THERMODYNAMIC CHARACTERIZATION OF METAL PHTHALOCYANINES-HUMAN SERUM ALBUMIN INTERACTIONS

Abstract

The temperature dependence of the binding constants for human serum albumin and sulfonated metal-phthalocyanines were estimated by fluorescence spectroscopy. Stern-Volmer’s analysis and Chipman’s fits provided binding data for van’t Hoff calculations of the thermodynamic parameters governing these interactions. Results show that the formations of the HSA- phthalocyanine complexes are favorable processes by both the ΔH° and ΔS°. However, the formation of HSA-AIPcS₄ has a stronger dependence than HSA-ZnPcS ₄on ΔS°.

Introduction

Chemical thermodynamics enables one to predict the spontaneous direction of chemical reactions and to determine the driving forces governing the interactions (1–3). Two thermodynamic parameters of interest are the enthalpy change (ΔH) and the entropy change (ΔS). The enthalpy change is a measure of heat exchange between the system and surroundings under constant pressure conditions. The entropy measures the change in randomness or disorder associated with the chemical event. The Gibbs free energy change (ΔG) ultimately determines the direction of the reaction. The relationship between ΔG, ΔH, and ΔS is given by:

$$\mathrm{\Delta }\mathrm{G}=\mathrm{\Delta }\mathrm{H}-\mathrm{T}\mathrm{\Delta }\mathrm{S}$$ (1)

where T is the temperature measured in Kelvin. A given reaction proceeds spontaneously in the forward direction when ΔG is negative and in the reverse direction if positive. If ΔG is zero, then the reaction is said to be in a state of dynamic equilibrium. This steady state condition refers to a system wherein the rate of the forward reaction is exactly equal to the rate of the reverse reaction.

The following equation depicts the interaction between a protein and ligand:

$$\mathrm{P}+\mathrm{L}=\text{PL}$$ (2)

The reaction quotient is given by the ratio of product concentration to reactant concentration at any time during the course of the reaction:

$$Q=[\mathit{\text{PL}}]/[P][L]$$ (3)

For a reversible reaction that has not reached equilibrium, the relationship between the free energy and reaction quotient (Q) is given by:

$$\mathrm{\Delta }\mathrm{G}=\mathrm{\Delta }\mathrm{G}°+\text{RT ln }\mathrm{Q}$$ (4)

where R is equal to the gas constant (8.31 J K⁻¹ mol⁻¹). Under steady-state conditions ΔG is zero and the reaction quotient becomes the equilibrium constant (K_eq) resulting in an expression that allows the investigator to study the energetics of chemical binding:

$$\mathrm{\Delta }\mathrm{G}°=-RT\text{ ln }{K}_{\mathit{\text{eq}}}=\mathrm{\Delta }\mathrm{H}°-\mathrm{T}\mathrm{\Delta }\mathrm{S}°$$ (5)

The K_eq measures the “tightness of binding” between the protein and ligand, such that large values of K_eq indicate the formation of a strongly bonded protein-ligand complex (PL). A thorough analysis of the thermodynamic parameters will elucidate key information about forces that mediate binding (4). Protein-ligand binding is governed by a number of weak forces such as hydrogen bonding, electrostatic interactions, hydrophobic interactions and van der Waals Forces. The magnitude and sign of ΔH and ΔS will provide information associated with these forces. The compensation between these thermodynamic quantities leads to the analysis of an additional parameter called the heat capacity change under constant pressure, ΔC_p of a system. Large heat capacity changes are often linked to the removal of a hydrophobic surface from contact with water (5, 6).

Phthalocyanines are organic dyes that are similar in structure to porpryins and are under extensive investigation for their application in photodynamic therapy (PDT). PDT is a noninvasive technique for shrinking both malignant and non-malignant solid tumors. The technique works by introducing a dye-like compound to the tumor tissue and then activating it with light of appropriate energy. Once activated, the dye (photosensitizer) transfers some of its energy to triplet oxygen ³O₂, which is abundant in cancerous tissue, and converts it to singlet oxygen, ¹O₂. Highly reactive ¹O₂ is capable of disrupting a wide range of interactions critical to cell survival. Tissue subjected to a light activated photosensitizer will ultimately succumb to cell death via necrosis or apoptosis (7–11).

