Biosensor Recognition of Thyroid-Disrupting Chemicals Using Transport Proteins

Abstract

Novel surface plasmon resonance-based biosensor assays for the bioeffect-related screening of chemicals with thyroid-disrupting activity are described. Two thyroid transport proteins (TPs), thyroxine binding globulin (TBG) and recombinant transthyretin (rTTR), were applied in an inhibition assay format in a Biacore 3000 using CM5 biosensor chips coated with l-thyroxine (T4), the main hormone of the thyroid system. Assay conditions were optimized for the natural thyroid hormones, and known thyroid disruptors and structurally related compounds were selected as model compounds to be tested in both assays for their relative potency (RP) compared to T4. The chosen compounds were halogenated phenols, halogenated bisphenols, bisphenol A, 3,5-dichlorobiphenyl, and its hydroxylated metabolite 4-hydroxy-3,5-dichlorobiphenyl (4-OH PCB 14). The TBG-based assay was highly specific for T4, and the rTTR-based assay was sensitive toward several compounds, the highest sensitivity (RP = 4.4) being obtained with 4-OH PCB 14, followed by tetrabromobisphenol A (RP = 1.5) and tetrachlorobisphenol A (RP = 0.75). For the bioeffect-related screening of known and identification of possible new thyroid disruptors, the TPs-based biosensor assays were more sensitive (IC(50) of 13.7 +/- 1.3 and 8.6 +/- 0.7 nM for the rTTR and the TBG-based assay, respectively), easier to perform, and faster alternatives (10 min/sample) than the currently used methods such as radioligand binding assays and immunoprecipitation-HPLC.

Fractal Analysis of Binding and Dissociation of Protein–Analyte Interactions on Biosensor Surfaces

“…Marchesini et al [1] have recently developed a novel surface plasmon resonance-based biosensor to screen chemicals which exhibit thyroid-disrupting activity. These authors detected two thyroid transport proteins (TPs), thyroxine binding globulin (TBG) and recombinant transtyretin (rTTR) in solution using a CM5 sensor chip coated with L-thyroxine (T4) in a Biacore 3000 biosensor system.…”

“…Lans et al [8] and Hallgren and Darnerud [9] indicate that polyhalogenated aromatic hydrocarbons (PHAHs) and several hydroxylated metabolites interact with high affinity with transthyretin (rTTR). Figure 1a shows the binding of 18.2 nM rTTR in solution to T4 immobilized on a CM5 sensor chip using the spacer E [1]. A single-fractal analysis is adequate to describe the binding and the dissociation kinetics.…”

“…Both, a single-and a dual-fractal analysis is used to model the binding and the dissociation kinetics. The binding and dissociation (where ever applicable) is presented for (a) rTTR in solution to T4 immobilized on a CM5 sensor chp surface using different spacers [1], and the interactions of G protein transducin (G t ) in solution to rhodopsin (Rho) immobilized on a sensor chip surface [2]. The fractal analysis emphasizes the importance of the biosensor surface on the interactions occurring on the surface, and helps provide physical insights into these interactions.…”

“…Biosensors have often been employed to analyze these types of interactions. In this article we use fractal analysis to analyze (a) the binding and dissociation (if applicable) kinetics of (a) recombinant transthyretin (rTTR) in solution to T4 (L-thyroxine (T4) immobilized on a sensor chip surface using different spacers [ [1], and (b) G t (G protein transducin) in solution to different densities of Rho (rhodopsin) immobilized on a sensor chip surface [2].…”

Surface‐Plasmon‐Resonance‐Based Chemical Proteomics: Efficient Specific Extraction and Semiquantitative Identification of Cyclic Nucleotide‐Binding Proteins from Cellular Lysates by Using a Combination of Surface Plasmon Resonance, Sequential Elution and Liquid Chromatography–Tandem Mass Spectrometry

“…SPR can be used as an affinity-based extraction technique in which the extraction process can be followed in real time, without the need to label proteins. [7][8][9][10] More importantly, SPR provides quantitative information about the amount of material bound to the sensor. When SPR is combined with MS, quantitative and qualitative information can be combined, and a powerful technique is obtained that is suitable for the determination of intact proteins [11][12][13][14] as well as digests.…”