Thermodynamic Study of Human Serum Albumin upon Interaction with

Hindawi Publishing Corporation Journal of Chemistry Volume 2013, Article ID 696394, 4 pages http://dx.doi.org/10.1155/2013/696394

Research Article Thermodynamic Study of Human Serum Albumin upon Interaction with Ytterbium (III) G. Rezaei Behbehani,1 L. Barzegar,1 M. K. Kiani Savad Koohi,2 M. Mohebbian,2 B. Samak Abedi,3 A. A. Saboury,4 and A. Divsalar4, 5 1

Chemistry Department, Faculty Of science, Islamic Azad University, Takestan Branch, Takestan, Iran Chemistry Department, Payame Noor University (PNU), Abhar, Iran 3 Department of Biology, Islamic Azad University, East Tehran Branch Tehran, Iran 4 Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran 5 Department of Biological Sciences, Tarbiat Moallem University, Tehran, Iran 2

Correspondence should be addressed to G. Rezaei Behbehani; [email protected] Received 16 December 2011; Accepted 3 April 2012 Academic Editor: Murat Senturk Copyright © 2013 G. Rezaei Behbehani et al. is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Complexation reaction between Yb3+ and human serum albumin is examined using isothermal titration calorimetry (ITC). e extension solvation theory was used to reproduce the enthalpies of HAS + Yb3+ interactions over the whole range of Yb3+ concentrations. e binding parameters recovered from this model were attributed to the structural change of HSA. e results show that Yb3+ ions bind to HSA with three equivalent affinity sites. It was found that in the high concentrations of the ytterbium ions, the HSA structure was destabilized.

1. Introduction HSA greatly increases the solubilising capacity of plasma for its cargo compounds, allowing them to be present at near millimolar concentrations that are signi�cantly in excess of their aqueous solubilities [1, 2]. Serum albumin, a highly abundant extracellular protein in blood plasma and tissue �uids, is a formidable multitasker. Due to its high concentration (around 0.6 mM in plasma) albumin makes a major contribution to the colloid osmotic pressure of plasma. e fascinating binding properties of albumin, a 66 kDa monomer, have been studied for over 40 years. ese investigations have been very fruitful but hampered by the complexity of the protein, which has multiple binding sites and is known to be rather �exible [3, 4]. When blood is unavailable, plasma expanders are commonly used to treat patients with signi�cant blood loss by restoring their circulatory volume [5]. HSA is naturally produced in the liver and secreted into the bloodstream at a high concentration [6, 7], where it binds a variety of

molecules [8]. e protein is composed of three homologous domains (I–III); each domain has two subdomains (A and B) possessing common structural elements [9]. It transports metals, fatty acids, cholesterol, bile pigments, and drugs. In general, albumin represents the major and predominant antioxidant in plasma, a body compartment known to be exposed to continuous oxidative stress. A large proportion of total serum antioxidant properties can be attributed to albumin [10]. e most important binding sites on HSA are sites I and II, which are also called warfarin binding sites and benzodiazepine binding sites [11]. e principal regions of ligand binding sites of albumin are located in hydrophobic cavities in subdomains IIA and IIIA, which exhibit similar chemistry. e IIIA subdomain is the most active in accommodating many ligands, such as digoxin, ibuprofen, and tryptophan [12]. e study of lanthanide series interactions with HSA protein in particular indicates the importance of the molecular shape of the complexes in addition to the suggested

2

Journal of Chemistry

hydrophobic, van der Waals, and electrostatic contributions. Ytterbium is a member of lanthanide series, originally known as rare earth metals. Ytterbium complex of tetraphenylporphyrin was used as �uorescence label of HSA [13–18]. In this work, we present the most comprehensive study on the interactions of Yb3+ ions with HSA for further understanding of the effects of Yb3+ ions on the stability and the structural changes of the HSA molecules.

