Search

Enzymatic catalysis is one means of accelerating the rate of hydrolysis of G-type organophosphorus nerve agents. Here, the stereospecificity of the catalysis of cyclosarin (GF, O -cyclohexyl methylphosphonofluoridate) hydrolysis by several enzymes was investigated. Stereospecificity was not evident at 3 mM GF but was evident at 0.5 mM GF. The differential effect was apparently due to fluoride-catalyzed racemization of the substrate. Alteromonas sp. JD6.5 organophosphorus acid anhydrolase (OPAA), Alteromonas haloplanktis OPAA and the wild-type phosphotriesterase (PTE) enzymes were all found to catalyze preferentially the hydrolysis of the (+)GF isomer, as determined by GC analysis of the remaining unreacted (−)GF isomer. Acetylcholinesterase inhibition experiments showed the purified (−)GF isomer to be approximately twice as toxic as the racemic mixture. One PTE mutant, H254G/H259W/L303T, was found to reverse the native PTE stereospecificity and preferentially catalyze the hydrolysis of the (−)GF isomer, as shown by its complementation of Alteromonas sp. JD6.5 OPAA and by GC analysis of the remaining (+)GF isomer. This procedure also permitted the individual preparation of either of the two GF isomers by enzymatic degradation followed by extraction of the remaining isomer.

Organophosphorus acid anhydrolases (OPAA; E.C.3.1.8.2) are a class of enzymes that hydrolyze a variety of toxic acetylcholinesterase-inhibiting organophosphorus (OP) compounds, including pesticides and fluorine-containing chemical nerve agents. In this paper, subphase conditions have been optimized to obtain stable OPAA Langmuir films, and the diisopropylfluorophosphate (DFP) hydrolysis reaction catalyzed by OPAA in aqueous solution and at the air-water interface was studied. OPAA-DFP interactions were investigated utilizing different spectroscopic techniques, that is, circular dichroism and fluorescence in aqueous solution and infrared reflection absorption spectroscopies at the air-water interface. The characterization of OPAA and its secondary structure in aqueous solution and as a monolayer at the air-water interface in the absence and in the presence of DFP dissolved in aqueous solution or in the aqueous subphase demonstrated significantly distinctive features. The research described herein demonstrated that OPAA can be used in an enzyme-based biosensor for DFP detection.

The paraoxon hydrolysis reaction catalyzed by organophosphorus hydrolase (OPH) monolayer at the air-water interface was studied. OPH-paraoxon interactions, occurring at the two-dimensional interface, by close-packed, highly orientated OPH monolayer, were investigated by several different surface chemistry techniques; e.g. surface pressure area isotherms, atomic force microscopy (AFM), and in situ epifluorescence microscopy. The characterization of OPH Langmuir and Langmuir-Blodgett films prepared in both the presence and absence of paraoxon, demonstrated significantly distinctive feature when compared with one another. Continuous growth of the OPH aggregates is a distinct phenomenon associated with hydrolysis, in addition to the pH changes in the local environment of the enzyme macromolecules.

Langmuir–Blodgett (LB) film deposition technique was employed for the immobilization of organophosphorus hydrolase (OPH). OPH enzyme was covalently bonded to a fluorescent probe, fluorescein isothiocyanate (FITC), and used as a biological recognition element. Under optimal experimental conditions, OPH monolayers were deposited onto the surface of silanized quartz slides as LB film and utilized as a bioassay for the detection of paraoxon. Two different methods were employed for detection of paraoxon: the fluorescence quenching of the fluorescence probe (FITC) covalently bonded to OPH and the UV–vis absorption spectrum of the paraoxon hydrolysis product. The UV–vis absorption measurement demonstrated a linear relationship between the absorbance at 400 nm and the concentration of paraoxon solutions over the range of 1.0 × 10−7–1.0 × 10−5 M (0.27–27 ppm). By observing the FITC fluorescence quenching, the concentration of paraoxon can be detected as low as 10−9 M (S/N = 3). The research described herein showed that the LB film bioassay had high sensitivity, rapid response time and good reproducibility.

