Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry assay for organophosphorus toxicants bound to human albumin at Tyr411

Abstract

Our goal was to determine whether chlorpyrifos oxon, dichlorvos, diisopropylfluorophosphate (DFP), and sarin covalently bind to human albumin. Human albumin or plasma was treated with organophosphorus agent (OP) at alkaline pH, digested with pepsin at pH 2.3, and analyzed by MADLI-TOF mass spectrometry. Two singly charged peaks, 1718 and 1831 m/z, corresponding to the unlabeled peptide fragments containing the active site Tyr 411 residue, were detected in all samples. The sequences of the two peptides were VRYTKKVPQVSTPTL and LVRYTKKVPQVSTPTL. The peptide-OP adducts of these peptides were also found. They had masses of 1854 and 1967 for chlorpyrifos oxon, 1825 and 1938 for dichlorvos, 1881 and 1994 for DFP, and 1838 and 1938 for sarin; these masses fit a mechanism whereby OP bound covalently to Tyr 411. The binding of DFP to Tyr 411 of human albumin was confirmed by electrospray tandem mass spectrometry and analysis of product ions. None of the OP-albumin adducts lost an alkoxy group, leading to the conclusion that aging did not occur. Our results show that OP pesticides and nerve agents bind covalently to human albumin at Tyr 411. The presence of Tyr 411 on an exposed surface of albumin suggests that an antibody response could be generated against OP-albumin adducts.

Pseudo-esterase Activity of Human Albumin

“…Albumin Esterase Activity-In our previous reports we had identified Tyr-411 as the residue in albumin that is labeled by soman, sarin, DFP, chlorpyrifos oxon, FP-biotin, and dichlorvos (16,18). Others have also identified tyrosine as the site of covalent binding of soman, sarin, cyclosarin, and tabun to albumin (20).…”

“…The esteratic site has been identified as Tyr-411 based on site-directed mutagenesis studies (17). Mass spectrometry (MS) 2 has identified Tyr-411 as the residue labeled by organophosphorus esters including diisopropylfluorophosphate (DFP), soman, sarin, dichlorvos, FP-biotin, and chlorpyrifos oxon (16,18) confirming the report by Sanger that a tyrosine in albumin is labeled by DFP (19),and reports that a tyrosine in albumin is labeled by the nerve agents soman, sarin, cyclosarin, and tabun (20). When albumin is labeled with 1 mol of DFP, albumin loses the fast phase of its esterase activity (the burst), supporting the conclusion that DFP and p-nitrophenyl acetate bind to the same site, namely to Tyr-411 (21).…”

Effect of propolis consumption on hepatotoxicity and brain damage in male rats exposed to chlorpyrifos

“…protein and free amino acid in the liver (Li et al, 2007). They suggested that albumin could be used as a biomarker of CPF-toxicity.…”

Immunodetection of Serum Albumin Adducts as Biomarkers for Organophosphorus Exposure

“…To test the lowest level of detection of the antibodies, we conducted a titration experiment by serially diluting an authentic HSA-GD protein standard. Previously, the amount of phosphonylated HSA Tyr 411 adducted with GD had been determined by tryptic digestion and LC-MS studies and was available as a standard (Li et al, 2007; O. Lockridge, personal communication).…”

“…An attempt to develop antibodies against phosphonylated BChE showed that the chemical instability of phosphonylated decapeptide haptens designed to mimic phosphonylated BChE precluded using this approach (MacDonald et al, 2010, and unpublished observations). In addition, despite the fact that AChE and BChE are prominent targets of OP exposure, these enzymes are present at low concentrations (e.g., BChE is present at 4 mg/ml in plasma) and the OP-BChE adduct is labile (Li et al, 2007). In contrast, serum albumin is the most abundant protein in blood plasma (40,000 mg/ml), and after OP exposure, OPs form a stable adduct (e.g., with human serum albumin [HSA] at Tyr 411) on the basis of LC-MS analysis of proteolytic digests .…”