Refinement of structural leads for centrally acting oxime reactivators of phosphylated cholinesterases

We present a systematic structural optimization of uncharged but ionizable N-substituted 2-hydroxyiminoacetamido alkylamine reactivators of phosphylated human acetylcholinesterase (hAChE) intended to catalyze the hydrolysis of organophosphate (OP)-inhibited hAChE in the CNS. Starting with the initial lead oxime RS41A identified in our earlier study and extending to the azepine analog RS194B, reactivation rates for OP-hAChE conjugates formed by sarin, cyclosarin, VX, paraoxon, and tabun are enhanced severalfold in vitro. To analyze the mechanism of intrinsic reactivation of the OP-AChE conjugate and penetration of the blood-brain barrier, the pH dependence of the oxime and amine ionizing groups of the compounds and their nucleophilic potential were examined by UV-visible spectroscopy, (1)H NMR, and oximolysis rates for acetylthiocholine and phosphoester hydrolysis. Oximolysis rates were compared in solution and on AChE conjugates and analyzed in terms of the ionization states for reactivation of the OP-conjugated AChE. In addition, toxicity and pharmacokinetic studies in mice show significantly improved CNS penetration and retention for RS194B when compared with RS41A. The enhanced intrinsic reactivity against the OP-AChE target combined with favorable pharmacokinetic properties resulted in great improvement of antidotal properties of RS194B compared with RS41A and the standard peripherally active oxime, 2-pyridinealdoxime methiodide. Improvement was particularly noticeable when pretreatment of mice with RS194B before OP exposure was combined with RS194B reactivation therapy after the OP insult.     

Phosphoryl oxime inhibition of acetylcholinesterase during oxime reactivation is prevented by edrophonium.

Reactivation of organophosphate (OP)-inhibited acetylcholinesterase (AChE) is a key objective in the treatment of OP poisoning. This study with native, wild-type, and mutant recombinant DNA-expressed AChEs, each inhibited by representative OP compounds, establishes a relationship between edrophonium acceleration of oxime-induced reactivation of OP-AChE conjugates and phosphoryl oxime inhibition of the reactivated enzyme that occurs during reactivation by pyridinium oximes LüH6 and TMB4. No such recurring inhibition could be observed with HI-6 as the reactivator due to the extreme lability of the phosphoryl oximes formed by this oxime. Phosphoryl oximes formed during reactivation of the ethoxy methylphosphonyl-AChE conjugate by LüH6 and TMB4 were isolated for the first time and their structures confirmed by (31)P NMR. However, phosphoryl oximes formed during the reactivation of the diethylphosphoryl-AChE conjugate were not sufficiently stable to be detected by (31)P NMR. The purified ethoxy methylphosphonyl oximes formed during the reactivation of ethoxy methylphosphonyl-AChE conjugate with LüH6 and TMB4 are 10- to 22-fold more potent than MEPQ as inhibitors of AChE and stable for several hours at pH 7.2 in HEPES buffer. Reactivation of both ethoxy methylphosphonyl- and diethylphosphoryl-AChE by these two oximes was accelerated in the presence of rabbit serum paraoxonase, suggesting that organophosphorus hydrolase can hydrolyze phosphoryl oxime formed during the reactivation. Our results emphasize that certain oximes, such as LüH6 and TMB4, if used in the treatment of OP pesticide poisoning may cause prolonged inhibition of AChE due to formation of phosphoryl oximes.     

Analysis and sequencing of the active-site peptide from native and organophosphate-inactivated acetylcholinesterase by electrospray ionization, quadrupole/time-of-flight (QTOF) mass spectrometry

A method to identify and sequence recombinant mouse acetylcholinesterase (rMoAChE) including the native and organophosphate-modified active-site peptides was developed using capillary liquid chromatography with electrospray ionization, quadrupole/time-of-flight mass spectrometry. Addition of 2-propanol to the reversed-phase gradient system and a decreased gradient slope improved the peptide resolution and the signal of the active-site peptide. The highest protein coverage and active-site peptide signal were achieved when the rMoAChE:chymotrypsin ratio of 5:1 was used with digestion at 37 degrees C. rMoAChE and the active-site peptide were identified and sequenced from chymotryptic digests of native, methyl paraoxon-, and ethyl paraoxon-inactivated rMoAChE showing unequivocally that the exact modification site was the active-site serine.     

