Article pubs.acs.org/JPCB
Binding Mode Investigations on the Interaction of Lead(II) Acetate with Human Chorionic Gonadotropin Hao Zhang,† Yang Liu,† Rui Zhang,† Rutao Liu,*,† and Yadong Chen‡ †
School of Environmental Science and Engineering, Shandong University, 27 Shanda Nanlu, Jinan 250100, P. R. China Laboratory of Molecular Design and Drug Discovery, School of Basic Science, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China
‡
S Supporting Information *
ABSTRACT: Lead exposure could induce endocrine disruption and hormonal imbalance of humans, resulting in detrimental effects on the reproductive system even at low doses. However, mechanisms of lead actions remain unknown. This article investigated lead interactions with human chorionic gonadotropin (HCG) as a conceivable mechanism of its reproductive toxicity by spectroscopic technique, isothermal titration calorimetry (ITC), molecular docking study, and enzyme-linked immunosorbent assay (ELISA). Fluorescence measurements showed that lead acetate dynamically quenched intrinsic fluorescence of HCG through collisional mechanism with the association constant (KSV) in the magnitude of 103 L/mol at the detected temperatures (298, 303, and 310 K). ITC and molecular docking results revealed lead acetate could bind into 5 binding sites of HCG through electrostatic effects (ΔH < 0, ΔS > 0) and hydrophobic forces (ΔH > 0, ΔS > 0). The conformational investigation of HCG by UV−vis absorption spectroscopy, circular dichroism spectroscopy, and ELISA indicated lead acetate changed the secondary structure of HCG by loosening and destruction of HCG skeleton and increasing the hydrophobicity around Tyr residues and resulted in the decreased bioactivities of HCG. This work presents direct interactions of lead with sex hormones and obtains a possible mechanism on lead induced reproductive toxicity at the molecular level.
1. INTRODUCTION
HCG (structure shown in Figure S1, Supporting Information), a sex hormone produced by the placenta, is a 36.7 kDa glycoprotein and consists of 237 amino acids with two noncovalently bound polypeptide chains (A-chain of 92 amino acids and B-chain of 145 amino acids),15,16 including 6 tyrosines (no tryptophan), which can emit the intrinsic fluorescence of HCG. HCG plays vital roles in pregnancy maintenance, inhibiting the growth of ovarian cancer and mammary carcinomas by regulating secretion of ovarian steroid and the levels of estrogen and progesterone.17,18 When lead accumulates in human bodies in excess of the maximal tolerated dose, and contacts with HCG, and the structure and function of HCG can be damaged due to the direct interactions with lead. In this work, the toxic effects of lead on HCG structure and the binding mechanism of lead with HCG were investigated at the molecular level using fluorescence spectroscopy, UV−vis absorption spectroscopy, circular dichroism spectroscopy, isothermal titration calorimetric (ITC) measurement, molecular docking study, and enzyme-linked immunosorbent assay (ELISA). The study explains direct binding interactions of lead
Lead belongs to the nonbiodegradable and toxic heavy metal, which can be widely spread into the environment by anthropogenic industrial processes and gradually accumulated into human bodies through respiratory and gastrointestinal tracts, causing adverse health problems.1−3 Although toxic effects of lead on the human reproductive system have been investigated for decades, the mechanisms of lead actions remain unclear.4,5 Studies have shown that lead can act on the hypothalamus−pituitary−gonadal axis,6 resulting in endocrine disruption and hormonal imbalance,7−9 such as human chorionic gonadotropin (HCG), human protamine,10 and disturbance in the levels of hormonal receptors and postreceptors, causing changes of physiological functions of target cells and tissues, and inducing the reproductive toxicity.10,11 However, there is a lack of report focusing on direct effects of lead on sex hormones, which may be another possible reason for reproductive damage caused by lead. Direct interactions between macromolecules (protein, DNA) and toxic ligands at the molecular level have been systematically investigated by our group to explore binding mechanisms of ligands with functional biomolecules,12−14 which is helpful to evaluate potential toxic mechanisms of xenobiotics in vivo. © 2014 American Chemical Society
Received: June 5, 2014 Revised: July 25, 2014 Published: August 6, 2014 9644
dx.doi.org/10.1021/jp505565s | J. Phys. Chem. B 2014, 118, 9644−9650
The Journal of Physical Chemistry B
Article
1HRP) was downloaded from the Protein Data Bank (http:// www.pdb.org/). The 3D structure of lead acetate was drawn and minimized using Builder module and the Energy Minimize module of MOE, respectively. 2.7. ELISA Assay of HCG. According to the user instruction of HCG ELISA kit, solid-phase antibody of HCG was coated into each of microtiter plate wells, then HCG solutions (50 μL, standards and samples) were added into wells and incubated at 37 °C for 30 min. After washing 5 times using wash solutions, the HRP-labeled HCG antibody (50 μL) was dropped into each well and incubated in the same conditions as before. After washing completely, the tetramethylbenzidine (TMB) substrate solutions (100 μL) were added into wells and incubated at 37 °C for 15 min in the dark. Then the reaction was terminated by the addition of sulfuric acid solutions (100 μL, 4 mol/L) and the absorbance of each wells was measured with a microplate reader (GF-M3000, Rainbow, China) at 450 nm. The standard curve of HCG biological activities was developed by preparing a series of standard solutions (62.5, 125, 250, 500, and 1000 IU/ L). The samples were prepared as follows: first, 100 μL of 2.7 × 10−5 mol/L HCG, 100 μL of 0.2 mol/L NaAc-HAc buffer and different amounts of lead acetate solutions were added into 1.5 mL eppendorf tubes and diluted to 1 mL with ultrapure water. Then, after 30 min reaction at 37 °C, each sample was prepared under 5,000 × 5-fold dilution with ultrapure water (including 5fold dilution during ELISA steps) to meet the measurement range of 0.71−1000 IU/L. Data (mean ± SD) were analyzed by Graphpad Prism software (Version 5.01, San Diego, USA), using one-way analysis of variance (ANOVA) as the statistical method, followed by Newman-Keuls multiple comparison test, considering p < 0.05 as significant.
