0.99065

10.0

0.99978

0.99840

0.99677

0.99492

0.99288

0.99068

20.0

0.99982

0.99848

0.99683

0.99498

0.99295

0.99074

30.0

1.00002

0.99855

0.99690

0.99507

0.99301

0.99088

(ii) Curcumin in 25 µmol·kg-1 HSA 0.00

0.99972

0.99830

0.99666

0.99483

0.99280

0.99059

0.05

0.99975

0.99834

0.99668

0.99485

0.99282

0.99061

0.10

0.99977

0.99836

0.99669

0.99487

0.99284

0.99063

0.20

0.99980

0.99839

0.99672

0.99489

0.99287

0.99065

11 0.50

0.99985

0.99844

0.99679

0.99494

0.99291

0.99070

1.00

0.99990

0.99850

0.99684

0.99499

0.99295

0.99075

• Standard uncertainties, u: u(T) = ±0.01 K, u(p) = 10 kPa, u(ρ)= ±5·10-2 kg·m-3, u(concentration of buffer solution) = ±1·10−3 mol · kg−1 , u(concentration of HSA solution (in buffer solution)) = ±1·10−7 mol · kg−1, u(concentration of drug solution (in HSA+ buffer solution) = ±1·10−4 mol · kg−1bB = concentration of drug solution in (HSA+buffer solution) • Concentration of sodium phosphate buffer (pH 7.4)=0.02 mol·kg-1. • 50 ml stock solution of 0.02 mol kg-1 sodium phosphate buffer (pH=7.4) was prepared by mixing 15.50 ml of 0.02 mol kg-1 solution of Na2HPO4 and 4.50 ml of 0.02 mol kg-1 solution of NaH2PO4. TABLE 3 Speeds of sound, u/m.s-1 as a function of molal concentration of drug at different temperatures and at pressure p = 0.1 MPa. bB/10-3 T/K -1 mol·kg 298.15 303.15 308.15 313.15 318.15 323.15

(i) Methimazole in 25 µmol·kg-1 HSA 0.0

1500.46

1512.66

1523.37

1532.36

1539.87

1546.00

1.0

1500.51

1512.71

1523.40

1532.39

1539.90

1546.03

5.0

1500.60

1512.77

1523.47

1532.45

1539.95

1546.09

10.0

1500.66

1512.85

1523.53

1532.51

1540.00

1546.13

20.0

1500.79

1512.93

1523.62

1532.60

1540.10

1546.21

30.0

1500.94

1513.06

1523.74

1532.71

1540.21

1546.31

(ii) Curcumin in 25 µmol·kg-1 HSA 0.00

1500.46

1512.66

1523.37

1532.36

1539.87

1546.00

0.05

1500.49

1512.69

1523.39

1532.40

1539.89

1546.02

0.10

1500.51

1512.71

1523.42

1532.42

1539.92

1546.05

0.20

1500.55

1512.75

1523.46

1532.45

1539.96

1546.10

0.50

1500.68

1512.88

1523.59

1532.56

1540.08

1546.19

1.00

1500.75

1512.95

1523.66

1532.65

1540.16

1546.29

•

Standard uncertainties, u: u(T) = ±0.1 K, u(p) = 10 kPa, u(u) = ±0.5 m·s-1, u(concentration of buffer solution) = ±1 × 10−3 mol · kg−1 , u(concentration of HSA solution (in buffer solution)) = ±1 × 10−7 mol · kg−1, u(concentration of drug solution (in HSA+ buffer solution) = ±1 × 10−4 mol·kg−1

•

bB = concentration of drug in (HSA+buffer solution)

12 •

Concentration of sodium phosphate buffer (pH 7.4)=0.02 mol·kg-1

•

50 ml stock solution of 0.02 mol kg-1 sodium phosphate buffer (pH=7.4) was prepared by mixing 15.50 ml of 0.02 mol kg-1 solution of Na2HPO4 and 4.50 ml of 0.02 mol·kg-1 solution of NaH2PO4.

TABLE 4 Apparent molar volumes, Vɸ·10-6/m3·mol-1 as a function of the drug concentration at different temperatures. bB/10-3 T/K mol·kg-1 298.15 303.15 308.15 313.15 318.15 323.15

(i) Methimazole in 25 µmol·kg-1 HSA 1.0

114.110

114.272

114.449

114.670

114.894

115.150

5.0

114.120

114.275

114.455

114.675

114.899

115.165

10.0

114.125

114.279

114.462

114.682

114.910

115.175

20.0

114.130

114.285

114.470

114.690

114.920

115.185

30.0

114.170

114.290

114.480

114.699

114.930

115.200

(ii) Curcumin in 25 µmol·kg-1 HSA 0.05

367.872

368.190

369.204

369.883

370.638

371.464

0.10

367.965

368.306

369.246

369.906

370.650

371.457

0.20

368.053

368.403

369.291

369.945

370.670

371.551

0.50

368.175

368.519

369.339

369.967

370.787

371.614

1.00

368.237

368.733

369.367

370.073

370.843

371.656

13 TABLE 5 Least Least-squares fit coefficients of equation
 =
 +   at different temperatures


 ×106/ (m3·mol−1) (i) Methimazole in 25 µmol·kg-1 HSA T/K

Sv ×10-4/ (m3· mol−2.kg)

