Thermal Study of a Newly Synthesized Cu ( II ) Complex Binding to Bovine ββ-Lactoglobulin

We have investigated the interactions between ββ-lactoglobulin, BLG, and new synthesized Cu(II) complex (2,2′-dibipyridine Cu(II) chloride) using isothermal titration calorimetry (ITC) methods at different temperatures of 298 and 310K. e heats of BLG + Cu(II) interactions are reported and analyzed in terms of the extended solvation theory for calculation of binding and thermodynamic parameters of the interaction.e results suggested that binding of Cu(II) complex on BLG resulted in signi�cant changes on the tertiary structure and conformation of protein via increasing of hydrophobicity and inducing partially unfolded structure in BLG which has a good agreement with the solvation parameters recovered by the extended solvation model suggesting destabilization of the protein.


Introduction
Milk proteins consist of caseins (78.3%), whey proteins (19%), and miscellaneous proteins (2.7%) [1].Whey proteins are widely used in formulated foods because they have high nutritional value and excellent functional properties.Whey proteins, which include serum albumin, immunoglobulins, -lactalbumin, and -lactoglobulin (BLG), are more reactive because they dissolve in the serum.BLG, the major protein in whey, is responsible for most of the bioactive properties of whey proteins.Due to their emulsifying, interfacial, and gelation properties, tryptic peptides from whey proteins are of great interest to the food industry.BLG composed of 162 amino acid residues and two disul�de bonds (Cys66-Cys160 and Cys106-Cys119) belongs to the lipocalin protein family [1,2].At physiological pH, BLG is mostly found as dimers, and at pH values below 3.5 and above 7.5, the protein tends to be monomeric [3].BLG solutions form gels in various conditions, when the native structure is sufficiently destabilised to allow aggregation.As milk is a known allergen, manufacturers need to prove the presence or absence of -lactoglobulin to ensure that their labelling satis�es the requirements of the aforementioned directive.Food testing laboratories can use ELISA (enzyme-linked immunosorbent assay) methods to identify and quantify -lactoglobulin in food products [4][5][6][7][8][9][10][11].
Copper is an essential trace element for all biological organisms, from bacterial cells to humans.ere are eight negative charges on the surface of BLG in neutral pH conditions, which may be sites for binding positively charged metal ions [12,13].
Since BLG is one of the most important milk carrier proteins and has great ability for binding to different drugs, specially cancer chemotherapy drugs, then it may has a central role in the molecular pharmacology of drugs used in cancer chemotherapy [14].en, in the present study, we have decided to investigate the binding of a new synthesized Cu(II) complex (2,2′-dibipyridine Cu(II) chloride), with milk carrier protein of BLG at different temperatures of 298 (room temperature) and 310 K (physiologic temperature) (upper fever temperature) using calorimetric (ITC) methods of the extended solvation theory.

Materials and Method
e isothermal titration calorimetric experiments were carried out on a VP-ITC ultrasensitive titration calorimeter (MicroCal, LLC, Northampton, MA).e microcalorimeter consists of a reference cell and a sample cell of 1.8 mL in volume, with both cells insulated by an adiabatic shield.All solutions were thoroughly degassed before use by stirring under vacuum.e sample cell was loaded with BLG solution (47 M), and the reference cell contained NaCl solution.e solution in the cell was stirred at 307 rpm by the syringe (equipped with micropropeller) �lled with Cu(II) complex solution (1.5 mM) to ensure rapid mixing.e titration of protein with Cu(II) complex solution involved 30 consecutive injections of the copper solution, the �rst being 5 L, and the remaining ones of 10 L.In all cases, each injection was done in 6 s at 3-minute intervals.To correct the thermal effects due to Cu(II) complex dilution, control experiments were done in which identical aliquots were injected into the NaCl solution in the absence of BLG.e measurements were performed at constant temperatures of 298.00 and 310.00 ± 0.02 K, and the temperature was controlled using a Poly-Science water bath.e data were collected automatically, and the heat of interaction between BLG + Cu(II) complex was subtracted from heat of Cu(II) complex dilution.e measured heats of binding have been shown graphically in Figure 1.