with HCG and provides experimental evidence for overall evaluation of reproductive toxicity induced by lead in vivo.
2. EXPERIMENTAL PROCEDURES 2.1. Reagents. HCG (∼6500 IU/mL, from urine of pregnant women, ProSpec, Israel) was dissolved in ultrapure water to form a 2.7 × 10−4 mol/L solution, and kept at 0−4 °C and diluted as required. HCG ELISA kit (0.71−1000 IU/L) was purchased from Kmaels Biotechnology, China. A stock solution (1.0 × 10−3 mol/L) of lead acetate (Kermel Chemical Reagent, China) was prepared by dissolving 0.0379 g of Pb(Ac)2 in 100 mL of water and filtered by 0.22 μm syringe filters. NaAc-HAc buffer (0.2 mol/L, pH 5.5) was utilized to stabilize acidity. Ultrapure water (18.25 MΩ) was used throughout the experiments. 2.2. Fluorescence Measurements. Fluorescence emission spectra, synchronous fluorescence spectra, and time-resolved fluorescence measurements were employed to determine the binding modes between HCG and lead acetate. (1). Steady-State Fluorescence Measurement. The steadystate fluorescence spectra were measured on an F-4600 fluorescence spectrophotometer (Hitachi, Japan) equipped with a micro quartz cell (1 mm width) using an excitation wavelength of 278 nm and 5 nm bandwidth for both excitation and emission. Scan speed was 1200 nm/min. The PMT (photo multiplier tube) voltage was set at 750 V. (2). Fluorescence Lifetime Measurements. Time-resolved fluorescence measurements of HCG were carried out using an FLS920 combined fluorescence lifetime and steady state spectrometer (Edinburgh, U.K.) at λex = 278 nm, λem = 330 nm after eliminating the influence of scattered light emitted by solvents. (3). Synchronous Fluorescence Measurements. The synchronous fluorescence spectra of HCG were measured (Δλ = 15 nm, λex = 275−315 nm) using the F-4600 fluorescence spectrophotometer (Hitachi, Japan) with an 1 mm width cuvette. 2.3. UV−vis Absorption Measurements. The absorption spectra of HCG influenced by different concentrations of lead were all measured on a UV-2450 spectrophotometer (Shimadzu, Kyoto, Japan) with 1 mm micro cuvettes. 2.4. Circular Dichroism (CD) Spectra Measurement. CD spectra of HCG were measured using a J-810 circular dichroism spectrometer (Jasco, Japan). The CD spectra were collected from 190 to 260 nm with the scan speed of 200 nm/ min, and three scans were made and averaged for each CD spectrum. The secondary structure contents of HCG obtained from CD spectra were analyzed using CDpro software (available at http://lamar.colostate.edu/∼sreeram/CDPro/). 2.5. Isothermal Titration Calorimetry (ITC) Experiments. ITC experiments were performed on a Microcal ITC200 microcalorimeter (Microcal Inc.,Northampton, MA) at 298 K by titrating 200 μL of HCG (0.027 mM) with approximately 40 μL of Pb2+ (1 mM) using stirring speed at 1000 rpm. Both of HCG and lead acetate were dissolved in 0.02 M NaAc-HAc buffer (pH 5.5), and the spacing time between each injection was set to 120 s to control thermodynamic equilibration. 2.6. Molecular Docking Investigations. Binding characters of HCG with lead acetate were confirmed by molecular docking studies using molecular operating environment software (MOE) (Version 2009, Chemical Computing Group Inc., Canada). The crystal structure of HCG (PDB code
3. RESULTS AND DISCUSSIONS 3.1. Quenching Mechanism of HCG Fluorescence. The fluorescence emission spectra of HCG affected by a series lead
Figure 1. Effects of Pb(Ac)2 on the fluorescence intensities of HCG. Conditions: HCG: 2.7 × 10−6 mol/L; Pb(Ac)2 /(10−5 mol/L) (a-e): 0, 1, 4, 7, 10; pH 5.5; T = 298 K.
concentrations at 298 K are illustrated in Figure 1. The absorbance value in the range of 278 to 450 nm is too small (
