Five Tyrosines and Two Serines in Human Albumin Are Labeled by the Organophosphorus Agent FP-Biotin

Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska 68198, USA.
Chemical Research in Toxicology (Impact Factor: 3.53). 09/2008; 21(9):1787-94. DOI: 10.1021/tx800144z
Source: PubMed

ABSTRACT 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, digested with trypsin or pepsin, and analyzed by mass spectrometry. The most reactive residue was always Tyr 411. Diethoxyphosphate-labeled Tyr 411 was stable for months at pH 7.4. These results will be useful in the development of specific antibodies to detect OP exposure and to engineer albumin for use as an OP scavenger.

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Available from: Florian Nachon, Sep 29, 2015
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    • "Though several tyrosines of albumin could react with CPO (Ding et al., 2008), the most reactive is tyrosine 411. To confirm the identity of the CPO and CPF modified tyrosine, the CPO-albumin and CPFalbumin samples were digested with pepsin and analyzed by MALDI- TOF mass spectrometry. "
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    ABSTRACT: Studies of human cases of self-inflicted poisoning suggest that chlorpyrifos oxon reacts not only with acetylcholinesterase and butyrylcholinesterase but also with other blood proteins. A favored candidate is albumin because in vitro and animal studies have identified tyrosine 411 of albumin as a site covalently modified by organophosphorus poisons. Our goal was to test this proposal in humans by determining whether plasma from humans poisoned by chlorpyrifos has adducts on tyrosine. Plasma samples from 5 self-poisoned humans were drawn at various time intervals after ingestion of chlorpyrifos for a total of 34 samples. All 34 samples were analyzed for plasma levels of chlorpyrifos and chlorpyrifos oxon (CPO) as a function of time post-ingestion. Eleven samples were analyzed for the presence of diethoxyphosphorylated tyrosine by mass spectrometry. Six samples yielded diethoxyphosphorylated tyrosine in pronase digests. Blood collected as late as 5 days after chlorpyrifos ingestion was positive for CPO-tyrosine, consistent with the 20-day half-life of albumin. High plasma CPO levels did not predict detectable levels of CPO-tyrosine. CPO-tyrosine was identified in pralidoxime treated patients as well as in patients not treated with pralidoxime, indicating that pralidoxime does not reverse CPO binding to tyrosine in humans. Plasma butyrylcholinesterase was a more sensitive biomarker of exposure than adducts on tyrosine. In conclusion, chlorpyrifos oxon makes a stable covalent adduct on the tyrosine residue of blood proteins in humans who ingested chlorpyrifos.
    Toxicology and Applied Pharmacology 04/2013; 269(3). DOI:10.1016/j.taap.2013.03.021 · 3.71 Impact Factor
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    • "OP-adducts on the active site serine of acetyl-and butyrylcholinesterase are well known, but are cleared from the circulation more rapidly than OP–albumin adducts (Read et al., 2010). Albumin is the most abundant protein in plasma and covalent attachment of OP to tyrosine 411 of human albumin has been demonstrated by in vitro studies (Ding et al., 2008; Li et al., 2007) as well as by studies of plasma from humans poisoned by dichlorvos (Li et al., 2010). In addition to albumin, other plasma proteins may also be modified by OP and contribute to the OP–tyrosine isolated from plasma. "
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    ABSTRACT: Inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activity is an established biomarker of exposure to organophosphorus poisons (OP). Inhibition of activity is due to covalent binding of the OP to the active site serine. Mass spectrometry has made it possible to monitor OP exposure by analyzing OP adducts on tyrosine in proteins that have no active site serine. Our goal was to test the hypothesis that OP-tyrosine may serve as a biomarker of OP exposure in mice. A MALDI-TOF mass spectrometry strategy to analyze diethoxyphosphate-tyrosine of m/z 318 was developed. The adduct was synthesized by incubating l-tyrosine with chlorpyrifos oxon at pH 8.1. The adduct eluted from a reverse phase HPLC column with 22-23% acetonitrile. The fragmentation spectrum of the m/z 318 precursor ion confirmed its identity as diethoxyphosphate-tyrosine. Diethoxyphosphate-tyrosine was isolated from chlorpyrifos oxon treated mouse albumin after digesting the protein with pronase. Mice (n=3 per group) were treated with a nontoxic dose of chlorpyrifos oxon (3 mg/kg) and a toxic dose (10 mg/kg transdermally). The pronase digested plasma yielded diethoxyphosphate-tyrosine up to 120 h after treatment with 3 mg/kg chlorpyrifos oxon and up to 144 h after 10 mg/kg. In contrast plasma AChE activity returned to normal after 24-72 h. In conclusion MALDI-TOF mass spectrometry can be used to diagnose exposure to chlorpyrifos oxon days after AChE inhibition assays are uninformative.
    Toxicology 03/2012; 295(1-3):15-22. DOI:10.1016/j.tox.2012.03.001 · 3.62 Impact Factor
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    • "The most reactive residue in human albumin is tyrosine 411, though other tyrosines are also labeled when conditions are maximized in vitro (Ding et al., 2008; Means and Wu, 1979). Tyrosine 411 can be found in peptic peptides VRY 411 TKKVPQVSTPTL and LVRY 411 TKKVPQVSTPTL, which are related by a missed cleavage. "
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    ABSTRACT: Studies in mice and guinea pigs have shown that albumin is a new biomarker of organophosphorus toxicant (OP) and nerve agent exposure. Our goal was to determine whether OP-labeled albumin could be detected in the blood of humans exposed to OP. Blood from four OP-exposed patients was prepared for mass spectrometry analysis by digesting 0.010 ml of serum with pepsin and purifying the labeled albumin peptide by offline high performance liquid chromatography. Dimethoxyphosphate-labeled tyrosine 411 was identified in albumin peptides VRY411TKKVPQVSTPTL and LVRY411TKKVPQVSTPTL from two patients who had attempted suicide with dichlorvos. The butyrylcholinesterase activity in these serum samples was inhibited 80%. A third patient whose serum BChE activity was inhibited 8% by accidental inhalation of dichlorvos had undetectable levels of adduct on albumin. A fourth patient whose BChE activity was inhibited 60% by exposure to chlorpyrifos had no detectable adduct on albumin. This is the first report to demonstrate the presence of OP-labeled albumin in human patients. It is concluded that tyrosine 411 of human albumin is covalently modified in the serum of humans poisoned by dichlorvos and that the modification is detectable by mass spectrometry. The special reactivity of tyrosine 411 with OP suggests that other proteins may also be modified on tyrosine. Identification of other OP-modified proteins may lead to an understanding of neurotoxic symptoms that appear long after the initial OP exposure. © The Author 2010. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: [email protected] /* */
    Toxicological Sciences 07/2010; 116(1):23-31. DOI:10.1093/toxsci/kfq117 · 3.85 Impact Factor
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