Results and Discussion
It has been reported previously [15][16][17][18][19][20][21][22][23] that the heats of interactions of biopolymers with ligands (BLG-A + Cu(II) complex in this case) in the aqueous solvent (Cu(II) complex + water in the present case) mixtures can be reproduced via the following equation: e parameters    and    are the indexes of the BLG stability as a result of interaction with Cu(II) complex in the low and high Cu(II) complex concentrations, respectively.If the binding of ligand at one site increases the affinity for ligand at another site, the macromolecule exhibits positive cooperativity.Conversely, if the binding of ligand at one site lowers the affinity for ligand at another site, the protein exhibits negative cooperativity.If the ligand binds at each site independently, the binding is noncooperative.  1 or   1 indicates positive or negative cooperativity of macromolecule for binding with ligand, respectively;   1 indicates that the binding is noncooperative.  ′ can be expressed as follows: ′ is the fraction of the bound Cu(II) complex to the binding sites, and   ′  1 −   ′ is the fraction of unbound Cu(II) complex.We can express   fractions as the total Cu(II) complex concentrations divided by the maximum concentration of the Cu(II) complex upon saturation of all BLG-A as follows: [Cu(II)] is the concentration of Cu(II) complex, and [complex] max is the maximum concentration of the Cu(II) complex upon saturation of all BLG.  and   are the relative contributions of unbound and bound Cu(II) complex to the heats of dilution in the absence of BLG and can be calculated from the heats of dilution of Cu(II) complex in buffer,  dilut , as follows: e heats of Cu(II) complex + BLG interactions, , were �tted to (1) over the whole Cu(II) complex compositions.
In the procedure, the adjustable parameter () 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 binding parameters for Cu(II) complex + BLG interactions recovered from (1) were 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).
values were calculated to be one, at different temperatures, which indicates that there are not any cooperativity in four binding sites of BLG (Table 1).
values (Table 1) for BLG + Cu(II) complex at 298 and 310 K are −3.49and −9.95, respectively, indicating that T 1: Binding parameters for BLG + Cu(II) complex interaction recovered from ( 1) and ( 2) at different temperatures of 298 and 310 K.    indicates that the binding is noncooperative in the four binding sites.e negative values of    or    indicate that the low and high concentrations of Cu(II) complex destabilize the BLG structure.e interaction is strong as indicated by equilibrium association constants.e interaction is entropy driven, indicating that the hydrophobic forces are dominant in this interaction.

Parameters
K    K   /L mol − . ×  4 ± 4 .7 ×  5  in the low concentrations of Cu(II) complex, BLG structure has been destabilized to allow aggregation.Negative values for    and    at both of temperatures indicate that Cu(II) complex destabilizes BLG structures and is a good support for signi�cant structural changes of BLG due to interaction with Cu(II) complex, predicted by the extended solvation model.
For a set of identical and independent binding sites to provide the number of binding sites () and the dissociation binding constant (  ), a plot of (Δ/ max )  versus (Δ/)  should be a linear plot by a slope of / and the vertical intercept of (−  /): and   are the concentrations of BLG and Cu(II), respectively.While q represents the heat value at a certain   and  max indicates the heat value upon saturation of all BLG, Δ   max − .e linearity of the plot has been examined by different estimated values for  max to �nd the best value for the correlation coefficient.e small relative standard coefficient errors (±0.001) and the high   values (0.99999) indicate the best linear plot of (Δ/ max )  against (Δ/)  .If the optimized  max is calculated per mole of BLG, then the standard molar enthalpy of binding for each binding site will be Δ ∘   max /.e change of the standard Gibbs free energy of binding (Δ ∘ ) is determined by using the association binding constant (  ), obtained from the inverse of   value, in (6), where  is the gas constant and  is the absolute temperature: e change in standard entropy (Δ ∘ ) of this binding can be calculated as ( 7) All calculated thermodynamic parameters are reported in Table 1.

Conclusion
Negative values of    and    calculated from the extended solvation theory show that low and high concentrations of Cu(II) complex induced some structural changes in lactoglobulin which lead to destabilization of the protein.Previous reports represent that gelation of globular proteins results from an aggregation process, which is generally triggered by a conformational change of the protein induced by a modi�cation of solvent conditions.In the aggregation reactions, either or both covalent (inter-and intramolecular disulphide bonds) and noncovalent bonds (hydrophobic, hydrogen, and ionic interactions) are involved.Since thermal denaturation of BLG is characterized by changes in the secondary and tertiary structures, exposing hydrophobic residues to the solvent and aggregation process due to hydrophobic interactions may subsequently take place [24][25][26][27][28][29][30].
e above interpretations are in agreement with the solvation parameters recovered from the extended solvation model (Table 1), suggesting destabilization of BLG upon interaction with Cu(II) complex.

F 1 :
Comparison between experimental heats of interactions between BLG and copper ion at 25 (○) and 37 ∘ C (Δ) and calculated heats obtained from (1).