2. Materials and Method Human serum albumin (HSA; MW = 66411 gr/mol), Tris salt, and Yb3+ ions were obtained from sigma chemical Co. e isothermal titration microcalorimetric experiments were performed with the four-channel commercial microcalorimetric system. Yb3+ solution (2 mM) was injected by use of a Hamilton syringe into the calorimetric titration vessel, which contained 1.8 mL HSA (75.2 𝜇𝜇M at 300 K and 69.7 𝜇𝜇M at 310 K). Injection of Yb3+ solution into the perfusion vessel was repeated 29 times, with 10 𝜇𝜇L per injection. e calorimetric signal was measured by a digital voltmeter that was part of a computerized recording system. e heat of each injection was calculated by the “ermometric Digitam 3” soware program. e heat of dilution of the Yb3+ solution was measured as described above except that HSA was excluded. e microcalorimeter was frequently calibrated electrically during the course of the study.

3. Results and Discussion We have shown previously that the heats of the ligand + HSA interactions in the aqueous solvent mixtures, can be calculated via the following equation [14–20]: 𝑞𝑞 𝑞 𝑞𝑞max 𝑥𝑥�𝐵𝐵 − 𝛿𝛿𝜃𝜃𝐴𝐴 󶀢󶀢𝑥𝑥�𝐴𝐴 𝐿𝐿𝐴𝐴 + 𝑥𝑥�𝐵𝐵 𝐿𝐿𝐵𝐵 󶀲󶀲

− 󶀢󶀢𝛿𝛿𝜃𝜃𝐵𝐵 − 𝛿𝛿𝜃𝜃𝐴𝐴 󶀲󶀲 󶀲󶀲𝑥𝑥�𝐴𝐴 𝐿𝐿𝐴𝐴 + 𝑥𝑥�𝐵𝐵 𝐿𝐿𝐵𝐵 󶀲󶀲 𝑥𝑥�𝐵𝐵 ,

(1)

𝑞𝑞 is the heat of Yb3+ + HSA interaction and 𝑞𝑞max represents the heat value upon saturation of all HSA. e parameters 𝛿𝛿𝜃𝜃𝐴𝐴 and 𝛿𝛿𝜃𝜃𝐵𝐵 are the indexes of HSA stability in the low and high Yb3+ concentrations, respectively. Cooperative binding requires that the macromolecule has more than one binding site since cooperativity results from the interactions between identical binding sites with the same ligand. If the binding of a ligand at one site increases the affinity for that ligand at another site, then the macromolecule exhibits positive cooperativity. Conversely, if the binding of a ligand at one site lowers the affinity for that ligand at another site, then the enzyme exhibits negative cooperativity. If the ligand binds at each site independently, the binding is noncooperative. 𝑝𝑝 𝑝 𝑝 or 𝑝𝑝 𝑝 𝑝 indicates positive or negative cooperativity of a macromolecule for binding with a ligand, respectively; 𝑝𝑝 𝑝𝑝 indicates that the binding is noncooperative. 𝑥𝑥�𝐵𝐵 can be expressed as follows: 𝑝𝑝𝑝𝑝𝐵𝐵 𝑥𝑥�𝐵𝐵 = . (2) 𝑥𝑥𝐴𝐴 + 𝑝𝑝𝑝𝑝𝐵𝐵

We can express 𝑥𝑥𝐵𝐵 fractions as the total Yb3+ concentrations divided by the maximum concentration of the Yb3+ upon saturation of all HSA as follows: 𝑥𝑥𝐵𝐵 =

󶁢󶁢Yb3+ 󶁲󶁲

󶁢󶁢Yb3+ 󶁲󶁲max

,

𝑥𝑥𝐴𝐴 = 1 − 𝑥𝑥 𝐵𝐵𝐵

(3)

[Yb3+ ] is the concentration of Yb3+ and [Yb3+ ]max is the maximum concentration of the Yb3+ upon saturation of all HSA. In general, there will be “g” sites for binding of Yb3+ per HSA molecule. 𝐿𝐿𝐴𝐴 and 𝐿𝐿𝐵𝐵 are the relative contributions due to the fractions of unbound and bound metal ions in the heat of dilution in the absence of HSA and can be calculated from the heats of dilution of Yb3+ in the buffer solution, 𝑞𝑞dilut , as follows: 𝐿𝐿𝐴𝐴 = 𝑞𝑞dilut + 𝑥𝑥𝐵𝐵 󶀥󶀥

