Locating the binding sites of Pb(II) ion with human and bovine serum albumins

Abstract

Lead is a potent environmental toxin that has accumulated above its natural level as a result of human activity. Pb cation shows major affinity towards protein complexation and it has been used as modulator of protein-membrane interactions. We located the binding sites of Pb(II) with human serum (HSA) and bovine serum albumins (BSA) at physiological conditions, using constant protein concentration and various Pb contents. FTIR, UV-visible, CD, fluorescence and X-ray photoelectron spectroscopic (XPS) methods were used to analyse Pb binding sites, the binding constant and the effect of metal ion complexation on HSA and BSA stability and conformations. Structural analysis showed that Pb binds strongly to HSA and BSA via hydrophilic contacts with overall binding constants of K(Pb-HSA) = 8.2 (±0.8)×10(4) M(-1) and K(Pb-BSA) = 7.5 (±0.7)×10(4) M(-1). The number of bound Pb cation per protein is 0.7 per HSA and BSA complexes. XPS located the binding sites of Pb cation with protein N and O atoms. Pb complexation alters protein conformation by a major reduction of α-helix from 57% (free HSA) to 48% (metal-complex) and 63% (free BSA) to 52% (metal-complex) inducing a partial protein destabilization.

Figure 1. Three-dimensional structures of HSA and BSA with tryptophan residues in green color.

Figure 2. FTIR spectra in the region of 1800–600 cm⁻¹ of hydrated films (pH 7.4) for free BSA (0.25 mM) and its Pb complexes (A) and for free HSA (0.25 mM) and its Pb complexes (B) with difference spectra (diff.) (bottom two curves) obtained at different Pb concentrations (indicated on the figure).

Figure 3. Second derivative resolution enhancement and curve-fitted amide I region (1700–1600 cm⁻¹) for free BSA and HSA (0.25 mM) and their Pb complexes with 0.5 mM Pb concentration.

Figure 4. Fluorescence emission spectra of Pb-BSA systems in 10 mM Tris-HCl buffer pH 7.4 at 25°C presented for (A) Pb–BSA: (a) free BSA (7.5 µM), (b–j) with Pb cationl at 1, 2.5, 5, 7.5, 10, 15, 20, 30, 40 and 60 µM; (B) Pb– HSA: (a) free HSA (7.5 µM), (b–i) Pb at 1, 2.5, 5, 7.5, 10, 15, 20, 30, 40 and 60 µM. Inset: F ₀/(F ₀−F) vs 1/[Pb] for A′ (Pb-BSA) and B′ (Pb-HSA).

Figure 5. Stern-Volmer plots of fluorescence quenching constant (Kq) for the Pb-BSA and Pb-HSA complexes at different Pb concentrations (A) Pb-BSA and (B) Pb-HSA.

Figure 6. The plot of Log (F₀−F/F) as a function of Log (Pb concentration).

Figure 7. XPS spectra of C atoms for the free HSA and BSA and their Pb complexes.

Figure 8. XPS spectra of O atoms for the free HSA and BSA and their Pb complexes.

Figure 9. XPS spectra of N atoms for the free HSA and BSA and their Pb complexes.