The secondary structure of organophosphorus hydrolase (OPH) has been studied with circular dichroism (CD) spectroscopy in the far-UV region. The effect of pH on the secondary structure of OPH solution was examined over the pH range from 3.56 to 9.60. As shown on the CD spectra, the secondary structure of OPH is well defined when the pH value is near the isoelectric point (7.6); however, it is destroyed when the pH values are increased or decreased further. This is explained by the loss of helical structure. The pH effect on CD spectra contributes to clarify the optimum pH needed to obtain a stable OPH Langmuir film at the air−water interface and its correlation to the secondary structure of the enzyme. Comparative study of the thermal treatment on the secondary structure of OPH in solution, Langmuir−Blodgett film, and dry film shows that the molecular arrangement plays a dominant role in the thermal stability of OPH. With use of the CDPro software package a quantitative estimation of the secondary structu...

Layer-by-layer (LbL) assembly has been utilized to fabricate an ultrathin film of polyelectrolytes. The architecture was composed of chitosan and organophosphorus hydrolase polycations along with thioglycolic acid-capped CdSe quantum dots (QDs) as the polyanion. The topography of the films was studied using epifluorescence microscopy imaging. The photoluminescence property of the functionalized QDs improved when sandwiched between the polycation layers. The enhanced optical property of QDs allowed easy monitoring of LbL growth and detection of paraoxon with high sensitivity. The presence of organophosphorus compounds was confirmed through UV−vis and emission spectroscopies.

In this paper, we describe the preparation and characterization of Langmuir and Langmuir-Blodgett (LB) monolayers of the enzyme organophosphorus acid anhydrolase (OPAA). Langmuir films of OPAA were characterized on different subphases, such as phosphate, ammonium carbonate, and bis-tris-propane buffers. Monolayers at the air-water interface were characterized by measuring the surface pressure and surface potential-area isotherms. In situ UV-vis absorption spectra were also recorded from the Langmuir monolayers. The enzyme activity at the air-water interface was tested by the addition of diisopropylfluorophosphate (DFP) to the subphase. LB films of OPAA were transferred to mica substrates to be studied by atomic force microscopy. Finally, a one-layer LB film of OPAA labeled with a fluorescent probe, fluorescein isothiocyanate (FITC), was deposited onto a quartz slide to be tested as sensor for DFP. The clear, pronounced response and the stability of the LB film as a DFP sensor show the potential of this system as a biosensor.

The aim of this study is to immobilize an enzyme, namely, organophosphorus hydrolase (OPH), and to detect the presence of paraoxon, which is an organophosphorus compound, using the layer-by-layer (LbL) deposition technique. To lift the OPH from the solid substrate, a pair of polyelectrolytes (positively charged chitosan (CS) and negatively charged poly(thiophene-3-acetic acid) (PTAA)) were combined. These species were made charged by altering the pH of the solutions. LbL involved alternate adsorption of the oppositely charged polyions from dilute aqueous solutions onto a hydrophilic quartz slide. This polyion cushion was held together by the electrostatic attraction between CS and PTAA. The growing process was monitored by fluorescence spectroscopy. OPH was then adsorbed onto the five-bilayer CS/PTAA system. This five-bilayer macromolecular structure compared to the solid substrate rendered stability to the enzyme by giving functional integrity in addition to the ability to react with paraoxon solutions. The ultimate goal is to use such a system to detect the presence of organophosphorus compounds with speed and sensitivity using the absorption and fluorescence detection methodologies.

Organophosphorus hydrolase (OPH), immobilized within polyelectrolyte multilayers deposited on glass beads (30−50 μm), showed sustained catalytic activity under ambient conditions for several months...

The composition of the subphase to obtain a stable organophosphorus hydrolase (OPH) monolayer was reexamined. The surface pressure−area (π−A) isotherms show that a subphase at pH 7.6 with 0.5 M KCl...

