Synthesis, characterization, and antibacterial activities of novel methacrylate polymers containing norfloxacin

A novel methacrylate monomer containing a quinolone moiety was synthesized and homopolymerized in N,N-dimethylformamide (DMF) by using azobisisobutyronitrile (AIBN) as an initiator. The new monomer was copolymerized with poly(ethylene glycol) methyl ether methacrylate (MPEGMA) in DMF using the same initiator. The monomer, homopolymer, and copolymer were characterized by elemental analysis, thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), size exclusion chromatography (SEC), FTIR, (13)C NMR, and (1)H NMR. The antibacterial activities of the monomer as well as polymers were investigated against Staphylococcus aureus and Escherichia coli, which are representative of Gram-positive and Gram-negative bacteria, respectively. All compounds showed excellent antibacterial activities against these two types of bacteria. The antibacterial activities were determined using the shaking flask method, where 25 mg/mL concentrations of each compound were tested against 10(5) CFU/mL bacteria solutions. The number of viable bacteria was calculated by using the spread plate method, where 100 microL of the incubated antibacterial agent in bacteria solutions were spread on agar plates and the number of viable bacteria was counted after 24 h of incubation period at 37 degrees C.     

Direct amidation of poly(gamma-glutamic acid) with benzylamine in dimethyl sulfoxide

Partially benzylamidated, amphipathic poly(gamma-glutamic acid) (BzPGA) was synthesized from poly(gamma-glutamic acid) (PGA) and benzylamine by direct amidation in dimethyl sulfoxide (DMSO). Benzylamine and PGA were heated in DMSO for 1 to 26 h at temperatures between 110 and 130 degrees C, producing derivatives of various degrees of benzylamidation as a function of the reaction time and temperature. Neither any carboxyl-activating agent nor catalyst is needed for the reaction to proceed. After purification by dialysis, the product was identified by 1H and 13C 1D and 2D NMR in DMSO-d(6). BzPGA prepared by the new direct amidation method was identical to that obtained with a conventional carbodiimide-mediated reaction in water. The one-pot amidation procedure described in the present article can probably be applied to the synthesis of amides from other amines and carboxylic acids.     

Chemoenzymatic synthesis of sucrose-containing aromatic polymers.

A chemoenzymatic approach was developed to prepare sucrose-containing aromatic polymers. The protease from Bacillus licheniformis catalyzed the transesterification of sucrose with a diester of terephthalic acid in pyridine to give the mono- and diester products. At 45 degrees C, >70% of sucrose was consumed after 1 day and sucrose diester began to form after 6 days when >95% of sucrose had been converted to sucrose monoester. The final yield of sucrose diester after 20 days was 13.8%. The sucrose monoester was identified as sucrose 1'-terephthalate and the diester products consisted of sucrose 6,1'-diterephthalate and sucrose 6',1'-diterephthalate in a ratio of 2:1. The sucrose diester products were polymerized with ethylene-glycol and ethylene-diamine to give poly(ethylene-terephthalate) and poly(ethylene-terephthalamide), with sucrose contained in the polymer backbone. The polycondensation reactions were carried out in dimethylsulfoxide (DMSO) at 70 degrees C using zinc acetate as a catalyst. The sucrose-containing polyester and polyamide were obtained at 65% yield for 24 h and at 73% yield for 12 h, respectively. End-group analysis of the polymers by (13)C-NMR or (1)H-NMR in DMSO provided a number average molecular weight of 3200 and 4300 Da, respectively. Structural analyses of the polymers were performed with (1)H-NMR, (13)C-NMR, and FTIR. On the basis of (13)C-NMR, acylation of the C1', C6, and C6' hydroxyls were maintained in the polymer backbones.     