σ/ (m3·mol−1)

298.15

114.102

2.400

0.017

303.15

114.273

3.936

0.002

308.15

114.450

5.746

0.003

313.15

114.670

6.139

0.025

318.15

114.876

8.100

0.028

323.15

115.162

10.949

0.010

(ii) Curcumin in 25 µmol·kg-1 HSA 298.15

367.933

0.345

0.071

303.15

368.240

0.514

0.059

308.15

369.233

0.652

0.033

313.15

369.887

0.784

0.016

318.15

370.634

0.826

0.026

323.15

371.472

0.906

0.041

Table 6 Isentropic compressibilities, κs·10-11/ m2·N-1 as a function of the drug concentration at different temperatures bB/10-3 T/K mol·kg-1 298.15 303.15 308.15 313.15 318.15 323.15

(i) Methimazole in 25 µmol·kg-1 HSA 0.0

44.430

43.778

43.236

42.808

42.479

42.236

1.0

44.426

43.774

43.230

42.806

42.476

42.233

5.0

44.420

43.769

43.227

42.801

42.472

42.229

10.0

44.415

43.763

43.222

42.796

42.468

42.226

14 20.0

44.406

43.755

43.214

42.789

42.460

42.219

30.0

44.388

43.744

43.204

42.779

42.451

42.207

(ii) Curcumin in 25 µmol·kg-1 HSA 0.00

44.430

43.778

43.236

42.808

42.479

42.236

0.05

44.426

43.774

43.233

42.806

42.476

42.234

0.10

44.424

43.772

43.228

42.804

42.474

42.232

0.20

44.421

43.768

43.220

42.801

42.471

42.222

0.50

44.411

43.761

43.216

42.793

42.462

42.214

1.00

44.405

43.752

43.211

42.785

42.456

42.204

TABLE 7 Apparent molar isentropic compressibilities,  ·10-7/bar-1·m3. mol-1 as a function of drug concentration at different temperatures. bB/10-3 T/K mol·kg-1

298.15

303.15

308.15

313.15

318.15

323.15

(i) Methimazole in 25µmol·kg-1 HSA 1.0

5.0654

4.9981

4.9510

4.9065

4.8772

4.8601

5.0

5.0672

4.9983

4.9535

4.9069

4.8783

4.8611

10.0

5.0674

4.9985

4.9545

4.9075

4.8790

4.8620

20.0

5.0667

4.9987

4.9555

4.9085

4.8798

4.8629

30.0

5.0675

4.9564

4.9094

4.8806

4.8635

4.9989 -1

(ii) (Curcumin in 25µmol·kg HSA )10

-5

0.05

16.2830

16.0573

15.9014

15.7928

15.6823

15.6477

0.10

16.2860

16.0610

15.9090

15.7950

15.6910

15.6489

0.20

16.3042

16.0790

15.9140

15.7980

15.7056

15.6596

0.50

16.3130

16.0910

15.9210

15.8050

15.7143

15.6630

1.00

16.3260

16.1067

15.9355

15.8100

15.7212

15.6670

15 TABLE 8  Least-squares fit coefficients of equation  =  +   at different temperatures

  ×1011/

T/K

-1

3

-1

( bar ·cm ·mol ) (i) Methimazole in 25 µmol·kg-1 HSA

Sk × 10-5/ ( bar-1·cm3.mol-2·kg)

σ/ ( bar-1·cm3·mol-1)

298.15

5.067

0.706

0.081

303.15

4.998

1.684

0.072

308.15

4.953

2.100

0.093

313.15

4.907

2.405

0.080

318.15

4.877

2.911

0.089

4.861

3.450

0.095

323.15 -1

(ii) Curcumin in 25 µmol·kg HSA 298.15

16.287

0.043

0.008

303.15

16.061

0.039

0.007

308.15

15.904

0.032

0.003

313.15

15.794

0.027

0.002

318.15

15.690

0.025

0.009

323.15

15.649

0.020

0.005

45.5 298.15K 303.15K 308.15K 313.15K 318.15K 323.15K

45.0

44.0

-11

2

s/10 m N

-1

44.5

43.5

43.0

42.5

42.0 0

5

10

15 -3

m/10 mol.kg

20

25

30

-1

Figure 1: Isentropic compressibilities ( ) of the methimazole in 25 µmol·kg-1 HSA as a function of concentration at temperatures: ■, 298.15 K; ●, 303.15 K;▲,308.15 K; ▼,313.15 K; ♦,318.15 K and◄, 323.15 K.

17 Highlights

and

• • •

,

values of Methimidzole/Curcumin in HSA solutions have been determined. have been discussed in terms of electrostatic and hydrophobic interactions.

have been discussed in terms of structure breaking behavior of drugs.