The ability of the enzyme organophosphorus acid anhydrolase (OPAA) to selectively hydrolyze the PF bond of fluorine containing organophosphates has been used to develop a biosensor for specific detection of these compounds. Hydrolysis rate of diisopropyl fluorophosphate (DFP), paraoxon and demeton-S, by soluble and immobilized OPAA was measured. These compounds were selected as representative substrates of OPAA hydrolysis of PF, PO and PS bonds, respectively. Results indicate that hydrolysis of phosphofluoridates such as DFP is dominant while hydrolysis of phosphotriesters such as paraoxon or of phosphothiolates such as demeton-S, is negligible. Two experimental approaches were used for biosensor development. In the first, OPAA was covalently immobilized on silica gel and used in batch-mode measurements with flat glass pH electrode to detect pH changes due to PX bond cleavage. In the second approach, the enzyme was covalently immobilized to the porous silica modified gate insulator of a pH-sensitive field effect transistor (pH-FET) and changes in pH relative to a second non-enzyme coated pH-FET were measured in stop-flow mode. Concentrations of DFP down to 25 μM with the glass electrode and 20 μM with the pH-FET were readily detected. No sensor response was observed with paraoxon or demeton-S indicating that such OPAA-based biosensors could be useful for direct and discriminative detection of fluorine containing organophosphorus neurotoxins (such as the G-type chemical warfare agents sarin GB and soman GD) in samples also containing multiple organophosphate pesticides.

Enzymes catalyzing the hydrolysis of highly toxic organophosphorus compounds (OPs) are classified as organophosphorus acid anhydrolases (OPAA; EC 3.1.8.2). Recently, the genes encoding OPAA from two species of Alteromonas were cloned and sequenced. Sequence and biochemical analyses of the cloned genes and enzymes have established Alteromonas OPAAs to be prolidases (E.C. 3.4.13.9), a type of dipeptidase hydrolyzing dipeptides with a prolyl residue in the carboxyl-terminal position (X-Pro). Alteromonas prolidases hydrolyze a broad range of G-type chemical warfare (CW) nerve agents. Efforts to over-produce a prolidase from A. sp.JD6.5 with the goal of developing strategies for long-term storage and decontamination have been successfully achieved. Large-scale production of this G-agent degrading enzyme is now feasible with the availability of an over-producing recombinant cell line. Use of this enzyme for development of a safe and non-corrosive decontamination system is discussed.

We report on the immobilization of an OPAA enzyme on luminescent porous silicon devices, and on the utilization of this new platform to hydrolyze p-nitrophenyl-soman.

Organophosphorus acid (OPA) anhydrolases offer considerable potential for safe, non-corrosive decontamination of chemical nerve agents. The Alteromonas sp. strain JD6.5 gene encoding an OPA anhydrolase (designated as OPAA-2), which hydrolyzes a wide variety of nerve agents, has been cloned in Escherichia coli . Employing agent-analog diisopropyl fluorophosphate (DFP) as a substrate, the effects of buffers, pH, temperature, and various protein stabilizing agents on OPAA-2 activity were studied. Ammonium carbonate, which is innocuous and inexpensive, proved to be a superior buffer for enzyme activity. Compared with enzyme assayed under standard conditions, enzyme activity with ammonium carbonate was six-fold greater. To evaluate effects of storage and reconstitution on enzyme activity, the cloned enzyme was lyophilized, rehydrated, and then assessed by measuring activity against DFP. Whereas almost 100% of the hydrolytic activity was recovered with enzyme reconstituted in (NH 4 ) 2 CO 3 -buffered distilled water or chlorinated drinking water, approximately 20% of the activity was recovered with ocean water. Enzyme stability in blast-containment foam or fire-fighting foam was also demonstrated by high activity in (NH 4 ) 2 CO 3 -buffered distilled water or drinking water. These findings suggest the potential of a foam-based enzyme system for field decontamination of chemical nerve agents.