Rates of heat-induced DNA purine alterations in synthetic polydeoxyribonucleotides

Damage to DNA by heat can occur at physiological conditions. The effects of the varying conformational states adopted by double-stranded DNA on the incidences and distributions of thermally induced hydrolytic purine alterations are unknown. The possible role of conformational changes on damage by heat to purines in DNA polymers was therefore investigated. Model compounds used were the synthetic alternating copolymer poly(dG-dC):poly(dG-dC) and the homopolymer poly(dG):poly(dC). Base damages were assayed by high performance liquid chromatography using polymers radioactively labeled in guanine. Conformational states were assayed by circular dichroic spectral changes. Incubation and heating of the polymers in 1 mM Mn2+ caused the spectral shift reported for the left-handed Z-DNA conformation in the alternating copolymer and the change reported for the triple helix in the homopolymer. After incubation at 85 degrees C., incidences of base damages were compared between the polymers. No deamination of guanine to xanthine was observed under any conditions. The presence of manganese reduced depurination in both polymers. Rates of guanine imidazole ring openings to yield 2,6-diamino-4-hydroxy-5-formamidopyrimidine were increased in the presence of the cation and constituted the chief form of purine damage in the homopolymer. Therefore, the distribution of heat-induced DNA alterations within the genome may be determined by DNA conformational states. This observed opening of purine imidazole rings in the presence of manganese ions may have mutagenic consequences and may be involved in carcinogenesis by metals.     

Degradation of polyesters by a novel marine Nocardiopsis aegyptia sp. nov.: application of Plackett-Burman experimental design for the improvement of PHB depolymerase activity

This is the first report on the degradation of poly(3-hydroxybutyrate) (PHB), and its copolymers poly(3-hydroxyvalerate) P(3HB-co-10-20% HV) by Nocardiopsis aegyptia, a new species isolated from marine seashore sediments. The strain excreted an extracellular PHB depolymerase and grew efficiently on PHB or its copolymers as the sole carbon sources. The degradation activity was detectable by the formation of a transparent clearing zone around the colony on an agar Petri plate after 25 days, or a clearing depth under the colony in test tubes within 3 weeks. The previous techniques proved that the bacterium was able to assimilate the monomeric components of the shorter alkyl groups of the polymers. Nocardiopsis aegyptia hydrolyzed copolymers 10-20% PHBV more rapidly than the homopolymer PHB. The bacterial degradation of the naturally occurring sheets of poly(3-hydroxybutyrate), and its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) was observed by scanning electron microscopy (SEM). The samples were degraded at the surface and proceeded to the inner part of the materials. Clear morphological alterations of the polymers were noticed, indicating the degradative capability of the bacterium. Plackett-Burman statistical experimental design has been employed to optimize culture conditions for maximal enzyme activity. The main factors that had significant positive effects on PHB depolymerase activity of Nocardiopsis aegyptia were sodium gluconate, volume of medium/flask and age of inoculum. On the other hand, MgSO4.7H2O, KH2PO4, K2HPO4 and NH4NO3 exhibited negative effects. Under optimized culture conditions, the highest activity (0.664 U/mg protein) was achieved in a medium predicted to be near optimum containing (in g/L): PHB, 0.5; C6H11O7Na, 7.5; MgSO4.7H2O, 0.35; K2HPO4, 0.35; NH4NO3, 0.5; KH2PO4, 0.35; malt extract, 0.5 and prepared with 50% seawater. The medium was inoculated with 1% (v/v) spore suspension of 7 days old culture. Complete clarity of the medium was achieved after 3 days at 30 degrees C.     

Nonspecific interaction of Escherichia coli pyrenyl RNA polymerase holoenzyme with synthetic polynucleotides as monitored by fluorescence spectroscopy

A derivative of RNA polymerase containing approximately 2 pyrene equiv per enzyme molecule has been used to study the interaction of RNA polymerase with poly[d(A-T)].poly[d(A-T)] and poly[d-(G-C)].poly[d(G-C)]. As monitored by fluorescence spectroscopy, pyrenyl RNA polymerase displays a unique set of conformational changes with each synthetic polynucleotide as a function of temperature. An increase in the fluorescence intensity was observed for both polynucleotides at 5 degrees C. A decrease was observed in the case of poly[d(A-T)].poly[d(A-T)] at 25 and 37 degrees C, whereas no discernible perturbation was observed in the case of poly[d(G-C)].poly[d(G-C)]. Different salt dependencies were observed for the interaction of pyrenyl RNA polymerase with these polynucleotides at 5 and 25 degrees C. Further characterization of these interactions as well as correlation of the observed fluorescence changes to the corresponding open and closed complexes was carried out with heparin. The interaction between pyrenyl RNA polymerase and poly[d-(A-T)].poly[d(A-T)] at 25 degrees C was quantified by using two different methods.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glycine derived polymerizable cosurfactant in the synthesis of functionalized poly(butyl acrylate) nanolatexes

