Solution speciation and human serum protein binding of indium(III) complexes of 8-hydroxyquinoline, deferiprone and maltol

Abstract

Solution speciation and serum protein binding of selected In(III) complexes bearing O,O and O,N donor sets were studied to provide comparative data for In(III) and analogous Ga(III) complexes. Aqueous stability of the In(III) complexes of maltol, deferiprone, 8-hydroxyquinoline (HQ) and 8-hydroxyquinoline-5-sulfonate (HQS) was characterized by a combined pH-potentiometric and UV-visible spectrophotometric approach. Formation of mono, bis and tris-ligand complexes was observed. The tris-ligand complexes of HQ (InQ₃) and deferiprone (InD₃) are present in solution in ca. 90% at 10 µM concentration at pH = 7.4, while the tris-maltolato complex (InM₃) displays insufficient stability under these conditions. Binding towards human serum albumin (HSA) and (apo)transferrin ((apo)Tf) of InQ₃, InD₃ and InM₃ complexes and Ga(III) analogue of InQ₃ (GaQ₃) together with InCl₃ was investigated by a panel of methods: steady-state and time-resolved spectrofluorometry, UV-visible spectrophotometry and membrane ultrafiltration. Moderate binding of InQ₃ to HSA was found (log K' = 5.0-5.1). InD₃ binds to HSA to a much lower extent in comparison to InQ₃. ApoTf is able to displace HQ, deferiprone and maltol effectively from their In(III) complexes. Protein binding of non-dissociated InQ₃ was also observed at high complex-to-apoTf ratios. Studies conducted with the InQ₃/GaQ₃ - HSA - Tf ternary systems revealed the more pronounced Tf binding of In(III) via ligand release, while the original GaQ₃ scaffold is preferably retained upon protein interactions and significant albumin binding occurs. Significant dissociation of InQ₃ was detected in human blood serum as well.

Keywords: Albumin; Fluorescence; Stability constant; Transferrin; Ultrafiltration.

Chart 1

General structure of the tris-ligand In(III) complexes and chemical structure and abbreviations of the bidentate ligands 8-hydroxyquinoline (HQ), 8-hydroyquinoline-5-sulfonate (HQS), 3-hydroxy-1,2-dimethylpyridin-4(1H)-one (deferiprone) and 3-hydroxy-2-methyl-4H-pyran-4-one (maltol)

Fig. 1

a UV–vis spectra recorded for the In(III) ‒ HQ (1:3) system at various pH values. b Computed molar spectra of the individual species. c Concentration distribution curves for the same system plotted together with the absorbance values at 368 nm (●) in addition to that of the metal-free ligand ( ×). {c_In(III) = 28.3 μM, c_L = 93.3 μM, T = 25.0 °C, I = 0.20 M (KCl), ℓ = 2 cm}

Fig. 2

Estimated distribution of the tris-ligand HQ complexes of In(III) (solid lines) and Ga(III) (dashed lines) at pH 7.4 at various total concentrations. The majority (≥ 98%) of the ligand-bound complexes is present as InQ₃ and GaQ₃, and practically 100% of the unbound metal ions is found in hydroxido complexes. {T = 25.0 °C, I = 0.20 M (KCl)}

Fig. 3

a Fluorescence spectra of InQ₃ alone (thick black line) and in the presence of increasing amounts of HSA (grey lines). b Experimental (●) and fitted (solid line) intensities of the same system at λ_EM = 530 nm. {c_complex = 18 μM; c_HSA = 0 – 124 μM; λ_EX = 367 nm; pH = 7.40}

Fig. 4

a Fluorescence emission spectra of apoTf in the presence of various amounts of InD₃, inset shows the intensity changes at 330 nm. b Comparison of the fluorescence intensities of apoTf by addition of InCl₃ (■), InM₃ (●) InD₃ (♦) and InQ₃ (▲) at 330 nm plotted against the ratio c_compound / c_apoTf. {c_apoTf = 1 μM, c_compound = 0 – ca. 30 μM; λ_EX = 280 nm; pH 7.40 equilibration time: 1 h (InD₃), 3 h (InCl₃, InM₃), 6 h (InQ₃)}

Fig. 5

a UV–vis absorbance and b fluorescence emission spectra recorded for InQ₃ in the absence and presence of various amounts of apoTf, the spectrum of free HQ (red dashed line) is plotted as well. c Changes of the absorbance at 310 nm (●) and 370 nm (▲) (red dashed and blue dotted lines denote the absorbance belonging to free HQ at 310 nm and 370 nm, respectively). d Fluorescence intensity at λ_EM = 530 nm of the same system (♦) (while the green dashed line shows the emission intensity of HQ). All the absorbance spectra are subtracted by the spectrum of apoTf. {c_complex = 19 μM; c_apoTf = 0 – 30 μM; c_HQ = 57 μM; λ_EX = 367 nm (c); ℓ = 2 cm (a, c), 1 × 1 cm (b, d); pH = 7.40, equilibration time: 6 h}

Fig. 6

Molar fractions of complex bound (InD₃, ▲,Δ) and free deferiprone (●,○) calculated on the basis of UV–vis spectra recorded for the InD₃ – apoTf system (filled symbols) and results of the ultrafiltration–UV–vis experiments (empty symbols) are plotted as well. {c_complex = 22 μM (UV–vis), 19.8 μM (ultrafiltration); pH = 7.40}

Fig. 7

Three-dimensional fluorescence spectra recorded for InQ₃ – HSA – Tf and GaQ₃ – HSA – Tf ternary systems at the indicated compositions. Numbers indicate the complex-to-HSA-to-Tf ratios. The ratio of the two proteins corresponds to their physiological ratio in the blood serum. {c_InQ3 = c_GaQ3 = 10 μM, c_HSA = 0 – 212 μM; c_Tf = 0 – 12.8 μM; pH = 7.40, equilibration time: 6 h; spectra are corrected by self-absorbance and inner filter effect}

Fig. 8

Changes of fluorescence intensity in the InQ₃ – HSA – Tf (■), InQ₃ – Tf (●) and InQ₃ – HSA (♦,---) ternary systems normalized for the emission intensity of the complex at λ_EM = 550 nm. Plotted values are derived from Fig. 7 (ternary system), Figure S5 (Tf) and Fig. 3 and the computed binding constant (logK’ = 5.1) calculated for the InQ₃ – HSA system. {c_InQ3 = 10 μM; pH = 7.40; λ_EX = 367 nm; equilibration time: 6 h; spectra are corrected by self-absorbance and inner filter effect}

Fig. 9

UV–vis absorbance spectra recorded for InQ₃ (a) and GaQ₃ (b) in the absence (black lines) and presence of threefold diluted blood serum (blue lines) plotted together with the spectrum of free HQ (grey dotted line). Absorbance spectra of the mixtures are subtracted by the spectrum of blood serum. {c_complexes = 10 μM; blood serum: filtered on 1.2 μm filter and threefold diluted with buffer; ℓ = 1 cm; pH = 7.40, equilibration time: 6 h}