Organophosphorus acid (OPA) anhydrolase enzymes have been found in a wide variety of prokaryotic and eukaryotic organisms. Interest in these enzymes has been prompted by their ability to catalyze the hydrolysis of toxic organophosphorus cholinesterase-inhibiting compounds, including pesticides and chemical nerve agents. The natural substrates for these enzymes are unknown. The gene (opaA) which encodes an OPA anhydrolase (OPAA-2) was isolated from an Alteromonas sp. strain JD6.5 EcoRI-lambda ZAPII chromosomal library expressed in Escherichia coli and identified by immunodetection with anti-OPAA-2 serum. OPA anhydrolase activity expressed by the immunopositive recombinant clones was demonstrated by using diisopropylfluorophosphate (DFP) as a substrate. A comparison of the recombinant enzyme with native, purified OPAA-2 showed they had the same apparent molecular mass (60 kDa), antigenic properties, and enzyme activity against DFP and the chemical nerve agents sarin, soman, and O-cyclohexyl methylphosphonofluoridate. The gene expressing this activity was found in a 1.74-kb PstI-HindIII fragment of the original 6.1-kb EcoRI DNA insert. The nucleotide sequence of this PstI-HindIII fragment revealed an open reading frame of 1,551 nucleotides, coding for a protein of 517 amino acid residues. Amino acid sequence comparison of OPAA-2 with the protein database showed that OPAA-2 is similar to a 647-amino-acid sequence produced by an open reading frame which appears to be the E. coli pepQ gene. Further comparison of OPAA-2, the E. coli PepQ protein sequence, E. coli aminopeptidase P, and human prolidase showed regions of different degrees of similarity or functionally conserved amino acid substitutions. These findings, along with preliminary data confirming the presence of prolidase activity expressed by OPAA-2, suggest that the OPAA-2 enzyme may, in nature, be used in peptide metabolism.

Amplification and detection of target DNA sequences are made possible in a polymerase chain reaction (PCR) by using a mixture of biotinylated and ruthenium(II) trisbipyridal (Ru(bpy)3(2+))-end-labelled primers. In this way, biotin for capture and Ru(bpy)3(2+) for detection are directly incorporated into the PCR product obviating subsequent probe hybridization. PCR of a bacterial DNA template from Alteromonas species strain JD6.5 using a cocktail of biotin- and Ru(bpy)3(2+)-labelled primers amplified a 1 kilobase region. Serial dilution of PCR product followed by magnetic separation with Streptavidin (SA)-coated magnetic beads and an electrochemiluminescence (ECL) assay using the semi-automated QPCR System 5000 demonstrated sensitive (pg range) DNA detection. ECL assay of probe hybridization to a human immunodeficiency virus (HIV) sequence also produced pg level sensitivity. Quantitative DNA determination by ECL assay correlated well with visual detection of DNA in electrophoretic gels. However, DNA detection by ECL assay was 10 to 100 times more sensitive than conventional ethidium bromide staining. The combination of DNA-based magnetic separation with ECL assay provides a very sensitive and rapid method of quantitating DNA which, owing to its rapid and facile nature, may have many applications in the research, environmental monitoring, industrial and clinical fields.

A polyelectrolyte architecture was fabricated that was composed of chitosan and organophosphorus hydrolase polycations along with thioglycolic acid-capped CdSe quantum dots (QDs) as the polyanion. This film was imaged by epifluorescence microscopy. UV−vis and emission spectroscopies were used to monitor the growth of the bilayer film due to the enhanced optical property of QDs. Photoluminescence of the functionalized QDs improved when sandwiched between the polycations layers. The presence of organophosphorus compounds was confirmed through photoluminescence spectroscopy.

Previously, a G-type nerve agent degrading enzyme activity was found in a halophilic bacterial isolate designated JD6.5. This organism was tentatively identified as an unknown species of the genus Alteromonas. In order to determine whether this type of enzyme activity was common in other species of Alteromonas, a screening program was initiated. A number of Alteromonas species and five halophilic bacterial isolates were cultured and their crude cell extracts screened for hydrolytic activity against several organophosphorus chemical agents and other related compounds. The samples were also screened for cross-reactivity with a monoclonal antibody raised against the purified enzyme from JD6.5 and for hybridization with a DNA probe based on its N-terminal amino acid sequence A wide spectrum of activities and reactivities were seen, suggesting a significant heterogeneity between the functionally similar enzymes that are present in these bacterial species. Enzymes of the type described here have considerable potential for the decontamination and demilitarization of chemical warfare agents.

