Erythrocytes were washed three times with two volumes of sodium/potassium phosphate buffer (0.1 M, pH 7.4). Then, the packed erythrocytes were diluted in 20 volumes of hypotonic sodium/potassium phosphate buffer (6.7 mM,
pH 7.4) to facilitate the hemolysis, followed by centrifugation at 30,000g (30 min, 4 °C). The supernatant was removed and the pellet resuspended in hypotonic phosphate buffer. After two additional washing cycles, the pellet was resuspended in sodium/potassium phosphate buffer (0.1 M, see more pH 7.4), passed through one more centrifugation at 30,000g (30 min, 4 °C) and were kindly removed. Next, the AChE activity was adjusted to the original activity by appropriate dilution with phosphate buffer (0.1 M, pH 7.4). Aliquots of the erythrocyte selleck compound ghosts were stored at −20 °C until use. Hemoglobin content present in ghost membranes was measured at 540 nm as the cyano-met-Hb form, but no hemoglobin was detected. Ghost erythrocyte acetylcholinesterase and human plasma butyrylcholinesterase activities were estimated by Ellman method (Ellman et al., 1961), using acetylthiocholine iodide as substrate. The rate of hydrolysis of acetylthiocholine iodide is measured at 412 nm through the release of the thiol compound that, when reacted with DTNB, produces the color-forming compound TNB. Whole
blood AChE was measured by Ellman method (Ellman et al., 1961) with modifications (Worek et al., 1999a and Worek et al., 1999b). For butyrylcholinesterase activity, the same protocol was used, but butyrylthiocoline iodide was used as the substrate. Ghost erythrocyte acetylcholinesterase and human plasma butyrylcholinesterase were exposed to IBTC in two different assay conditions in order to identify a possible protective or a reactivation capacity of IBTC:
(a) Protection: the enzyme was exposed to methamidophos (MAP) 25 μM and IBTC (10–100 μM) at the same time into a total incubation period of 60 min. The Bay 11-7085 different protocols aim to test the prophylactic and therapeutic effect of IBTC on MAP-induced AChE inhibition. The protein content was determined as described previously (Lowry et al., 1951) using bovine serum albumin (BSA) as standard. Docking simulations of the oximes with Mus musculus AChE were carried out using AutoDock Vina 1.1.1 ( Trott and Olson, 2010), followed by redocking with Autodock 4.0.1. The non-aged MAP-inhibited Mus musculus AChE obtained from the RCSB Protein Data Bank (http://www.rcsb.org/pdb/) was used as macromolecule (PDB code 2jge). IBTC was constructed using the program Avogadro 0.9 and their geometry were optimized with the MMFF 94 force field. Both ligand and macromolecule are previously prepared using AutoDock Tools ( Morris et al., 2009) and Chimera 1.5 ( Pettersen et al., 2004). All rotatable bonds within the ligands were allowed to rotate freely, and the receptor was considered rigid.