ABSTRACT
Pesticides represent some of the most common man-made chemicals in the world. Despite their unquestionable utility in the agricultural field and in the prevention of pest infestation in public areas of cities, pesticides and their biotransformation products are toxic to the environment and hazardous to human health. Esterase-based biosensors represent a viable alternative to the expensive and time-consuming systems currently used for their detection. In this work, we used the esterase-2 from Alicyclobacillus acidocaldarius as bioreceptor for a biosensing device based on an automated robotic approach. Coupling the robotic system with a fluorescence inhibition assay, in only 30 s of enzymatic assay, we accomplished the detection limit of 10 pmol for 11 chemically oxidized thio-organophosphates in solution. In addition, we observed differences in the shape of the inhibition curves determined measuring the decrease of esterase-2 residual activity over time. These differences could be used for the characterization and identification of thio-organophosphate pesticides, leading to a pseudo fingerprinting for each of these compounds. This research represents a starting point to develop technologies for automated screening of toxic compounds in samples from industrial sectors, such as the food industry, and for environmental monitoring.
Subject(s)
Biosensing Techniques/methods , Organophosphates/chemistry , Organophosphorus Compounds/chemistry , Robotics/methods , Alicyclobacillus/chemistry , Biological Assay/methods , Environmental Monitoring/methods , Esterases/chemistry , Fluorescence , Limit of Detection , Pesticides/chemistryABSTRACT
Organophosphates are organic substances that contain a phosphoryl or a thiophosphoryl bond. They are mainly used around the world as pesticides, but can also be used as chemical warfare agents. Their detection is normally entrusted to techniques like GC- and LC-MS that, although sensitive, do not allow their identification on site and in real time. We have approached their identification by exploiting the high-affinity binding of these compounds with the esterase 2 from Alicyclobacillus acidocaldarius. Using an in silico analysis to evaluate the binding affinities of the enzyme with organophosphate inhibitors, like paraoxon, and other organophosphate compounds, like parathion, chlorpyriphos, and other organophosphate thio-derivatives, we have designed fluorescence spectroscopy experiments to study the quenching of the tryptophan residues after esterase 2 binding with the organophosphate pesticides. The changes in the fluorescence signals permitted an immediate and quantitative identification of these compounds from nano- to picomolar concentrations. A fluorescence based polarity-sensitive probe (ANS) was also employed as a means to understand the extent of the interactions involved, as well as to explore other ways to detect organophosphate pesticides. Finally, we designed a framework for the development of a biosensor that exploits fluorescence technology in combination with a sensitive and very stable bio-receptor.
Subject(s)
Biosensing Techniques , Chemical Warfare Agents/isolation & purification , Esterases/chemistry , Organophosphates/isolation & purification , Bacillus/enzymology , Chemical Warfare Agents/toxicity , Humans , Insecticides , Organophosphates/toxicity , Paraoxon , Spectrometry, FluorescenceABSTRACT
Heparin Binding Hemagglutinin A (HBHA) is hitherto the sole virulence factor associated with tuberculosis dissemination from the lungs, the site of primary infection, to epithelial cells. We have previously reported the solution structure of HBHA, a dimeric and elongated molecule. Since oligomerisation of HBHA is associated with its ability to induce bacterial agglutination, we investigated this process using experimental and modelling techniques. We here identified a short segment of HBHA whose presence is mandatory for the stability of folded conformation, whose denaturation is a reversible two-state process. Our data suggest that agglutination-driven cell-cell interactions do not occur via association of HBHA monomers, nor via association of HBHA dimers and open the scenario to a possible trans-dimerisation process.
Subject(s)
Lectins/chemistry , Lectins/metabolism , Mycobacterium tuberculosis/metabolism , Agglutination , Amino Acid Sequence , Antigens, Bacterial/chemistry , Antigens, Bacterial/metabolism , Bacterial Adhesion/physiology , Lectins/physiology , Light , Models, Molecular , Molecular Sequence Data , Mycobacterium tuberculosis/physiology , Protein Conformation , Protein Folding , Protein Multimerization , Protein Stability , Scattering, Radiation , TemperatureABSTRACT
Organophosphates (OPs) constitute the largest class of insecticides used worldwide and certain of them are potent nerve agents. Consequently, enzymes degrading OPs are of paramount interest, as they could be used as bioscavengers and biodecontaminants. Looking for a stable OPs catalyst, able to support industrial process constraints, a hyperthermophilic phosphotriesterase (PTE) (SsoPox) was isolated from the archaeon Sulfolobus solfataricus and was found to be highly thermostable. The solved 3D structure revealed that SsoPox is a noncovalent dimer, with lactonase activity against "quorum sensing signals", and therefore could represent also a potential weapon against certain pathogens. The structural basis of the high thermostability of SsoPox has been investigated by performing a careful comparison between its structure and that of two mesophilic PTEs from Pseudomonas diminuta and Agrobacterium radiobacter. In addition, the conformational stability of SsoPox against the denaturing action of temperature and GuHCl has been determined by means of circular dichroism and fluorescence measurements. The data suggest that the two fundamental differences between SsoPox and the mesophilic counterparts are: (a) a larger number of surface salt bridges, also involved in complex networks; (b) a tighter quaternary structure due to an optimization of the interactions at the interface between the two monomers.
Subject(s)
Archaeal Proteins/chemistry , Sulfolobus solfataricus/chemistry , Circular Dichroism , Models, Molecular , Protein Conformation , Spectrometry, Fluorescence , Spectrophotometry, Ultraviolet , Static ElectricityABSTRACT
The conformational stability of the rat thyroid transcription factor 1 homeodomain, TTF-1HD, has been investigated by means of circular dichroism (CD) and differential scanning calorimetry (DSC) measurements at pH 5.0 as a function of KCl concentration. Thermal unfolding of TTF-1HD is a reversible two-state transition. The protein is not stable against temperature, showing a denaturation temperature of 32 degrees C in the absence of salt and 50 degrees C at 75 mM KCl. The binding energetics of TTF-1HD to its target DNA sequence has been characterized by means of isothermal titration calorimetry (ITC) measurements, complemented with CD data. At 25 degrees C, pH 5.0 and 75 mM KCl, the binding constant amounts to 1.5 x 10(8)M(-1) and the binding enthalpy change amounts to -41 kJ mol(-1). The process is enthalpy driven, but also the entropy change is favorable to complex formation. To gain a molecular level understanding of the interactions determining the association of TTF-1HD to the target DNA sequence structural information would be requested, but it is not yet available. Therefore, structural models of two complexes, TTF-1HD with the target DNA sequence and TTF-1HD with a modified DNA sequence, have been constructed by using as a template the NMR structure of the complex between NK-2 HD and its target DNA, and by performing molecular dynamics simulations 3.5 ns long. Analysis of these models allows one to shed light on the origin of the DNA binding specificity characteristic of TTF-1HD.