The approach of employing N-glycinylmaleamic acid (NGMA) as an efficient cosurfactant to provide microemulsion polymerization of butyl acrylate using a weight ratio of sodium dodecyl sulfate (SDS)/butyl acrylate (BA) at 相似文献   

New 6-butylamino-6-deoxycellulose and 6-deoxy-6-pyridiniumcellulose derivatives with highest regioselectivity and completeness of reaction

This paper investigates the nucleophilic substitution (S(N)) reactions of tosylcellulose with butylamine and pyridine, respectively. The S(N) reactions of tosylcellulose 1 (DS(Total) 2.02; DS(C-6) 1.0) with butylamine carried out at 25, 50, 75 and 100 degrees C in both dimethyl sulfoxide (DMSO) and pure butylamine showed that the regioselectivity of substitution at C-6 of cellulose is temperature dependent: the highest regioselectivity at C-6 can be reached at 25 and 50 degrees C; substitution at C-2 also occurred at 75 and 100 degrees C. The substitution speed in pure butylamine is greater than that in the presence of DMSO. A complete and regioselective substitution at C-6 with a DS of 1.0 was obtained under the conditions of 50 degrees C, 40 h in butylamine. The substitution reactions of 1 with pyridine carried out at 25, 50, 75 and 100 degrees C for 24h in DMSO did not occur. In contrast to this the S(N) reactions done in pure pyridine showed that a temperature- and steric-dependent, regioselective substitution took place at C-6 at temperatures from 25 to 145 degrees C. The highest regioselectivity and completeness at C-6 can be obtained at 100 degrees C for 90 h, whereas at 145 degrees C substitution also occurs at C-2. The results were proved by 1H NMR and 13C NMR spectroscopy.     

Formation and stability of repairable pyrimidine photohydrates in DNA

Ultraviolet irradiation of poly(dG-dC) and poly(dA-dU) in solution produces pyrimidine hydrates that are repaired by bacterial and mammalian DNA glycosylases [Boorstein et al. (1989) Biochemistry 28, 6164-6170]. Escherichia coli endonuclease III was used to quantitate the formation and stability of these hydrates in the double-stranded alternating copolymers poly(dG-dC) and poly(dA-dU). When poly(dG-dC) was irradiated with 100 kJ/m2 of 254-nm light at pH 8.0, 2.2% of the cytosine residues were converted to cytosine hydrate (6-hydroxy-5,6-dihydrocytosine) while 0.09% were converted to uracil hydrate (6-hydroxy-5,6-dihydrouracil). To measure the stability of these products, poly(dG-dC) was incubated in solution for up to 24 h after UV irradiation. Cytosine hydrate was stable at 4 degrees C and decayed at 25, 37, and 55 degrees C with half-lives of 75, 25, and 6 h. Uracil hydrate produced in irradiated poly(dA-dU) was stable at 4 degrees C and at 25 degrees C and decayed with a half-life of 6 h at 37 degrees C and less than 0.5 h at 55 degrees C. Uracil hydrate and uracil were also formed in irradiated poly(dG-dC). These experiments demonstrate that UV-induced cytosine hydrate may persist in DNA for prolonged time periods and also undergo deamination to uracil hydrate, which in turn undergoes dehydration to yield uracil. The formation and stability of these photoproducts in DNA may have promoted the evolutionary development of the repair enzyme endonuclease III and analogous DNA glycosylase/endonuclease activities of higher organisms, as well as the development of uracil-DNA glycosylase.     

Rates of heat-induced pyrimidine alterations in synthetic polydeoxyribonucleotides

Hydrolytic damages to DNA can occur at physiological conditions. The possible role of DNA conformation on the distribution of such alterations of pyrimidines was investigated. Model compounds used were the synthetic alternating copolymer poly(dG-dC):poly(dG-dC) and the homopolymer poly(dG):poly(dC). Base damages were assayed by paper chromatography using polymers radioactively labeled in cytosine. Conformational changes were assayed by circular dichroic spectral changes. Incubation and heating of the polymers in 1 mM MnCl₂ caused the spectral shift reported for the left-handed Z-DNA conformation in the alternating copolymer and the change reported for the triple helix in the homopolymer. After incubation in 85°C, incidences of base damages were compared between the polymers. The presence of manganese reduced depyrimidination in both polymers. Rates of cytosine deamination to uracil were substantial and did not vary among the various conformational states.     