A moderately halophilic bacterial isolate has been found to possess high levels of enzymatic activity against several highly toxic organophosphorus compounds. The predominant enzyme, designated organophosphorus acid anhydrase 2, has been purified 1,000-fold to homogeneity and characterized. The enzyme is a single polypeptide with a molecular weight of 60,000. With diisopropylfluorophosphate as a substrate, the enzyme has optimum activity at pH 8.5 and 50 degrees C, and it is stimulated by manganese and cobalt.

In this paper, we first report a novel biosensor for the detection of paraoxon based on (CdSe)ZnS core-shell quantum dots (QDs) and an organophosphorus hydrolase (OPH) bioconjugate. The OPH was coupled to (CdSe)ZnS core-shell QDs through electrostatic interaction between negatively charged QDs surfaces and the positively charged protein side chain and ending groups (-NH2). Circular dichroism (CD) spectroscopy showed no significant change in the secondary structure of OPH after the bioconjugation, which indicates that the activity of OPH was preserved. Detectable secondary structure changes were observed by CD spectroscopy when the OPH/QDs bioconjugate was exposed to organophosphorus compounds such as paraoxon. Photoluminescence (PL) spectroscopic study showed that the PL intensity of the OPH/QDs bioconjugate was quenched in the presence of paraoxon. The overall quenching percentage as a function of paraoxon concentration matched very well with the Michaelis-Menten equation. This result indicated that the quenching of PL intensity was caused by the conformational change in the enzyme, which is confirmed by CD measurements. The detection limit of paraoxon concentration using OPH/QDs bioconjugate was about 10(-8) M. Although increasing the OPH molar ratio in the bioconjugates will slightly increase the sensitivity of biosensor, no further increase of sensitivity was achieved when the molar ratio of OPH to QDs was greater than 20 because the surface of QDs was saturated by OPH. These properties make the OPH/QDs bioconjugate a promising biosensor for the detection of organophosphorus compounds.

The goal of our research is to combine porous silicon and enzymes in order to build hybrid platforms for extremely selective chemical sensing applications. For this, a new synthetic route to covalently anchor bio-molecules on photo-luminescent porous silicon (PL PSi) while preserving the optical properties of the matrix was developed. The hydride terminated porous silicon surface was covalently functionalized with t-butyloxycarbonyl protected amine by light-assisted hydrosysilation. Protein cross-linker chemistry was then used to extend the linker and immobilize various enzymes. The glu-coronidase enzyme/p-nitro-phenyl-beta-glucoronide substrate test system provided a proof of concept for an enzyme-based porous silicon detector. The enzymatic activity and the luminescence of the porous silicon platform were both retained after the functionali-zation procedure and, charge transfer between the products of the enzymatic breakdown and the silicon quantum dots was demonstrated. The organophosphorous hydrolase enzyme OPAA was then immobilized and tested on p-nitrophenyl-soman, a surrogate substrate for soman. The production of the hydrolysis product, p-nitrophenol, correlated with the reversible luminescence quenching of the porous silicon matrix demonstrating the relevance of the enzyme-based platform for detection applications. This detection scheme, although indirect, takes advantage of the extreme specificity of enzymes. The approach is general and can be implemented for a series of target molecules.

The reactivation of organophosphate (OP)-inhibited acetylcholinesterase (AChE) by oximes results inevitably in the formation of highly reactive phosphyloximes (POX), which may re-inhibit the enzyme. An impairment of net reactivation by stable POX was found with 4-pyridinium aldoximes, e.g. obidoxime, and a variety of OP compounds. In this study the effect of organophosphorus hydrolase (OPH), organophosphorus acid anhydrolase (OPAA) and diisopropylfluorophosphatase (DFPase) on obidoxime-induced reactivation of human acetylcholinesterase (AChE) inhibited by different OPs was investigated. Reactivation of paraoxon-, sarin-, soman- and VX-inhibited AChE by obidoxime was impaired by POX-induced re-inhibition whereas no deviation of pseudo first-order kinetics was observed with tabun, cyclosarin and VR. OPH prevented (paraoxon) or markedly reduced the POX-induced re-inhibition (VX, sarin, soman), whereas OPAA and DFPase were without effect. Additional experiments with sarin-inhibited AChE indicate that the POX hydrolysis by OPH was concentration-dependent. The activity of OP-inhibited AChE was not affected by OPH in the absence of obidoxime. In conclusion, OPH may be a valuable contribution to the therapeutic regimen against OP poisoning by accelerating the degradation of both the parent compound, OP, and the reaction product, POX.