Direct analysis of the kinetic profiles of organophosphate-acetylcholinesterase adducts by MALDI-TOF mass spectrometry

A sensitive matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry procedure has been established for the detection and quantitation of acetylcholinesterase (AChE) inhibition by organophosphate (OP) compounds. Tryptic digests of purified recombinant mouse AChE (mAChE) were fractionally inhibited by paraoxon to form diethyl phosphoryl enzyme. The tryptic peptide of mAChE that contains the active center serine residue resolves to a molecular mass of 4331.0 Da. Phosphorylation of the enzyme by paraoxon results in covalent modification of the active center serine and a corresponding increase in molecular mass of the tryptic peptide by 136 Da. The relative abundance of AChE peptides containing a modified active center serine strongly correlates with the fractional inhibition of the enzyme, achieving a detection range of phosphorylated to nonphosphorylated enzyme of 5-95%. Modifications of AChE by OP compounds resulting in dimethyl, diethyl, and diisopropyl phosphoryl adducts have been monitored with subpicomole amounts of enzyme. The individual phosphorylated adducts of AChE that result from loss of one alkyl group from the inhibited enzyme (the aging reaction) and the reappearance of unmodified AChE (spontaneous reactivation) have been resolved by the kinetic profiles and relative abundance of species. Further, the tryptic peptide containing the active center serine of AChE, isolated from mouse brain by anion-exchange and affinity chromatography, has been monitored by mass spectrometry. Native brain AChE, purified from mice treated with sublethal doses of metrifonate, has demonstrated that enzyme modifications resulting from OP exposure can be detected in a single mouse brain. For dimethyl phosphorylated AChE, OP exposure has been monitored by the ratio of tryptic peptide peaks that correspond to unmodified (uninhibited and/or reactivated), inhibited, and aged enzyme.     

Evaluation of peracid activated organophosphates in studies of insecticide resistance conferred by insensitive acetylcholinesterases

Peracid activated organophosphorus (OP) insecticides were evaluated against electric eel and a range of insect acetylcholinesterases (AChE). Incubation of chlorpyrifos and sulprofos with meta-chloroperoxybenzoic acid (MCPBA) produced active metabolites capable of distinguishing between AChE enzymes conferring OP susceptibility and resistance in populations of the tobacco whitefly, Bemisia tabaci Gennadius, and the beet armyworm, Spodoptera exigua Hubner. These products were also active against enzymes present in the lygus bug Lygus hesperus Knight and the greenhouse whitefly, Trialeurodes vaporariorum Westwood. Although profenofos was active against electric eel AChE, incubation of the OP with MCPBA did not enhance its anti-AChE properties toward the eel or insect AChEs.     

Antibodies to liposomal phosphatidylserine and phosphatidic acid

Polyclonal antisera to phosphatidylserine or phosphatidic acid were induced in rabbits by injecting liposomes containing phosphatidylserine or phosphatidic acid and lipid A. Adsorption of antisera with liposomes containing different phospholipids revealed that some degree of reactivity with one or more phospholipids other than the immunizing phospholipid was often observed. However, cross-reactivity with other phospholipids was not a universal phenomenon, and one antiserum to phosphatidylserine failed to cross-react (i.e., was not adsorbed) with liposomes containing other phospholipids. All of the antisera were inhibited by soluble phosphorylated haptens (e.g., phosphocholine but not choline), but one of the antisera to phosphatidylserine was inhibited both by phosphoserine and by serine alone. Liposomal membrane composition influenced the activity of antiserum to phosphatidylserine. Regardless of whether unsaturated (beef brain) or saturated (dimyristoyl) phosphatidylserine was used in the immunizing liposomes, the antisera reacted more vigorously with liposomes containing unsaturated than saturated phosphatidylserine. We conclude that liposomes containing lipid A can serve as vehicles for stimulating polyclonal antisera to phosphatidylserine and phosphatidic acid. Although cross-reactivity with certain other phospholipids can be observed, sera from selected animals apparently can exhibit a high degree of specific activity to the immunizing phospholipid antigen.     

Immunochemical studies of mammalian brain acetylcholinesterase.

Abstract— Specific antibodies were raised in rabbits to acetylcholinesterase (AChE) from bovine caudate nucleus and the‘native’(14S + 18S) and globular (11S) forms of AChE from eel electric tissue. All AChE preparations were purified by affinity chromatography to a specific activity of 100–400 mmol acetylthiocholine hydrolyzed/mg protein/h. Antigenic specificities of the different enzyme forms were studied by immunodiffusion, Immunoelectrophoresis and micro-complement fixation. Minor differences in antigenic determinants were observed between the different molecular forms of electric tissue AChE. In crossover experiments using both eel AChE and bovine caudate AChE antisera there was complete absence of cross reactivity between the mammalian brain AChE and the different molecular forms of the electric tissue enzyme. Brain AChE activity was inhibited up to 50% in the presence of its antiserum.     