One of the key problems with the application of PDT is selectivity. FDA approved photosensitizer accumulates preferentially in cancerous-to-normal tissue at a current ratio of about 2:1. Human serum albumin (HSA) is widely recognized as a transport protein for various drugs including photosensitizers (12,13). Elucidating the nature of the interactions between HSA and various photosensitizers is critical to improving the selectivity of PDT. This work investigated the energetics of tetrasulfonic acids of aluminum phthalocyanine chloride (AIPcS₄) and zinc II phthalocyanine (ZnPcS₄) binding to HSA.

Experimental

Materials & Equipment

All glassware was scrupulously cleaned with detergent, soaked in 3.0 M HCl solution, and rinsed with distilled water prior to use. Adjustments to buffer pH were made using an Orion Expandable Ion Analyzer EA 920 pH meter. The pH meter was calibrated between two buffer solutions prior to measurements. Tetrasulfonic acids of aluminum phthalocyanine chloride (AIPcS₄) and zinc II phthalocyanine (ZnPcS₄) were purchased (Frontier Scientific, Logan, Utah). Photosensitizers should be handled only while wearing proper eye protection, safety gloves, and garments. All other chemicals were purchased (Sigma-Aldrich Corp., St. Louis, MO). A Varian Cary 100 spectrophotometer was used for UV/Vis measurements. The emission spectra were recorded with a Varian Cary Eclipse Bio-Melt Fluorescence Spectrophotometer using 10 mm pathlength cells.

Results and Discussion

An absorption spectrum of ZnPcS₄ was recorded with the UV/Vis spectrophotometer to determine the wavelength of maximum absorbance (λ_max = 636 nm). This wavelength was used to excite the photosensitizer and record a fluorescence emission spectrum. Figure 1 shows the fluorescence (middle curve) of the photosensitizer as well as the emission spectrum for HSA (bottom curve). HSA did not produce a signal over the spectral region shown in Figure 1 because the only intrinsic florophore in the protein is a single tryptophan residue (Tip²¹⁴), which emits at around 350 nm. Note the large increase in emission associated with combining the HSA and the photosensitizer (top curve). The large signal observed at about 688 nm suggests the formation of a complex that substantially enhances the emission characteristics of the photosensitizer. This wavelength was used to measure the binding of HSA to the photosensitizer over a range of several temperatures.

Figure 1.

Fluorescence signal of HSA (bottom curve), ZnPcS₄ (middle curve), and ZnPcS₄ mixed with HSA (top curve).

The concentration of ZnPcS₄ was held constant at 800 nM and titrated with HSA. A fluorescence spectrum was recorded over the temperature range of 20.0 to 55.0°C in increments of 5.0°C. The signal at 688 nm continued to increase with increasing concentration of the protein and trended toward an asymptotic point that indicated the completion of reaction. Figure 2 shows a Chipman’s Fit (Equation #6) of the emission maximum verses the concentration of HSA at 40.0°C. All data recorded for the titration of ZnPcS₄ with HSA were fitted similarly to obtain temperature dependent dissociation constants, K_d. The natural log of binding constants, K_B were calculated (1/K_d) and plotted as a function of inverse temperature for a van’t Hoff analysis. The dissociation constants where determined by Chipman’s Analysis (14):

$$F=\frac{F_{\mathit{0}}+F_{\mathrm{\infty }}{K}_d[L]}{1+K_d[L]}$$ (6)

where F₀, F_∞, and [L] represents the signal in the absence of protein, the asymptotic signal, and the protein concentration respectively.

Figure 2.

Chipman’s fit for the fluorescence of 800 nM ZnPcS₄ titrated with HSA as a function of temperature.

It can be seen in Table 1 that an increase in the dissociation constant occurs with increasing temperature. This is expected because high temperatures disrupt the weak forces responsible for the formation of the protein-ligand complex.

Table 1.

The data for ZnPcS₄ titrated with HSA as a function of temperature. Data for van’t Hoff Plot of ZnPcS₄-HSA Interaction.

