Thermodynamic Study of Human Serum Albumin upon Interaction with Ytterbium (III)

Abstract

Complexation reaction between Yb³⁺ and human serum albumin is examined using isothermal titration calorimetry (ITC). The extension solvation theory was used to reproduce the enthalpies of HAS + Yb³⁺ interactions over the whole range of Yb³⁺ concentrations. The binding parameters recovered from this model were attributed to the structural change of HSA. The 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 significantly in excess of their aqueous solubilities [1, 2]. Serum albumin, a highly abundant extracellular protein in blood plasma and tissue fluids, 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. The fascinating binding properties of albumin, a 66 kDa monomer, have been studied for over 40 years. These investigations have been very fruitful but hampered by the complexity of the protein, which has multiple binding sites and is known to be rather flexible [3, 4].

When blood is unavailable, plasma expanders are commonly used to treat patients with significant 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]. The 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].

The most important binding sites on HSA are sites I and II, which are also called warfarin binding sites and benzodiazepine binding sites [11]. The principal regions of ligand binding sites of albumin are located in hydrophobic cavities in subdomains IIA and IIIA, which exhibit similar chemistry. The IIIA subdomain is the most active in accommodating many ligands, such as digoxin, ibuprofen, and tryptophan [12].

The 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 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 fluorescence 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=66411gr/mol), Tris salt, and Yb³⁺ ions were obtained from sigma chemical Co. The 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 μM at 300 K and 69.7 μM at 310 K). Injection of Yb³⁺ solution into the perfusion vessel was repeated 29 times, with 10 μL per injection. The calorimetric signal was measured by a digital voltmeter that was part of a computerized recording system. The heat of each injection was calculated by the “Thermometric Digitam 3” software program. The heat of dilution of the Yb³⁺ solution was measured as described above except that HSA was excluded. The 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]:𝑞=−𝑞𝛿_max𝜃𝐵𝑥^𝐵−𝛿^𝜃𝐴𝑥^𝐴𝐿_𝐴+𝑥^𝐵𝐿_𝐵−𝛿^𝜃𝐴𝑥^𝐴𝐿_𝐴+𝑥^𝐵𝐿_𝐵𝑥^𝐵,(1)𝑞 is the heat of Yb³⁺ + HSA interaction and 𝑞_max represents the heat value upon saturation of all HSA. The parameters 𝛿^𝜃𝐴 and 𝛿^𝜃𝐵 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. 𝑝>1 or 𝑝<1 indicates positive or negative cooperativity of a macromolecule for binding with a ligand, respectively; 𝑝=1 indicates that the binding is noncooperative. 𝑥^𝐵 can be expressed as follows:𝑥^𝐵=𝑝𝑥^𝐵𝑥_𝐴+𝑝𝑥_𝐵.(2)

We can express 𝑥_𝐵 fractions as the total Yb³⁺ concentrations divided by the maximum concentration of the Yb³⁺ upon saturation of all HSA as follows:𝑥_𝐵=Yb³⁺Yb³⁺_max,𝑥_𝐴=1−𝑥_𝐵,(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. 𝐿_𝐴and 𝐿_𝐵 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, 𝑞_dilut, as follows:𝐿_𝐴=𝑞_dilut+𝑥_𝐵𝜕𝜕𝑞𝑥^dilut_𝐵,𝐿^𝐵=𝑞_dilut+𝑥_𝐴𝜕𝜕𝑞𝑥^dilut_𝐵.(4)

The heat of Yb³⁺ + HSA interactions, 𝑞, was fitted to (1) across the whole Yb³⁺ compositions. In the fitting procedure, p was changed until the best agreement between the experimental and calculated data was approached (Figure 1). The optimized 𝛿^𝜃𝐴 and 𝛿^𝜃𝐵 values are recovered from the coefficients of the second and third terms of (1). The small relative standard coefficient errors and the high 𝑟² values (0.999) support the method. The binding parameters for Yb³⁺ + HSA interactions recovered from (1) was listed in Table 1. The agreement between the calculated and experimental results (Figure 1) is striking and gives considerable support to the use of (1). 𝛿^𝜃𝐴 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. 𝛿^𝜃𝐵 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 nonspecific interactions in that the nonspecific 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. 𝑝 value for Yb³⁺+ HSA interactions at 300 and 310 K is 1, indicating that the interaction is noncooperative.

Table 1 

Binding parameters for HAS + Yb³⁺ interactions. The big association equilibrium constant values show that there is strong interaction between Yb³⁺ and HSA. The 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. The large and negative 𝛿^𝜃𝐵 values show that HSA has been destabilized in the high concentrations of Yb³⁺ ions.

Figure 1 

Comparison between the experimental heat (▲) at 300 K and (⚪) at 310 K for Yb3+ + HSA interactions and the calculated data (lines) via (1). The [Yb3+] are the concentrations of [YbCl3] solution in μM.

According to the recent data analysis method, a plot of (Δ𝑞/𝑞_max)𝑀₀ versus (Δ𝑞/𝑞)𝐿₀should be a linear plot by a slope of 1/g and the vertical intercept of 𝐾_𝑑/𝑔, which 𝑔 and 𝐾_𝑑, can be obtained [14–20]:Δ𝑞𝑞_max𝑀₀=Δ𝑞𝑞𝐿⁰1𝑔−𝐾𝑔^𝑑,(5) where 𝑔 is the number of binding sites, 𝐾_𝑑 is the dissociation equilibrium constant, 𝑀₀ and 𝐿₀ are concentrations of HSA and Yb³⁺, respectively, Δ𝑞=𝑞_max−𝑞, q represents the heat value at a certain Yb³⁺ ion concentration, and 𝑞_max represents the heat value upon saturation of all HSA. If 𝑞 and 𝑞_max are calculated per mole of HSA then the molar enthalpy of binding for each binding site (ΔH) will be Δ𝐻=𝑞_max/𝑔. Dividing the 𝑞_max amount (12500 and 17200 μJ equal to 92.34 and 137.09 kJ mol⁻¹) by 𝑔=3, therefore, gives Δ𝐻=30.78and 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 (Δ𝐺^∘) should be calculated according to (6), whose value can be used in (7) for calculating the change in standard entropy (Δ𝑆^∘) of binding process:Δ𝐺^∘=−𝑅𝑇ln𝐾_𝑎,(6)Δ𝐺^∘=Δ𝐻^∘−𝑇Δ𝑆^∘,(7) where 𝐾_𝑎 is the association binding constant (the inverse of the dissociation binding constant, 𝐾_𝑑). The 𝐾_𝑎 values are obtained as 5.59 × 10⁵ and 9.17 × 10⁵ L·mol⁻¹ at 300 and 310 K, respectively Hence,𝑇=3Δ0𝑆0_∘KΔ𝐺^∘=−33kJmol⁻¹,𝑇=310K=0Δ.2𝐺1_∘kJmol⁻¹K⁻¹,Δ𝑆_∘=−34.24kJmol⁻¹,=0.25KJmol⁻¹K⁻¹.(8)

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 Δ𝐺^∘, Δ𝐻^∘, and Δ𝑆^∘ indicate that the process is endothermic and entropy driven. This 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

The financial support of the Islamic Azad University of Takestan is gratefully acknowledged.