In this paper, we describe the preparation and characterization of Langmuir and Langmuir-Blodgett (LB) monolayers of the enzyme organophosphorus acid anhydrolase (OPAA). Langmuir films of OPAA were characterized on different subphases, such as phosphate, ammonium carbonate, and bis-tris-propane buffers. Monolayers at the air-water interface were characterized by measuring the surface pressure and surface potential-area isotherms. In situ UV-vis absorption spectra were also recorded from the Langmuir monolayers. The enzyme activity at the air-water interface was tested by the addition of diisopropylfluorophosphate (DFP) to the subphase. LB films of OPAA were transferred to mica substrates to be studied by atomic force microscopy. Finally, a one-layer LB film of OPAA labeled with a fluorescent probe, fluorescein isothiocyanate (FITC), was deposited onto a quartz slide to be tested as sensor for DFP. The clear, pronounced response and the stability of the LB film as a DFP sensor show the potential of this system as a biosensor.

The GV (2-dialkylaminoalkyl N,N-dialkylphosphonamidofluoridate) nerve agent has a toxicity intermediate to G and V-type nerve agents and is not catalyzed by either organophosphorus acid anhydrolases (OPAA) or organophosphorus hydrolase (OPH) enzymes. We have screened and identified a number of Alteromonas strains possessing catalytic activity using a GV compound as substrate. The enzyme from one of these strains, A. undina, has been purified to homogeneity by ammonium sulfate fractionation and Q Sepharose anion exchange chromatography. The activity of GV-hydrolyzing enzyme peak is distinct from that of A. undina OPAA following the Q Sepharose column chromatography. The SDS-polyacrylamide gel electrophoresis of the GV-hydrolyzing enzyme fraction revealed a single polypeptide of ^ 20kDa. To our knowledge, this is the first report of enzymatic detoxification of GV.

The primary objective was to identify and clone novel chromosomal DNA fragments for use as B. anthracis-specific markers. Towards this goal, 300 random primers (RAPD technology, randomly amplified polymorphic DNA) were screened to identify polymorphic loci on the anthrax chromosome. Five such DNA fragments uniquely amplifying from anthrax chromosome were identified and isolated. These fragments were cloned in pCR vector and sequenced. Database (genebank) analysis of one of the cloned probe, VRTC899, revealed the presence of specific chromosomal DNA probe, Ba813 from anthrax. This prove also contains flanking DNA with no homology to known sequences. Availability of signature DNA probes for detection of antrax-causing agent in environmental samples is critical for field application of DNA-based sensor technologies. In conclusion, we have demonstrated application of RAPD technology for identification of anthrax-specific signature sequences. This strategy can be extended to identify signature sequences from other BW agents.© (2001) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

Subphase conditions have been optimized to obtain stable organophosphorous hydrolase (OPH-EC 3.1.8.1) as Langmuir films. The Langmuir film was characterized by surface pressure and surface potential-area isotherms and UV-Vis spectroscopy in situ. The interaction of an organophosphorous compound, namely Paraoxon, with the OPH film was investigated for various surface pressures. The stability of the monolayer and the evidence of the enzyme activity at air-water interface support the use of enzyme LB films as biosensor.

The enzyme OPAA hydrolyzes p-nitrophenyl phosphotriesters bearing substituents at the phosphorus center ranging in size from methyl to phenyl. The enzyme exhibits stereoselectivity toward the hydrolysis of chiral substrates with a preference for the Sp enantiomer.

