Use of Hupresin To Capture Red Blood Cell Acetylcholinesterase for Detection of Soman Exposure

Abstract

Toxicity from acute exposure to nerve agents and organophosphorus toxicants is due to irreversible inhibition of acetylcholinesterase (AChE) in the nervous system. AChE in red blood cells is a surrogate for AChE in the nervous system. Previously we developed an immunopurification method to enrich red blood cell AChE (RBC AChE) as a biomarker of exposure. The goal of the present work was to provide an alternative RBC AChE enrichment strategy, by binding RBC AChE to Hupresin affinity gel. AChE was solubilized from frozen RBC by addition of 1% Triton X-100. Insoluble debris was removed by centrifugation. The red, but not viscous, RBC AChE solution was loaded on a Hupresin affinity column. Hemoglobin and other proteins were washed off with 3 M NaCl, while retaining AChE bound to Hupresin. Denatured AChE was eluted with 1% trifluoroacetic acid. The same protocol was used for 20 mL of RBC AChE inhibited with a soman model compound. The acid denatured protein was digested with pepsin and analyzed by liquid chromatography tandem mass spectrometry on a 6600 Triple-TOF mass spectrometer. A targeted method identified the aged soman adduct on serine 203 in peptide FGESAGAAS. It was concluded that Hupresin can be used to enrich soman-inhibited AChE solubilized from 8 mL of frozen human erythrocytes, yielding a quantity sufficient for detecting soman exposure.

Purification of recombinant human butyrylcholinesterase on Hupresin®

“…It can be released with denaturing agents such as 1% trifluoroacetic acid or 50% acetonitrile [24]. This limits the application of Hupresin ® for purification of HuAChE to projects that can make use of denatured enzyme, such as detection of nerve agent exposure by mass spectrometry[24]. CHEMFORASE is synthesizing and testing new affinity ligands that will be useful for purifying AChE.…”

“…Hupresin ® has been used to isolate sarin-modified BChE tetramers from human plasma [19] and soman-modified AChE dimers from human red blood cells [24]. The yield of sarin-modified BChE was sufficiently high that the modified active site peptide could be detected by mass spectrometry.…”

Bone Morphogenetic Protein 4 Alleviates DSS-Induced Ulcerative Colitis Through Activating Intestinal Stem Cell by Target ID3

“…The preparation of a protein sample was performed as the method previously described (León et al, 2013). Data acquisition was performed with a Triple-TOF 6600 mass spectrometer (Sciex, United States) fitted with a Nanospray III source (Sciex, United States) (Onder et al, 2018). The ion spray voltage was 2,300 V, declustering potential was 80 V, curtain gas was 35 psi, nebulizer gas was 5 psi, and interface heater temperature was 150 • C. Peptides were introduced into the mass spectrometer using Nona 415 liquid chromatography (Sciex, United States).…”

Comparison of Hupresin^® to Procainamide-Sepharose for Purification of Butyrylcholinesterase and Acetylcholinesterase

“…HuAChE is not released from Hupresin ® by nondenaturing buffers. It can only be released with denaturing agents such as 1% trifluoroacetic acid or 50% acetonitrile 33 . This limits the application of Hupresin ® for purification of HuAChE to projects that can make use of denatured enzyme, such as…”

“…Hupresin ® has been used to isolate sarin-modified BChE tetramers from human plasma 3 and soman-modified AChE dimers from human red blood cells 33 . The yield of sarin-modified BChE was sufficiently high that the modified active site peptide could be detected by mass spectrometry.…”