Human albumin is thought to hydrolyze esters because multiple equivalents of product are formed for each equivalent of albumin. Esterase activity with p-nitrophenyl acetate has been attributed to turnover at tyrosine 411. However, p-nitrophenyl acetate creates multiple, stable, acetylated adducts, a property contrary to turnover. Our goal was to identify residues that become acetylated by p-nitrophenyl acetate and determine the relationship between stable adduct formation and turnover. Fatty acid-free human albumin was treated with 0.5 mm p-nitrophenyl acetate for 5 min to 2 weeks, or with 10 mm p-nitrophenyl acetate for 48 h to 2 weeks. Aliquots were digested with pepsin, trypsin, or GluC and analyzed by mass spectrometry to identify labeled residues. Only Tyr-411 was acetylated within the first 5 min of reaction with 0.5 mm p-nitrophenyl acetate. After 0.5–6 h there was partial acetylation of 16–17 residues including Asp-1, Lys-4, Lys-12, Tyr-411, Lys-413, and Lys-414. Treatment with 10 mm p-nitrophenyl acetate resulted in acetylation of 59 lysines, 10 serines, 8 threonines, 4 tyrosines, and Asp-1. When Tyr-411 was blocked with diisopropylfluorophosphate or chlorpyrifos oxon, albumin had normal esterase activity with β-naphthyl acetate as visualized on a nondenaturing gel. However, after 82 residues had been acetylated, esterase activity was almost completely inhibited. The half-life for deacetylation of Tyr-411 at pH 8.0, 22 °C was 61 ± 4 h. Acetylated lysines formed adducts that were even more stable. In conclusion, the pseudo-esterase activity of albumin is the result of irreversible acetylation of 82 residues and is not the result of turnover.

Acetylcholinesterase (AChE) is the physiologically important target for organophosphorus toxicants (OP) including nerve agents and pesticides. Butyrylcholinesterase (BChE) in blood serves as a bioscavenger that protects AChE in nerve synapses from inhibition by OP. Mass spectrometry methods can detect exposure to OP by measuring adducts on the active site serine of plasma BChE. Genetic variants of human AChE and BChE do exist, but loss of function mutations have been identified only in the BCHE gene. The most common AChE variant, His353Asn (H322N), also known as the Yt blood group antigen, has normal AChE activity. The most common BChE variant, Ala567Thr (A539T) or the K-variant in honor of Werner Kalow, has 33% reduced plasma BChE activity. The genetic variant most frequently associated with prolonged response to muscle relaxants, Asp98Gly (D70G) or atypical BChE, has reduced activity and reduced enzyme concentration. Early studies in young, healthy males, performed at a time when it was legal to test nerve agents in humans, showed that individuals responded differently to the same low dose of sarin with toxic symptoms ranging in severity from minimal to moderate. Additionally, animal studies indicated that BChE protects from toxicants that have a higher reactivity with AChE than with BChE (e.g., nerve agents) but not from toxicants that have a higher reactivity with BChE than with AChE (e.g., OP pesticides). As a corollary, we hypothesize that individuals with genetic variants of BChE may be at increased risk of toxicity from nerve agents but not from OP pesticides.

Exposure to organophosphorus (OP) agents can lead to learning and memory deficits. Disruption of axonal transport has been proposed as a possible explanation. Microtubules are an essential component of axonal transport. In vitro studies have demonstrated that OP agents react with tubulin and disrupt the structure of microtubules. Our goal was to determine whether in vivo exposure affects microtubule structure. One group of mice was treated daily for 14 days with a dose of chlorpyrifos that did not significantly inhibit acetylcholinesterase. Beta-tubulin from the brains of these mice was diethoxyphosphorylated on tyrosine 281 in peptide GSQQY₂₈₁RALTVPELTQQMFDSK. A second group of mice was treated with a single sublethal dose of chlorpyrifos oxon (CPO). Microtubules and cosedimenting proteins from the brains of these mice were visualized by atomic force microscopy nanoimaging and by Coomassie blue staining of polyacrylamide gel electrophoresis bands. Proteins in gel slices were identified by mass spectrometry. Nanoimaging showed that microtubules from control mice were decorated with many proteins, whereas microtubules from CPO-treated mice had fewer associated proteins, a result confirmed by mass spectrometry of proteins extracted from gel slices. The dimensions of microtubules from CPO-treated mice (height 8.7 ± 3.1 nm and width 36.5 ± 15.5 nm) were about 60% of those from control mice (height 13.6 ± 3.6 nm and width 64.8 ± 15.9 nm). A third group of mice was treated with six sublethal doses of CPO over 50.15 h. Mass spectrometry identified diethoxyphosphorylated serine 338 in peptide NS₃₃₈NFVEWIPNNVK of beta-tubulin. In conclusion, microtubules from mice exposed to chlorpyrifos or to CPO have covalently modified amino acids and abnormal structure, suggesting disruption of microtubule function. Covalent binding of CPO to tubulin and to tubulin-associated proteins is a potential mechanism of neurotoxicity.