𝜕𝜕𝜕𝜕dilut 󶀵󶀵 , 𝜕𝜕𝜕𝜕𝐵𝐵

𝐿𝐿𝐵𝐵 = 𝑞𝑞dilut + 𝑥𝑥𝐴𝐴 󶀥󶀥

𝜕𝜕𝜕𝜕dilut 󶀵󶀵 . 𝜕𝜕𝜕𝜕𝐵𝐵 (4)

e heat of Yb3+ + HSA interactions, 𝑞𝑞, was �tted to (1) across the whole Yb3+ compositions. In the �tting procedure, p was changed until the best agreement between the experimental and calculated data was approached (Figure 1). e optimized 𝛿𝛿𝜃𝜃𝐴𝐴 and 𝛿𝛿𝜃𝜃𝐵𝐵 values are recovered from the coefficients of the second and third terms of (1). e small relative standard coefficient errors and the high 𝑟𝑟2 values (0.999) support the method. e binding parameters for Yb3+ + HSA interactions recovered from (1) was listed in Table 1. e agreement between the calculated and experimental results (Figure 1) is striking and gives considerable support to the use of (1). 𝛿𝛿𝜃𝜃𝐴𝐴 value for Yb3+ + HSA interactions in the low concentrations of the metal ions at 300 and 310 K is positive, indicating that in the low concentrations of the metal ions the HSA structure is stabilized. 𝛿𝛿𝜃𝜃𝐵𝐵 value for Yb3+ + HSA interactions in the high concentrations of the metal ions at 300 and 310 K is negative, indicating that in the high concentrations of the metal ions the HSA structure is destabilized, resulting in an decrease in its activity. Destabilization by a ligand indicates that the ligand binds preferentially (either at more sites or with higher affinity) to the unfolded (denatured) enzyme or to a partially folded intermediate form of the enzyme. Such effects are characteristic of nonspeci�c interactions in that the nonspeci�c ligand binds weakly to many different groups at the protein, so that binding becomes a function of ligand concentration, which is increased through unfolding events. 𝑝𝑝 value for Yb3+ + HSA interactions at 300 and 310 K is 1, indicating that the interaction is noncooperative. According to the recent data analysis method, a plot of (Δ𝑞𝑞𝑞𝑞𝑞max )𝑀𝑀0 versus (Δ𝑞𝑞𝑞𝑞𝑞𝑞𝑞𝑞0 should be a linear plot by a slope of 1/g and the vertical intercept of 𝐾𝐾𝑑𝑑 /𝑔𝑔, which 𝑔𝑔 and 𝐾𝐾𝑑𝑑 , can be obtained [14–20]: Δ𝑞𝑞 Δ𝑞𝑞 1 𝐾𝐾 𝑀𝑀 = 󶀥󶀥 󶀵󶀵 𝐿𝐿0 − 𝑑𝑑 , 𝑞𝑞max 0 𝑞𝑞 𝑔𝑔 𝑔𝑔

(5)

where 𝑔𝑔 is the number of binding sites, 𝐾𝐾𝑑𝑑 is the dissociation equilibrium constant, 𝑀𝑀0 and 𝐿𝐿0 are concentrations of HSA


3 value can be used in (7) for calculating the change in standard entropy (Δ𝑆𝑆∘ ) of binding process:

o 18

Δ𝐺𝐺∘ = −𝑅𝑅𝑅𝑅 𝑅𝑅 𝑅𝑅𝑎𝑎 ,

16 14

Δ𝐺𝐺∘ = Δ𝐻𝐻∘ − 𝑇𝑇𝑇𝑇𝑇∘ ,

12

(7)

where 𝐾𝐾𝑎𝑎 is the association binding constant (the inverse of the dissociation binding constant, 𝐾𝐾𝑑𝑑 ). e 𝐾𝐾𝑎𝑎 values are obtained as 5.59 × 105 and 9.17 × 105 L⋅mol−1 at 300 and 310 K, respectively Hence,

