Butyrylcholinesterase for protection from organophosphorus poisons; catalytic complexities and hysteretic behavior

We report a highly sensitive square wave voltammetric biosensor for the determination of carbofuran using an acetylcholinesterase (AChE) enzyme immobilized iron oxide–chitosan nanocomposite film modified glassy carbon electrode (AChE/Fe3O4–CH/GCE). The Fe3O4–CH nanocomposite was prepared by a simple solution mixing process and its formation was confirmed by FT-IR spectroscopy. The effective enzyme immobilization onto the nanocomposite matrix was confirmed by electrochemical impedance, scanning electron and atomic force microscopy studies. Various experimental parameters such as effect of scan rate, inhibition time and substrate concentration were optimized. The nanocomposite-based biosensor could detect carbofuran as low as 3.6 × 10−9 M. The reproducibility of the AChE/Fe3O4–CH/GCE was ascertained by performing intra-assay and inter-assay experiments using cyclic voltammetry. The practical application of the biosensor was ascertained by the determination of carbofuran from cabbage samples and by comparing the results with those obtained by the standard high-performance liquid chromatography (HPLC) method.

Electrochemical biosensing of carbofuran based on acetylcholinesterase immobilized onto iron oxide–chitosan nanocomposite

Serum albumin (Alb) is the most abundant protein in blood plasma. Alb reacts with many carcinogens and/or their electrophilic metabolites. Studies conducted over 20 years ago showed that Alb forms adducts with the human carcinogens aflatoxin B1 and benzene, which were successfully used as biomarkers in molecular epidemiology studies designed to address the role of these chemicals in cancer risk. Alb forms adducts with many therapeutic drugs or their reactive metabolites such as β-lactam antibiotics, acetylsalicylic acid, acetaminophen, nonsteroidal anti-inflammatory drugs, chemotherapeutic agents, and antiretroviral therapy drugs. The identification and characterization of the adduct structures formed with Alb have served to understand the generation of reactive metabolites and to predict idiosyncratic drug reactions and toxicities. The reaction of candidate drugs with Alb is now exploited as part of the battery of screening tools to assess the potential toxicities of drugs. The use of gas chromatography-...

http://pubs.acs.org/doi/pdf/10.1021/acs.chemrestox.6b00366

Biomonitoring Human Albumin Adducts: The Past, the Present, and the Future.

History and perspectives of bioanalytical methods for chemical warfare agent detection

We report a new approach for electrochemical quantification of enzymatic inhibition and phosphorylation for biomonitoring of exposure to organophosphorus (OP) pesticides and nerve agents based on a magnetic bead (MB) immunosensing platform. The principle of this approach is based on the combination of MB immunocapture-based enzyme activity assay and competitive immunoassay of the total amount of enzyme for simultaneous detection of enzyme inhibition and phosphorylation in biological fluids. Butyrylcholinesterase (BChE) was chosen as a model enzyme. In competitive immunoassay, the target BChE in a sample competes with the BChE immobilized on the MBs to bind to the limited sites of anti-BChE antibody labeled with quantum dots (QD–anti-BChE), followed by stripping voltammetric analysis of the bound QD conjugate on the MBs. This assay shows a linear response over the total BChE concentration range of 0.1–20 nM. Simultaneous real time BChE activity was measured on an electrochemical carbon nanotube-based senso...

Magnetic electrochemical sensing platform for biomonitoring of exposure to organophosphorus pesticides and nerve agents based on simultaneous measurement of total enzyme amount and enzyme activity.

Tyrosine 411 of human albumin is an established site for covalent attachment of 10-fluoroethoxyphosphinyl-N-biotinamidopentyldecanamide (FP-biotin), diisopropylfluorophosphate, chlorpyrifos oxon, soman, sarin, and dichlorvos. This work investigated the hypothesis that other residues in albumin could be modified by organophosphorus agents (OP). Human plasma was aggressively treated with FP-biotin; plasma proteins were separated into high and low abundant portions using a proteome partitioning antibody kit, and the proteins were digested with trypsin. The FP-biotinylated tryptic peptides were isolated by binding to monomeric avidin beads. The major sites of covalent attachment identified by mass spectrometry were Y138, Y148, Y401, Y411, Y452, S232, and S287 of human albumin. Prolonged treatment of pure human albumin with chlorpyrifos oxon labeled Y138, Y150, Y161, Y401, Y411, and Y452. To identify the most reactive residue, albumin was treated for 2 h with DFP, FP-biotin, chlorpyrifos oxon, or soman, digest...

https://pubs.acs.org/doi/pdf/10.1021/tx800144z

Five tyrosines and two serines in human albumin are labeled by the organophosphorus agent FP-biotin.

Carbofuran is a pesticide whose acute toxicity is due to inhibition of acetylcholinesterase. Butyrylcholinesterase (BChE) in plasma is inhibited by carbofuran and serves as a biomarker of poisoning by carbofuran. The goal was to develop a method to positively identify poisoning by carbofuran. Sera from an attempted murder and an attempted suicide were analyzed for the presence of carbofuran adducts on BChE. The BChE from 1 ml of serum was rapidly purified on a 0.2 ml procainamide-Sepharose column. Speed was essential because the carbofuran-BChE adduct decarbamylates with a half-life of about 2 h. The partially purified BChE was boiled to denature the protein, thus stopping decarbamylation and making the protein vulnerable to digestion with trypsin. The labeled peptide was partially purified by HPLC before analysis by LC/MS/MS in the multiple reaction monitoring mode on the QTRAP 2000 mass spectrometer. Carbofuran was found to be covalently bound to Ser 198 of human BChE in serum samples from two poisoning cases. Multiple reaction monitoring triggered MS/MS spectra positively identified the carbofuran-BChE adduct. In conclusion a mass spectrometry method to identify carbofuran poisoning in humans has been developed. The method uses 1 ml of serum and detects low-level exposure associated with as little as 20% inhibition of plasma butyrylcholinesterase.

Larry Tong

Papers

Five tyrosines and two serines in human albumin are labeled by the organophosphorus agent FP-biotin.

Carbofuran poisoning detected by mass spectrometry of butyrylcholinesterase adduct in human serum.

Fast affinity purification coupled with mass spectrometry for identifying organophosphate labeled plasma butyrylcholinesterase