Linkage isomerization reaction of intrastrand cross-links in trans-diamminedichloroplatinum(II)-modified single-stranded oligonucleotides.

The stability of trans-(Pt(NH3)2[d(CGAG)-N7-G,N7-G]) adducts, resulting from cross-links between two guanine residues at d(CGAG) sites within single-stranded oligonucleotides by trans-diamminedichloro-platinum(II), has been studied under various conditions of temperature, salt and pH. The trans-(Pt(NH3)2[d(C GAG)-N7-G,N7-G]) cross-links rearrange into trans-(Pt(NH3)2[d(CGAG)-N3-C,N7-G]) cross-links. The rate of rearrangement is independent of pH, in the range 5-9, and of the nature and concentration of the salt (NaCl or NaCIO4) in the range 10-400 mM. The reaction rate depends upon temperature, the t1/2 values for the disappearance of the (G,G) intrastrand cross-link ranging from 120 h at 30 degrees C to 70 min at 80 degrees C. The linkage isomerization reaction occurs in oligonucleotides as short as the platinated tetramer d(CGAG). Replacement of the intervening residue A by T has no major effect on the reaction. The C residue adjacent to the adduct on the 5' side plays a key-role in the reaction; its replacement by a G, A or T residue prevents the reaction occuring. No rearrangement was observed with the C residue adjacent to the adduct on the 3' side. It is proposed that the linkage isomerization reaction results from a direct attack of the base residue on the platinum(II) square complex.     

The thermodynamics of drug-DNA interactions: ethidium bromide and propidium iodide

We report the first calorimetrically-derived characterization of the thermodynamics of ethidium bromide (EB) and propidium iodide (PI) binding to a series of nucleic acid host duplexes. Our spectroscopic and calorimetric measurements yield the following results: 1) At low salt (16mM Na+) and 25 degrees C. PI binds more strongly than EB to a given host duplex. The magnitude of this PI preference depends only marginally on base sequence, with AT base pairs showing a greater PI preference than GC base pairs. 2) The enhanced binding of PI relative to EB at low salt and 25 degrees C reflects a more favorable entropic driving force for PI binding. 3) The PI binding preference diminishes at higher salt concentrations (216mM). In other words, the binding preference is electrostatic in origin. 4) The salt dependence of the binding constants (delta lnKb/delta ln[Na+]) reveal that PI binds as a dication while EB binds as a monocation. 5) PI and EB both exhibit impressive enthalpy-entropy compensations when they bind to the deoxy homopolymers poly dA.poly dT and poly dA.poly dU. We have observed a similar enthalpy-entropy compensation for netropsin binding to the poly dA.poly dT homopolymer duplex. We therefore conclude that the compensation phenomenon is an intrinsic property of the host duplex rather than reflecting a property of the binding ligand. 6) When either PI or EB bind to the corresponding ribo homopolymer (poly rA.poy rU) we do not observe the enthalpy-entropy compensation that characterizes the binding to the deoxy homopolymer. 7) EB and PI both bind more strongly to poly d(AT).poly d(AT) than to poly d(AU).poly d(AU). Specifically, the absence of the thymine methyl group in poly d(AU).poly d(AU) reduces the binding constant of both drugs by a factor of four. This reduction in binding is due to a less favorable entropy change. In this paper we present and discuss possible molecular origins for our observed thermodynamic and extra-thermodynamic data. In particular, we evoke solvent effects involving both the drugs and the host duplexes when we propose molecular interpretations which are consistent with our thermodynamic data.     

Polyhydroxybutyrate-enhanced transformation of log-phase Escherichia coli

Transformation of Escherichia coli plays an important role in recombinant DNA technology. Most current transformation protocols require that the cells be treated to attain a particular physiological state known as "competence," and this makes transformation procedures lengthy and arduous. Here we describe a protocol for transforming log-phase E. coli using dimethyl sulfoxide (DMSO) solutions of poly-(R)-3-hydroxybutyrate (PHB) to facilitate the transfer of plasmid DNA into cells, and certain reagents and temperature shocks to promote DNA uptake. The protocol was optimized using factorial design techniques across variables that included PHB molecular weight and concentration, DMSO concentration, monovalent and divalent salts, glucose, cold and heat shocks, cell density, and pH. Using 10 ng DNA, the optimized protocol produces approximately 1000 colony-forming units (CFUs) from 100 microL early log-phase cell culture or approximately 300 CFU from a 21-24 h single colony, sufficient for many applications. The total volume of the transformation reaction mixture is only 150 microL suggesting that the procedure may be adapted for use in microplates or automated transformation technologies.     