Inhibition of acetylcholinesterase by chromophore-linked fluorophosphonates

Fluorophosphonate (FP) head groups were tethered to a variety of chromophores (C) via a triazole group and tested as FPC inhibitors of recombinant mouse (rMoAChE) and electric eel (EEAChE) acetylcholinesterase. The inhibitors showed bimolecular inhibition constants (k_i) ranging from 0.3 × 10⁵ M^?1 min^?1 to 10.4 × 10⁵ M^?1 min^?1. When tested against rMoAChE, the dansyl FPC was 12.5-fold more potent than the corresponding inhibitor bearing a Texas Red as chromophore, whereas the Lissamine and dabsyl chromophores led to better anti-EEAChE inhibitors. Most inhibitors were equal or better inhibitors of rMoAChE than EEAChE. 3-Azidopropyl fluorophosphonate, which served as one of the FP head groups, showed excellent inhibitory potency against both AChE’s (? 1 × 10⁷ M^?1 min^?1) indicating, in general, that addition of the chromophore reduced the overall anti-AChE activity. Covalent attachment of the dabsyl-FPC analog to rMoAChE was demonstrated using size exclusion chromatography and spectroscopic analysis, and visualized using molecular modeling.     

Crystal structures of aged phosphonylated acetylcholinesterase: nerve agent reaction products at the atomic level.

Organophosphorus acid anhydride (OP) nerve agents are potent inhibitors which rapidly phosphonylate acetylcholinesterase (AChE) and then may undergo an internal dealkylation reaction (called "aging") to produce an OP-enzyme conjugate that cannot be reactivated. To understand the basis for irreversible inhibition, we solved the structures of aged conjugates obtained by reaction of Torpedo californica AChE (TcAChE) with diisopropylphosphorofluoridate (DFP), O-isopropylmethylphosponofluoridate (sarin), or O-pinacolylmethylphosphonofluoridate (soman) by X-ray crystallography to 2.3, 2.6, or 2.2 A resolution, respectively. The highest positive difference density peak corresponded to the OP phosphorus and was located within covalent bonding distance of the active-site serine (S200) in each structure. The OP-oxygen atoms were within hydrogen-bonding distance of four potential donors from catalytic subsites of the enzyme, suggesting that electrostatic forces significantly stabilize the aged enzyme. The active sites of aged sarin- and soman-TcAChE were essentially identical and provided structural models for the negatively charged, tetrahedral intermediate that occurs during deacylation with the natural substrate, acetylcholine. Phosphorylation with DFP caused an unexpected movement in the main chain of a loop that includes residues F288 and F290 of the TcAChE acyl pocket. This is the first major conformational change reported in the active site of any AChE-ligand complex, and it offers a structural explanation for the substrate selectivity of AChE.     

Active site mutant acetylcholinesterase interactions with 2-PAM, HI-6, and DDVP

We used mouse recombinant wild-type acetylcholinesterase (AChE; EC 3.1.1.7), butyrylcholinesterase (BChE; EC 3.1.1.8), and AChE mutants with mutations (Y337A, F295L, F297I, Y72N, Y124Q, and W286A) that resemble residues found at structurally equivalent positions in BChE, to find the basis for divergence between AChE and BChE in following reactions: reversible inhibition by two oximes, progressive inhibition by the organophosphorus compound DDVP, and oxime-assisted reactivation of the phosphorylated enzymes. The inhibition enzyme-oxime dissociation constants of AChE w.t. were 150 and 46 microM, of BChE 340 and 27 microM for 2-PAM and HI-6, respectively. Introduced mutations lowered oxime binding affinities for both oximes. DDVP progressively inhibited cholinesterases yielding symmetrical dimethylphosphorylated enzyme conjugates at rates between 104 and 105/min/M. A high extent of oxime-assisted reactivation of all conjugates was achieved, but rates by both oximes were up to 10 times slower for phosphorylated mutants than for AChE w.t.     