T (°C)	1/T (K−1) • 10−3	Kd	ln(KB)
20	3.41	0.541	14.430
25	3.36	0.576	14.367
30	3.30	0.746	14.108
35	3.25	0.748	14.106
40	3.19	0.866	13.959
45	3.14	0.994	13.822
50	3.09	1.24	13.600
55	3.05	1.35	13.515

A van’t Hoff plot for HSA-ZnPcS₄ binding is shown in Figure 3. The van’t Hoff equation is in the form of a linear curve (Equation 7) with the enthalpy change and entropy change provided by the slope (−ΔH°/R) and the y-intercept (ΔS°/R) respectively.

$$\text{ln }{\mathrm{K}}_{\mathrm{B}}=\frac{-\mathrm{\Delta }H°}{\mathit{\text{RT}}}+\frac{\Delta S°}{R}$$ (7)

Figure 3.

The van’t Hoff analysis of ZnPcs₄, binding with HAS.

Using the gas constant R = 8.314 J mol⁻¹ K⁻¹ (1.987 cal mol⁻¹ K⁻¹), the enthalpy change and entropy change for the formation of HSA-ZnPcS₄ complex are −21.2 ± 0.2 kJ/mol and 47.9 ± 0.5 J mol⁻¹ K⁻¹ respectively.

A similar experiment was conducted for investigating the interaction of HSA with AIPcS₄. The data for this system is provided in Table 2. A Stern-Volmer analysis was used to fit this data because a decrease in the emission signal for HSA (quenching Trp²¹⁴) was observed as a function of ligand concentration. The Stern-Volmer plot (Equation 8) is shown in Figure 4. The quenching or binding constant is given by:

$$\frac{{\mathrm{F}}_0}{\mathrm{F}}=\frac{1}{1+{\mathrm{K}}_{\mathrm{B}}[L]}$$ (8)

where F₀ and F are the fluorescence intensities in the absence and presence of quenching ligand respectively (15–17).

Table 2.

The data for AIPcS₄ titrated with HSA as a function of temperature. Data for van’t Hoff Plot of AIPcS₄-HSA Interaction.

T (K)	1/T (K−1)•10−3	Kd (µM)	ln(KB)
293	3.41	0.847	13.982
298	3.36	0.885	13.938
303	3.30	0.855	13.972
308	3.25	0.909	13.910
313	3.19	0.952	13.865
318	3.14	0.971	13.845
323	3.09	1.02	13.796
328	3.05	1.13	13.693

Figure 4.

Stern-Volmer analysis of HSA-AIPcS₄ interaction at 55.0 °C.

The enthalpy and entropy changes obtained from the van’t Hoff analysis shown in Figure 5 are −5.9 ± 0.1 kJ mol⁻¹ and 96.4 ± 0.4 kJ mol⁻¹ K⁻¹ respectively.

Figure 5.

The van’t Hoff analysis of AIPcS₄ binding with HSA.

Reactions of both ZnPcS₄ and AIPcS₄ with serum albumin are thermodynamically favorable with regard to ΔH° and ΔS°. The larger negative ΔH° for ZnPcS₄ binding to HSA suggests that a combination of hydrogen-bonding and electrostatic interactions may be contributing to the stability of the complex. Electrostatic interactions, however, are generally believed to contribute little to the ΔH° (18, 19). The effects of ionic strength on these interactions may provide additional information regarding the difference in magnitude of ΔH° for the HSA complexed phthalocyanines of Al³⁺ and Zn²⁺. Solution dynamics seem to play a stronger role for binding between AIPcS₄-HSA as indicated by a larger ΔS°. The magnitude of ΔS° for AIPcS₄-HSA binding makes it the more thermodynamically favorable interaction. The effects of pH and ionic strength may provide additional insight into the mechanism of HSA interactions with sulfonated metal-phthalocyanines.

Table 3.

Thermodynamics of Sulfonated Phthalo-cyanines Binding to Human Serum Albumin.

ZnPcS4-HSA		AIPcS4-HSA
ΔH° (kJ·mol−1)	ΔS° (J·mol−1 K−1)	ΔH° (kJ·mol−1)	ΔS° (J·mol−1 K−1)
−21.2 ± 0.2	47.9 ± 0.5	−5.9 ± 0.1	96.4 ± 0.4