Effects of maternal identity and incubation temperature on snapping turtle (Chelydra serpentina) metabolism

Individual variation in physiological traits may have important consequences for offspring survivorship and adult fitness. Variance in offspring phenotypes is due to interindividual differences in genotype, environment, and/or maternal effects. This study examined the contributions of incubation environment, maternal effects, and clutch identity to individual variation in metabolic rates in the common snapping turtle, Chelydra serpentina. We measured standard metabolic rate, as determined by oxygen consumption, for 246 individuals representing 24 clutches at 15 degrees and 25 degrees C, and we measured standard metabolic rates additionally for 34 individuals at 20 degrees and 30 degrees C. Standard metabolic rate for 34 snapping turtles measured at 15 degrees, 20 degrees, 25 degrees, and 30 degrees C increased with increasing temperature. Mean standard metabolic rate for 246 individuals was 0.247 microL O(2) min(-1) g(-1) at 15 degrees C and 0.919 microL O(2) min(-1) g(-1) at 25 degrees C. At 15 degrees C, mass at hatching, individual mass, and egg mass had no significant effects on metabolic rate, but at 25 degrees C, mass at hatching, individual mass, and egg mass did have significant effects on metabolic rate. Incubation temperature had no significant effect on metabolic rate at 15 degrees, but it did have a significant effect at 25 degrees C. Clutch identity had a significant effect on metabolic rate at both 15 degrees and 25 degrees C. Interindividual variation in standard metabolic rate due to incubation temperature, and especially clutch identity, could have large effects on energy budgets. Results suggest that there were both environmental and genetic effects on standard metabolic rate.     

Acetylation of α-chitin in ionic liquids

Acetylation of α-chitin using acetic anhydride in an ionic liquid, 1-allyl-3-methylimidazolium bromide (AMIMBr), was performed. First, a mixture of chitin and AMIMBr (2% w/w) was heated at 100 °C for 24 h for dissolution. Then, acetic anhydride (5–20 equiv) was added to the solution and the mixture was heated with stirring at desired temperatures for 24 h. The product was precipitated by the addition of the reaction mixture into methanol. The IR spectrum of the product indicated the progress of acetylation. The degrees of substitution (DS), which were determined from the IR spectra, increased with increasing the amounts of acetic anhydride used for the reaction. The highest DS was 1.86, which was obtained by the reaction using 20 equiv of acetic anhydride at 100 °C. The product with this DS value was soluble in DMSO, and thus the structure of the product was further confirmed by ¹H NMR spectroscopy in DMSO-d₆. The DS value estimated by the integrated ratio of signals due to acetyl protons to a signal due to anomeric protons was in good agreement with that determined from the IR spectrum.     

Recovery from potentially lethal damage of irradiated L5178Y-R and L5178Y-S cells: the effect of temperature and benzamide

Mouse lymphoma L5178 Y-S and Y-R cells differing in radiosensitivity by 1.5 times were treated with benzamide, an inhibitor of poly(ADP-ribosylation), for 24 h before and 18 h after X-irradiation, and incubated after irradiation at 25 degrees C and 37 degrees C. Clonogenic capacity of LY-S cells incubated at 25 degrees C exceeded that of the same cells incubated at 37 degrees C; the clonogenic capacity of LY-R cells did not vary with the postirradiation incubation temperature. Benzamide increased equally the radiosensitivity of LY-R cells incubated at both temperatures, whereas that of LY-S cells was only increased at 37 degrees C. Repair of potentially lethal damages to LY-S cells incubated at 25 degrees C was independent of the effectiveness of poly(ADP-ribosylation).     