Structural changes of phenylalanine 338 and histidine 447 revealed by the crystal structures of tabun-inhibited murine acetylcholinesterase

Organophosphorus compounds (OPs) interfere with the catalytic mechanism of acetylcholinesterase (AChE) by rapidly phosphorylating the catalytic serine residue. The inhibited enzyme can at least partly be reactivated with nucleophilic reactivators such as oximes. The covalently attached OP conjugate may undergo further intramolecular dealkylation or deamidation reactions, a process termed "aging" that results in an enzyme considered completely resistant to reactivation. Of particular interest is the inhibition and aging reaction of the OP compound tabun since tabun conjugates display an extraordinary resistance toward most reactivators of today. To investigate the structural basis for this resistance, we determined the crystal structures of Mus musculus AChE (mAChE) inhibited by tabun prior to and after the aging reaction. The nonaged tabun conjugate induces a structural change of the side chain of His447 that uncouples the catalytic triad and positions the imidazole ring of His447 in a conformation where it may form a hydrogen bond to a water molecule. Moreover, an unexpected displacement of the side chain of Phe338 narrows the active site gorge. In the crystal structure of the aged tabun conjugate, the side chains of His447 and Phe338 are reversed to the conformation found in the apo structure of mAChE. A hydrogen bond between the imidazole ring of His447 and the ethoxy oxygen of the aged tabun conjugate stabilizes the side chain of His447. The displacement of the side chain of Phe338 into the active site gorge of the nonaged tabun conjugate may interfere with the accessibility of reactivators and thereby contribute to the high resistance of tabun conjugates toward reactivation.     

Biochemical monitoring of acetylcholinesterase sensitivity to organophosphorus insecticides in glassy-winged sharpshooter Homalodisca coagulata Say (Homoptera: Cicadellidae) and smoke-tree sharpshooter H. lacerta Fowler

The glassy-winged sharpshooter Homalodisca coagulata Say (Homoptera: Cicadellidae) is a new pest to California agriculture. It is the principal vector of several plant pathogenic diseases, particularly Pierce's Disease in grapevines, and oleander leaf scorch. A microplate-based assay is described that measures the sensitivity of acetylcholinesterase (AChE) activity to inhibition by organophosphorus (OP) insecticides in this important pest. The technique provides users with an accurate measure of the efficacy of OP binding to this target site, and is a valuable tool for monitoring field populations of the insect to determine whether the use of OP insecticides has selected for resistant individuals. The technique will also measure AChE sensitivity in the smoke-tree sharpshooter, H. lacerta Fowler. This species is native to California and is regarded only as a minor pest. Both inhibition and kinetic measurements for the AChE enzymes in these sharpshooters demonstrate the close phylogenetic relationships between the two species.     

Role of the N-terminal region of the regulatory light chain in the dephosphorylation of myosin by myosin light chain phosphatase.

Myosin regulatory light chain (RLC) is phosphorylated at various sites at its N-terminal region, and heterotrimeric myosin light chain phosphatase (MLCP) has been assigned as a physiological phosphatase that dephosphorylates myosin in vivo. Specificity of MLCP toward the various phosphorylation sites of RLC was studied, as well as the role of the N-terminal region of RLC in the dephosphorylation of myosin by MLCP. MLCP dephosphorylated phosphoserine 19, phosphothreonine 18, and phosphothreonine 9 efficiently with almost identical rates, whereas it failed to dephosphorylate phosphorylated serine 1/serine 2. Deletion of the N-terminal seven amino acid residues of RLC markedly decreased the dephosphorylation rate of phosphoserine 19 of RLC incorporated in the myosin molecule, whereas this deletion did not significantly affect the dephosphorylation rate of isolated RLC. On the other hand, deletion of only four N-terminal amino acid residues showed no effect on dephosphorylation of phosphoserine 19 of incorporated RLC. The inhibition of dephosphorylation by deletion of the seven N-terminal residues was also found with the catalytic subunit of MLCP. Phosphorylation at serine 1/serine 2 and threonine 9 did not influence the dephosphorylation rate of serine 19 and threonine 18 by MLCP. These results suggest that the N-terminal region of RLC plays an important role in substrate recognition of MLCP.     

