A Thermodynamic Study on the Binding of Human Serum Albumin with New synthesized Anticancer Pd(II) Complex

Introduction

Human serum albumin (HSA) is the most abundant protein in the systemic circulation, with HSA comprising 60% in plasma.  This protein can play a dominant role on the drug disposition and the efficiency. HSA has a high affinity to an extraordinarily diverse range of materials, such as drugs, metabolites, fatty acids and metal ions.^1-4  HSA can bind and carry through the bloodstream many drugs, which are poorly soluble in water and it is responsible for the maintenance of blood pH, the drug disposition and efficacy, and the contribution of colloid osmotic blood pressure.^4-6 The unique feature of albumin is its ability to bind a wide variety of compounds, mainly because of the availability of hydrophobic pockets inside the protein network and the flexibility of the albumins to adapt its shape.^7-8 The crystallographic analysis of HSA revealed that this protein is a single-chain 66 kDa protein, which is largely α-helical, and consists of three structurally homologous domains that assemble to form a heart-shaped molecule.Each domain is a product of two subdomains, which are predominantly helical and extensively cross-linked by several disulfide bridges.^9-11

The development of palladium anticancer drugs has not been promising and their design has mainly been based on the structure-activity relationship used for platinum anticancer drugs as well as good models for the analogous Pt(II) complexes in solution. Pd (II) complexes are expected to have lower kidney toxicity than cisplatin due to the inability of proteins in the kidney tubules to replace the tightly bound chelate ligands of Pd (II) with sulfydril groups.^12-14 Studies have shown that Pd (II) complexes are very effective in inhibiting proliferation in several tumor cell lines.^15-17

This work represents the most comprehensive study on the interactions between a new design anticancer drug of Pd₂Py₂ with carrier blood protein of HSA and provides new evidence for validity of the extended solvation model and more insights into the interactions of Pd₂Py₂ with HSA for further understanding of the effects of metal based drugs on the stability and the structural changes of carrier proteins.

Materials And Method

HSA was obtained from Sigma-Aldrich (Taiwan, China) and Pd₂Py₂was synthesized in our laboratory.¹⁷ Protein concentrations were determined from absorbance measurements at 277 nm in 1 cm quartz cuvettes. All other materials and reagents were of analytical grade, and solutions were made in 50 mM buffer phosphate using double-distilled water.

The isothermal titration calorimetric experiments were carried out on a VP-ITC ultra sensitive titration calorimeter (MicroCal, LLC, Northampton, MA). The microcalorimeter consists of a reference cell and a sample cell of 1.8mL in volume, with both cells insulated by an adiabatic shield. All solutions were thoroughly degassed before use by stirring under vacuum. The sample cell was loaded with HSA solution (60 M ) and the reference cell contained buffer solution. The solution inthe cell was stirred at 307 rpm by the syringe (equipped with micro propeller)filled withPd₂Py₂solution (1.2mM) to ensure rapid mixing. Injections werestarted after baseline stability had been achieved. The titration of HSA withPd₂Py₂ solution involved 30 consecutive injections of Pd₂Py₂ solution, thefirst injection was 5 μL and the remaining ones were 10 μL. In all cases, each injection wasdone in 6 s at 3-min intervals. To correct the thermal effects due to Pd₂Py₂ dilution, control experiments were done in which identical aliquotswere injected into the buffer solution with the exception of HSA.  In the ITC experiments, the enthalpy changes associated with processes occurring at a constant temperature are measured. The measurements were performed at a constant temperature of 27.0±0.02 ^◦C and the temperature was controlled using a Poly-Science water bath.

Result And Discussion

We have shown previously that the heart of the macromolecules + ligands interactions can be reproduced by Eq. 1in the aqueous solvent system.^18-29

qis the heat of HSA + Pd₂Py₂interaction at certain ligand concentrations and q_max represents the heat value upon saturation of all HSA. The parameters

exhibit the HSA + Pd₂Py₂ stability in the low and high Pd₂Py₂ concentrations respectively. The positive values of

show that HSA is stabilized by Pd₂Py₂. can be expressed as follows

[Pd₂Py₂]is the concentration of ligand after every injection and [Pd₂Py₂]_max is the maximum concentration of Pd₂Py₂ upon saturation of all HSA.The heats of HSA+Pd₂Py₂ interactions, q, were fitted to Eq.1 over the whole Pd₂Py₂compositions.  In the fitting procedure, p was changed until the best agreement between the experimental and calculated data was approached. If the binding of ligand at one site increases the affinity for ligand at another site, the macromolecule exhibits positive cooperativity (p>1). Conversely, if the binding of ligand at one site lowers the affinity for ligand at another site, exhibits negative cooperativity (p<1). If the ligand binds at each site independently, the binding is non-cooperative (p=1).L_A and L_B are the unbound and bound ligand contributions to the heats of dilution in the absence of HSA. L_A and L_Bcan be calculated from heats of dilution of Pd₂Py₂ in water, ,  as follows

The optimized

values are recovered from the coefficients of the second and third terms of Eq. 1. The small relative standard coefficient errors and the high r²values (0.99999) support the method (fig. 1). The binding parameters for HSA+Pd₂Py₂interactions recovered from Eq. 1 were listed in Table 2.

 

Table 1:  heats of HSA+ Pd2Py2 interaction, q , at 300 K  in 50mM phosphate buffer solution of pH=7, is the heat of dilution of  with water. The precision is ± 0.1 µJ or better. Click here to View table

 

Table 2: binding parameters for HSA+ interaction recovered from Eq. 1at pH=7. The positive value of  δθA show that stabilizes the HSA structure in the low concentration of Pd2Py2. Click here to View table

 

Figure 1: Comparison between the experimental heats, q, (O), for HSA+  interactions and calculated data (lines) via Eq. 1.  is concentration of  in μM at pH=7. Click here to View Figure

 

For a set of identical and independent binding sites, it is possibleto use Eq. (5) for calculation of K_d and ”g” in a very simple way as follows

Where . q represents the heat value at a certain ligand and biomolecule concentration.M₀ and L₀ are total concentrations of HSA and ligand, respectively.q_max represents the heat value upon saturation of all HSAmolecule.K_d is the dissociation equilibrium constant for the equilibrium

If q and q_max are calculated per mole of biomacromolecule then the molar enthalpy of binding for each binding site ( ) will be:

The standard Gibbs free energy, ∆G°, can be calculated from association constant 

Where  is the appearance association equilibrium constant as a function of Pd- anti-cancer concentration. Therefore for the first time, we managed to calculate  and  values with using one set of experimental data in one temperature. All thermodynamic parameters of ligand binding to HSA are summarized in Table 2.

Conclusion

It has been confirmed that the extended coordination model, via equation 1 will satisfactorily reproduce the heats of HSA+Pd₂Py₂ interaction. Prediction of number of binding sites on HSA molecule, structural changes, determining the binding enthalpies and associated binding constants for such a complicated system accurately, make this theory the most powerful one. and values are positive and negative respectively  (table 2), indicate that considerable conformational changes in HSA were  observed due to the interaction with  Pd₂Py₂.The negative value of  shows that the HSA structure is destabilized in the high concentration of Pd₂Py₂. The negativevalue of molar enthalpy (-17889kJ mol^-1 at 300 K) suggest that the binding process is only enthalpy-driven, indicating that electrostatic interaction is more important in the interaction.

Acknowledgements

Financial support from the Universities of Imam Khomeini (Qazvin) and Tehran are gratefully acknowledged. 

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