Numerous organophosphorus (OP) compounds of importance in agriculture, medicine, military defense, and research have been shown to be potent inhibitors of cholinesterases and other enzymes with active serine residues in their active sites. Enzymes that catalyze the hydrolysis of OP compounds have been under investigation for over 50 years. These enzymes result in the detoxification of a variety of these highly toxic compounds, including chemical warfare (CW) nerve agents and pesticides. For military operations, enzyme-based decontamination systems offer considerable potential for replacing current materials that are toxic, highly corrosive, flammable, and a danger to the environment.

The Russian-VX (R-VX) is the principle V-type nerve agent in the chemical warfare (CW) arsenal of the Former Soviet Union. We here report the enzymatic hydrolysis of the P-S bond of Russian-VX by organophosphorus hydrolase (OPH) from Pseudomonas diminuta. While the Michaelis constant, K(m) for R-VX (474 microM), was similar to that for VX (434 microM), the Vmax for R-VX (2.1 mumoles/mg/min) was about four-fold higher compared to that for VX (0.56 mumoles/mg/min). A 50% inhibition in the rate of the enzymatic hydrolysis of R-VX was observed in the presence of 0.5% ethanol, isoamyl-alcohol, or isopropanol. The presence of acetonitrile, diethylene glycol, or methanol had marginal effects. These results comprise the first demonstration of enzymatic detoxification of R-VX.

Organophosphorus acid anhydrolases (OPAA) catalyzing the hydrolysis of a variety of toxic organophosphorus cholinesterase inhibitors offer potential for decontamination of G-type nerve agents and pesticides. The gene (opa) encoding an OPAA was cloned from the chromosomal DNA of Alteromonas haloplanktis ATCC 23821. The nucleotide sequence of the 1.7-kb DNA fragment contained the opa gene (1.3 kb) and its flanking region. We report structural and functional similarity of OPAAs from A. haloplanktis and Alteromonas sp JD6.5 with the enzyme prolidase that hydrolyzes dipeptides with a prolyl residue in the carboxyl-terminal position. These results corroborate the earlier conclusion that the OPAA is a type of X-Pro dipeptidase, and that X-Pro could be the native substrate for such an enzyme in Alteromonas cells.

A highly active organophosphorus acid anhydrolase from Alteromonas undina was purified to homogeneity and found to be composed of a single polypeptide chain with a molecular weight of 53,000. With diisopropylfluorophosphate as a substrate, the purified enzyme has a specific activity of ∼575 μmol/min/mg of protein. The enzyme has optimum activity at pH 8.0 and 55�C and is stimulated by sulfhydryl reducing agents and manganese. It is capable of rapidly hydrolyzing a wide range of nerve agents and several chromogenic phosphinates.

Messenger RNA activity for α and β chains of hemoglobin has been detected in the RNA of free polyribosomes from splenic erythroblasts following phenylhydrazine injection of mice. This activity accumulates rapidly from 66 hours after injection, reaching a maximum at 114 hours. After polyacrylamide gel electrophoresis, the messenger RNA activity for both α and β chains is found in a single RNA band having an estimated molecular weight of 150,000. In contrast, globin messenger RNA isolated from rabbit reticulocytes has an estimated molecular weight of 190,000. When rabbit reticulocyte lysate was mixed with mouse spleens prior to homogenization, the RNA isolated was deficient in the 190,000-dalton reticulocyte mRNA, but contained an excess of the 150,000-dalton RNA which directed the synthesis in vitro of predominantly rabbit α and β chains. Thus, we infer that the globin mRNA obtained from mouse spleen erythroblasts has been modified by ribonuclease activity during its isolation. This modified mRNA is also deficient in polyadenylic acid, as judged by its behavior on oligo(dT) cellulose chromatography. We conclude that globin mRNA lacking a portion of its untranslated structure may still retain considerable mRNA template activity. Moreover, since globin messenger RNA activity is isolated at a specific time of cellular differentiation in the erythropoietic mouse spleen, this system appears well suited for the study of regulation of transcription.