Generation of a monoclonal antibody that has reduced binding activity to VX-inactivated butyrylcholinesterase (BuChE) compared to BuChE by phage display

Organophosphate (OP) nerve agents are known as the most toxic chemical warfare agents that act by inhibiting the enzyme acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). Because BuChE is present at a level of about 3,900 times higher than AChE in plasma, most OP agents first react with BuChE in plasma, suggesting that OP-inactivated BuChE (OP-iBuChE) may act as a biomarker of OP exposure. In this study, we generated an anti-BuChE monoclonal antibody (mAb) that has reduced binding activity to VX-inactivated BuChE compared to native BuChE by phage display. We performed subtractive biopanning of a synthetic human Fab library against native BuChE and soman-iBuChE or VX-iBuChE. As the results, we isolated four Fab clones that showed differential binding activities to VX-iBuChE and native BuChE in ELISAs. To confirm the antigen-binding specificity of the selected clones, the Fabs were converted to IgG1s, and the IgG antibodies were expressed in HEK293F cells and purified. One of them (A2) showed approximately 30% reduced binding activity to VX-iBuChE compared to BuChE in a dose-dependent manner, whereas the other three antibodies showed almost the same binding activities to VX-iBuChE and BuChE. In addition, the A2 antibody did not show reduced binding activity to sarin-iBuChE or soman-iBuChE compared to native BuChE. The results indicate that A2 antibody shows reduced binding activity only to VX-iBuChE. A2 antibody may be applied to specific diagnosis of VX exposure.     

A Globular, Not Asymmetric, Form of Acetylcholinesterase Is Expressed in Chick Motor Neurons: Down-Regulation Toward Maturity and After Denervation

Abstract: In vertebrate neuromuscular junctions, the postsynaptic specializations include the accumulation of acetylcholinesterase (AChE) at the synaptic basal lamina and the muscle fiber. Several lines of evidence indicate that the presynaptic motor neuron is able to synthesize and secrete AChE at the neuromuscular junctions. By using anti-AChE catalytic subunit, anti-butyrylcholinesterase (BuChE) catalytic subunit, and anti-AChE collagenous tail monoclonal antibodies, we demonstrated that the motor neurons of chick spinal cord expressed AChE in vivo and the predominant AChE was the globular form of the enzyme. Neither asymmetric AChE nor BuChE was detected in the motor neurons. The molecular mass of AChE catalytic subunit in the motor neuron was ∼105 kDa, which was similar to that of the globular enzyme from low-salt extracts of muscle; both of them were ∼5 kDa smaller than the asymmetric AChE from high-salt extracts of muscle. The level of AChE expression in the motor neurons decreased, as found by immunochemical and enzymatic analysis, during the different stages of the chick's development and after nerve lesion. Thus, the AChE activity at the neuromuscular junctions that is contributed by the presynaptic motor neurons is primarily the globular, not the asymmetric, form of the enzyme, and these contributions decreased toward maturity and after denervation.     

A theoretical model of the competition between hydrolase and carboxylesterase in protection against organophosphorus poisoning

A system of coupled non-linear differential equations describing interactions between organophosphorus compounds (OPs), OP hydrolase, acetylcholinesterase (AChE), and carboxylesterase (CaE) in a single compartment was derived incorporating irreversible combination of OP with AChE, hydrolytic breakdown of OP, and irreversible combination of OP with CaE. The equations were then uncoupled, providing non-linear differential equations on AChE, CaE and OP concentrations. One steady state solution of the AChE equation provided theoretical expressions for the amounts of OP hydrolyzed, bound with CaE, and bound with AChE. Assuming that the LD50 of an OP reflects the dose that depletes AChE to a 'minimal essential' level and that a single compartment model is applicable in vivo, the steady state solution becomes an equation predicting the LD50 from rate constants, initial enzyme levels, and the allowable AChE depletion. Normalization by initial AChE concentration produced a dimensionless relationship describing an 'OP toxicity surface' that clearly demonstrates regions where hydrolysis and CaE offer protection against OP poisoning. The surface can be used to theoretically predict an LD50 given only kinetic rate constants and effective whole-body AChE and CaE levels. Predictions of LD50s of seven OPs in rats were compared with published data. The relationship was found to adequately predict published LD50s spanning 5 orders of magnitude. The OP toxicity surface relationship provides a conceptual tool for use in OP toxicity research but should be particularly useful in predicting the relative protective effects of catalytic and stoichiometric scavenger mechanisms for an OP.     

