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

Complexation reaction between Yb and human serum albumin is examined using isothermal titration calorimetry (ITC). e extension solvation theory was used to reproduce the enthalpies of HAS + Yb interactions over the whole range of Yb concentrations. e binding parameters recovered from this model were attributed to the structural change of HSA. e results show that Yb 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.


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 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][14][15][16][17][18].In this work, we present the most comprehensive study on the interactions of Yb 3+ ions with HSA for further understanding of the effects of Yb 3+ ions on the stability and the structural changes of the HSA molecules.

Materials and Method
Human serum albumin (HSA; MW = 66411 gr/mol), Tris salt, and Yb 3+ ions were obtained from sigma chemical Co. e isothermal titration microcalorimetric experiments were performed with the four-channel commercial microcalorimetric system.Yb 3+ solution (2 mM) was injected by use of a Hamilton syringe into the calorimetric titration vessel, which contained 1.8 mL HSA (75.2 M at 300 K and 69.7 M at 310 K).Injection of Yb 3+ solution into the perfusion vessel was repeated 29 times, with 10 L 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 Yb 3+ solution was measured as described above except that HSA was excluded.e microcalorimeter was frequently calibrated electrically during the course of the study.

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][15][16][17][18][19][20]: is the heat of Yb 3+ + HSA interaction and  max represents the heat value upon saturation of all HSA.e parameters    and    are the indexes of HSA stability in the low and high Yb 3+ 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.  1 or   1 indicates positive or negative cooperativity of a macromolecule for binding with a ligand, respectively;  = 1 indicates that the binding is noncooperative. �  can be expressed as follows: We can express   fractions as the total Yb 3+ concentrations divided by the maximum concentration of the Yb 3+ upon saturation of all HSA as follows: [Yb 3+ ] is the concentration of Yb 3+ and [Yb 3+ ] max is the maximum concentration of the Yb 3+ upon saturation of all HSA.In general, there will be "g" sites for binding of Yb 3+ per HSA molecule.  and   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 Yb 3+ in the buffer solution,  dilut , as follows: e heat of Yb 3+ + HSA interactions, , was �tted to (1) across the whole Yb 3+ compositions.In the �tting procedure, p was changed until the best agreement between the experimental and calculated data was approached (Figure 1).e optimized    and    values are recovered from the coefficients of the second and third terms of (1).e small relative standard coefficient errors and the high  2 values (0.999) support the method.e binding parameters for Yb 3+ + 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).   value for Yb 3+ + 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.   value for Yb 3+ + 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. value for Yb 3+ + 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 (Δ/ max ) 0 versus (Δ/) 0 should be a linear plot by a slope of 1/g and the vertical intercept of   /, which  and   , can be obtained [14][15][16][17][18][19][20]: where  is the number of binding sites,   is the dissociation equilibrium constant,  0 and  0 are concentrations of HSA To compare all thermodynamic parameters in metal binding process for HSA, the change in standard Gibbs-free energy (Δ ∘ ) should be calculated according to (6), whose value can be used in (7) for calculating the change in standard entropy (Δ ∘ ) of binding process: Δ ∘  Δ ∘ − Δ ∘ , where   is the association binding constant (the inverse of the dissociation binding constant,   ).e   values are obtained as 5.59 × 10 5 and 9.17 ( It means that the binding process is spontaneous resulted by entropic driven.All thermodynamic parameters for the interaction between HSA and Yb 3+ ion are summarized in Table 1.All thermodynamic parameters of the complex formation including Δ ∘ , Δ ∘ , and Δ ∘ indicate that the process is endothermic and entropy driven.is issue shows the predominant role of hydrophobic forces in the interaction between Yb 3+ 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.
Δ   max − , q represents the heat value at a certain Yb 3+ ion concentration, and  max represents the heat value upon saturation of all HSA.If  and  max are calculated per mole of HSA then the molar enthalpy of binding for each binding site (ΔH) will be Δ   max /.Dividing the  max amount (12500 and 17200 J equal to 92.34 and 137.09 kJ mol −1 ) by   3, therefore, gives Δ  30.78 and 45.7 kJ mol −1 at 300 and 310 K, respectively.