10 ९ ঴+

(6)

8

𝑇𝑇𝑇𝑇𝑇𝑇 K

6

Δ𝐺𝐺∘ = −33 kJ mol−1 ,

Δ𝑆𝑆∘ = 0.21 kJ mol−1 K−1 ,

4

𝑇𝑇𝑇𝑇𝑇𝑇 K

2

Δ𝐺𝐺∘ = −34.24 kJ mol−1 ,

(8)

Δ𝑆𝑆∘ = 0.25 KJ mol−1 K−1 .

0

0

50

100

150

200

250

300

঴.

F 1: Comparison between the experimental heat (▴) at 300 K and (∘) at 310 K for Yb3+ + HSA interactions and the calculated data (lines) via (1). e [Yb3+ ] are the concentrations of [YbCl3 ] solution in 𝜇𝜇M. T 1: Binding parameters for HAS + Yb3+ interactions. e big association equilibrium constant values show that there is strong interaction between Yb3+ and HSA. e entropy driven of the interaction indicates that minimizing the number of hydrophobic side chains exposed to water is the principal driving force behind the folding process. e large and negative 𝛿𝛿𝜃𝜃𝐵𝐵 values show that HSA has been destabilized in the high concentrations of Yb3+ ions. Parameters

−1

𝐾𝐾𝑎𝑎 /L⋅mol p 𝛿𝛿𝜃𝜃𝐴𝐴 𝛿𝛿𝜃𝜃𝐵𝐵

ΔH/kJ mol−1 −1

ΔG/kJ mol

ΔS/kJ mol−1 K−1

T = 300 K 5

T = 310 K 5

5.59 × 10 ± 210 1 ± 0.01 29.04 ± 0.14 −53.22 ± 0.16

9.17 × 10 ± 210 1 ± 0.01 14.05 ± 0.14 −71.57 ± 0.16

0.21 ± 0.02

0.25 ± 0.02

30.78 ± 0.09 −33 ± 0.07

45.7 ± 0.09

−34.24 ± 0.07

and Yb3+ , respectively, Δ𝑞𝑞 𝑞 𝑞𝑞max − 𝑞𝑞, q represents the heat value at a certain Yb3+ ion concentration, and 𝑞𝑞max represents the heat value upon saturation of all HSA. If 𝑞𝑞 and 𝑞𝑞max are calculated per mole of HSA then the molar enthalpy of binding for each binding site (ΔH) will be Δ𝐻𝐻 𝐻𝐻𝐻max /𝑔𝑔. Dividing the 𝑞𝑞max amount (12500 and 17200 𝜇𝜇J equal to 92.34 and 137.09 kJ mol−1 ) by 𝑔𝑔 𝑔𝑔, therefore, gives Δ𝐻𝐻 𝐻 30.78 and 45.7 kJ mol−1 at 300 and 310 K, respectively. To compare all thermodynamic parameters in metal binding process for HSA, the change in standard Gibbs-free energy (Δ𝐺𝐺∘ ) should be calculated according to (6), whose

It means that the binding process is spontaneous resulted by entropic driven. All thermodynamic parameters for the interaction between HSA and Yb3+ ion are summarized in Table 1. All thermodynamic parameters of the complex formation including Δ𝐺𝐺∘ , Δ𝐻𝐻∘ , and Δ𝑆𝑆∘ indicate that the process is endothermic and entropy driven. is issue shows the predominant role of hydrophobic forces in the interaction between Yb3+ and HSA. Structure of HSA has a hydrophobic core in which side chains are concealed from water, which stabilizes the folded state, and polar side chains interact with surrounding water molecules.

Acknowledgment e �nancial support of the Islamic Azad �niversity of Takestan is gratefully acknowledged.

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