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 -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.Human albumin has been reported to have esterase activity with p-nitrophenyl acetate (1, 2), ␣-naphthyl acetate, phenyl acetate, 1-naphthyl N-methylcarbamate (3), -naphthyl acetate (4), aspirin (5), ketoprofen glucuronide (6), carprofen acylglucuronide (7), cyclophosphamide (8), nicotinate esters (9), longand short-chain fatty acid esters (10), octanoyl ghrelin (11), organophosphorus pesticides (12), carbaryl (13), o-nitrotrifluoroacetanilide (14), o-nitroacetanilide (15), and nerve agents (16).One site in albumin is rapidly acetylated by p-nitrophenyl acetate, showing a burst of product, but up to 5.2 molar equivalents are incorporated when albumin is treated with a 9-fold excess of p-nitrophenyl [14 C]acetate (1). The 5 equivalents of label are not removable by extensive dialysis. 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 acti...
Butyrylcholinesterase (BChE) is an efficient bioscavenger of highly toxic organophosphorus poisons and nerve agents. However, BChE administered into the periphery does not provide significant protection of the central nervous system (CNS) due to rejection by the blood brain barrier. In this study, we evaluated the feasibility of delivering BChE to the CNS by packing it into a block ionomer complex of nanoscale size with a cationic poly(L-lysine)-graft-poly(ethylene oxide) (PLL-g-PEO) copolymer. The multimolecular structure of BChE/PLL-g-PEO complexes was further reinforced by formation of cross-links between the polymer chains. The resulting cross-linked complexes were stable against dilution without significant loss of BChE enzymatic activity. In some cases the BChE was labeled with fluorescent IRDye 800CW before it was incorporated into nanoparticles. BChE/PLL-g-PEO complexes were injected into mice intramuscularly and intravenously. In vivo imaging showed incorporation of the fluorescently labeled BChE in brain. Activity assays showed that BChE remained active in the brain at 72 hour post injection. It was concluded that nanocomplexes can deliver the 340 kDa BChE tetramer to the brain.
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