Oxime-assisted acetylcholinesterase catalytic scavengers of organophosphates that resist aging

The cholinesterases, acetylcholinesterase (AChE) and butyrylcholinesterase, are primary targets of organophosphates (OPs). Exposure to OPs can lead to serious cardiovascular complications, respiratory compromise, and death. Current therapy to combat OP poisoning involves an oxime reactivator (2-PAM, obidoxime, TMB4, or HI-6) combined with atropine and on occasion an anticonvulsant. Butyrylcholinesterase, administered in the plasma compartment as a bio-scavenger, has also shown efficacy but is limited by its strict stoichiometric scavenging, slow reactivation, and a propensity for aging. Here, we characterize 10 human (h) AChE mutants that, when coupled with an oxime, give rise to catalytic reactivation and aging resistance of the soman conjugate. With the most efficient human AChE mutant Y337A/F338A, we show enhanced reactivation rates for several OP-hAChE conjugates compared with wild-type hAChE when reactivated with HI-6 (1-(2'-hydroxyiminomethyl-1'-pyridinium)-3-(4'-carbamoyl-1-pyridinium)). In addition, we interrogated an 840-member novel oxime library for reactivation of Y337A/F338A hAChE-OP conjugates to delineate the most efficient oxime-mutant enzyme pairs for catalytic bio-scavenging. Combining the increased accessibility of the Y337A mutation to oximes within the space-impacted active center gorge with the aging resistance of the F338A mutation provides increased substrate diversity in scavenging potential for aging-prone alkyl phosphate inhibitors.     

Mechanistic studies of phosphoserine phosphatase, an enzyme related to P-type ATPases

Phosphoserine phosphatase belongs to a new class of phosphotransferases forming an acylphosphate during catalysis and sharing three motifs with P-type ATPases and haloacid dehalogenases. The phosphorylated residue was identified as the first aspartate in the first motif (DXDXT) by mass spectrometry analysis of peptides derived from the phosphorylated enzyme treated with NaBH(4) or alkaline [(18)O]H(2)O. Incubation of native phosphoserine phosphatase with phosphoserine in [(18)O]H(2)O did not result in (18)O incorporation in residue Asp-20, indicating that the phosphoaspartate is hydrolyzed, as in P-type ATPases, by attack of the phosphorus atom. Mutagenesis studies bearing on conserved residues indicated that four conservative changes either did not affect (S109T) or caused a moderate decrease in activity (G178A, D179E, and D183E). Other mutations inactivated the enzyme by >80% (S109A and G180A) or even by >/=99% (D179N, D183N, K158A, and K158R). Mutations G178A and D179N decreased the affinity for phosphoserine, suggesting that these residues participate in the binding of the substrate. Mutations of Asp-179 decreased the affinity for Mg(2+), indicating that this residue interacts with the cation. Thus, investigated residues appear to play an important role in the reaction mechanism of phosphoserine phosphatase, as is known for equivalent residues in P-type ATPases and haloacid dehalogenases.     

磷酸酪氨酸蛋白专一抗体的制备和纯化

以偶联磷酸化酪氨酸的牛血清白蛋白（ＢＳＡ）作为免疫原免疫兔获得抗血清．自抗血清中分离获得抗体．自酪胺合成磷酸酪胺,并偶联到溴化氰活化的Ｓｅｐｈａｒｏｓｅ４Ｂ上．抗体经磷酸酪胺－Ｓｅｐｈａｒｏｓｅ４Ｂ亲和柱纯化,所得抗体专一性强,Ｄｏｔｂｌｏｔ显示：抗体仅对酪氨酸磷酸化的蛋白质包括酪氨酸磷酸化的血清白蛋白,溶菌酶,卵清蛋白起抗原抗体反应,而不识别非酪氨酸磷酸化的溶菌酶,卵清蛋白,也不识别作为免疫原的骨架成分ＢＳＡ,也不识别丝氨酸磷酸化的卵清蛋白和苏氨酸磷酸化的卵清蛋白．     

Research into selective biomarkers of erythrocyte exposure to organophosphorus compounds

Flowcytometric procedures provide distinct advantages over the colorimetric methods currently in use to monitor erythrocytes for exposure of patients to organophosphorus (OP) pesticides and chemical warfare agents; therefore, they warrant exploration. Two types of fluorescent probes—one to detect the total acetylcholinesterase on erythrocytes (RBC-AChE) and the other to distinguish between the active and OP-inhibited RBC-AChE—have been explored. Our studies demonstrate that a fluorescently conjugated fasciculin can be used to monitor total, active, and OP-inhibited RBC-AChE. However, a fluorescently tagged potent inhibitor of AChE, TZ2PIQ-A6 with a K_d of 33 fM, did not distinguish between the active and OP-inhibited RBC-AChE, nor did three different biotinylated OP compounds. The biotin-fluorescent avidin approach is not a viable procedure for monitoring RBC-AChE. Western blot studies indicate that there are at least 20 serine hydrolases on the surface of red blood cells (RBCs). Plans currently under way for the development of more specific probes to distinguish between active and OP-inhibited RBC-AChE are discussed.