Relative amounts and rates of synthesis of alpha- and beta-globin mRNAs were determined during splenic erythropoiesis in mice. At times after injection of mice with phenylhydrazine, alpha- and beta-globin mRNAs were separated by gel electrophoresis and quantitated by densitometric scanning of stained gels. At 66 hr after injection, the ratio of beta to alpha mRNA is about 1.2. By 138 hr, total globin mRNA is 5-fold greater in spleen cells, and the beta to alpha mRNA ratio approaches 2. This ratio remains around 1 in reticulocytes throughout this period. Analyses of globin products directed by these mRNAs from spleen cells and reticulocytes in the ascites cell-free system reflect the beta to alpha mRNA ratio observed by electrophoresis. Relative rates of synthesis of globin mRNAs were estimated after incubation of spleen cells with either [3H] uridine or [3H] adenosine. Although synthesis of both mRNAs is maximal at 114 hr and then declines sharply, beta mRNA is synthesized at a greater rate than alpha mRNA at every developmental stage. In contrast to the excess accumulation of beta mRNA in spleen cells, synthesis of alpha- and beta-globin chains remains balanced throughout erythroid development. These data suggest that during erythropoiesis in this system, equal synthesis of alpha and beta globin involves regulation at both transcriptional and post-transcriptional levels.

DNA sequence analysis of a cloned beta-globin gene from a Chinese patient with beta-thalassemia revealed a single nucleotide substitution (A leads to G) within the ATA box homology and 28 base pairs upstream from the cap site. The patient was homozygous for this particular allele based on restriction mapping at nine different polymorphic sites in the beta-globin gene cluster. Upon transient expression in HeLa cells this gene directed the production of 3-5-fold less beta-globin mRNA than the normal beta-gene. In RNA isolated from the patient's erythroid cells beta-RNA was 10-fold less abundant relative to alpha-RNA than normal, indicating close approximation of the heterologous cell expression results and the in vivo state. These findings support the validity of such transient expression assays for analysis of phenotypes associated with naturally occurring mutant genes and establish the functional significance of nucleotide substitutions at position -28 for human beta-globin gene transcription.

To perform a systematic analysis of beta-thalassemia genes among Chinese, we have determined the DNA haplotype in the beta-globin gene region of 37 Chinese beta-thalassemia chromosomes. Only four haplotypes were found. Blot hybridization analysis of erythroid RNA from patients homozygous for haplotypes 1, 2, and 3 demonstrated different patterns, suggesting that a different mutation was associated with each haplotype. The mutation associated with haplotype 1 was a C----T substitution at IVS-2, position 654. This mutation produces a new donor splice site and leads to formation of a beta-globin RNA with an insertion of 73 nucleotides. The mutation associated with haplotype 2 was a nucleotide insertion of A between codons 71 and 72, which results in a frameshift and premature termination of beta-globin synthesis. Haplotype analysis suggests that these two mutations may account for up to 85% of beta-thalassemia genes in this ethnic group. The haplotype 3 gene contained a transcriptional "TATA" box mutation that has been previously reported. Oligonucleotide hybridization demonstrated that the mutation associated with haplotype 4 was the same IVS-1 position 5 substitution commonly observed among beta-thalassemia genes in Asian Indians. Since haplotype 4 of Chinese differs at polymorphic sites on either side of the IVS-1 position 5 mutation from the haplotype associated with this mutation in Indians, the mutation presumably arose independently in these two populations.

A beta-globin gene cloned from a person with beta-thalassemia contained a T----C substitution within the conserved sequence AATAAA that forms a portion of the recognition signal for endonucleolytic cleavage and polyadenylation of primary mRNA transcripts. By Northern blot analysis a novel beta-globin RNA species, 1500 nucleotides in length, was detected in erythroid RNA. Nuclease protection studies of erythroid RNA, as well as RNA generated upon transient expression of the cloned mutant gene in HeLa cells, located the 3' terminus of this novel, polyadenylated RNA 900 nucleotides downstream of the normal poly(A) addition site, within 15 nucleotides of the first AATAAA in the 3'-flanking region of the beta-globin gene. These findings define the in vivo terminus of an elongated RNA and establish that human beta-globin transcription may extend at least 900 nucleotides 3' of the normal polyadenylation site.