In vitro hydrolytic degradation of hydroxyl-functionalized poly(alpha-hydroxy acid)s

The in vitro hydrolytic degradation of hydroxyl-functionalized poly(alpha-hydroxy acid)s was investigated. Benzyl-ether-protected hydroxyl-functionalized dilactones (S)-3-benzyloxymethyl-(S)-6-methyl-1,4-dioxane-2,5-dione (1a) and (S)-3-benzyloxymethyl-1,4-dioxane-2,5-dione (1b) were copolymerized in a melt with various amounts of L-lactide using benzyl alcohol and SnOct2 as the initiator and catalyst, respectively. The benzyl groups were removed by hydrogenation to yield polyesters with hydroxyl functional groups, poly(lactic acid-co-hydroxymethyl glycolic acid) and poly(lactic acid-co-glycolic acid-co-hydroxymethyl glycolic acid) (2a and 2b). Degradation of the hydroxyl-functionalized polyesters and poly(lactic-co-glycolic acid) (50/50) was studied by incubation of pellets of these polymers in phosphate buffer (174 mM, pH 7.4) at 37 degrees C. Polymer degradation was monitored by mass-loss measurements and by gel permeation chromatography, differential scanning calorimetry, and 1H NMR analysis. The degradation times ranging from less than 1 day (for the homopolymer of 2a) to 2 months (copolymer of 25% 2a and 75% lactide) were found. The degradation rates increased with increasing hydroxyl density of the polymers, which was associated with a switch from bulk to surface erosion. NMR and thermal analysis showed that the moieties with the hydroxyl groups were preferentially removed from the degrading polymer. In conclusion, this study shows that the degradation rate of polyesters containing 2a and 2b can be tailored from a few days to 2 months, making them very suitable for biomedical and pharmaceutical applications.     

Determination of the kinetics of deuteration of DNA.RNA hybrids by ultraviolet spectroscopy

The kinetics of the hydrogen-deuterium exchange reactions of poly(dA).poly(rU) and poly(rA).poly(dT) has been examined, at pH 7.0 and at various temperatures in the 15-35 degrees C range, by stopped-flow ultraviolet spectrophotometry. For comparison, the deuteration kinetics of poly[d(A-T)].poly[d(A-T)] and poly(rA).poly(rU) has been reexamined. At 20 degrees C, the imino deuteration (NH----ND) rates of the two hybrid duplexes were found to be 1.5 and 1.8 s-1, respectively. These are nearly equal to the imino deuteration rates of poly[d(A-T)].poly[d(A-T)] (1.1 s-1) and poly(rA).poly(rU) (1.5 s-1) but appreciably higher than that of poly(dA).poly(dT) (0.35 s-1). It has been suggested that a DNA.RNA hybrid, an RNA duplex, and the AT-alternating DNA duplex have in general higher base-pair-opening reaction rates than the ordinary DNA duplex. The amino deuteration (NH2----ND2) rates, on the other hand, have been found to be 0.25, 0.28, and 0.33 s-1, respectively, for poly(dA).poly(rU), poly(rA).poly(dT), and poly[d(A-T)].poly[d(A-T)], at 20 degrees C. These are appreciably higher than that for poly(rA).poly(rU) (0.10 s-1). In general, the equilibrium constants (K) of the base-pair opening are considered to be greatest for the DNA.RNA hybrid duplex (0.05 at 20 degrees C), second greatest for the RNA duplex (0.02 at 20 degrees C), and smallest for the DNA duplex (0.005 at 20 degrees C), although the AT-alternating DNA duplex has an exceptionally great K (0.07 at 20 degrees C). From the temperature effect on the K value, the enthalpy of the base-pair opening was estimated to be 3.0 kcal/mol for the DNA.RNA hybrid duplex.     

Acetone precipitation--an improved procedure for the isolation of soybean agglutinin

Isolation of soybean agglutinin (SBA) by the salt fractionation involves excessive amounts of (NH4)2SO4. We have found that SBA could be fractionally precipitated from an aqueous extract by adding acetone (40% final concentration). It is stable under these conditions for minimum 2 h at 5 degrees C and 25 degrees C. Incorporating these results, an improved procedure for the isolation of SBA has been developed. The SBA isolated by this method is obtained in better yield, has 6000 HU/mg protein and is identical to that isolated by the (NH4)2SO4 method as ascertained by chromatographic and electrophoretic comparisons and hapten inhibition assays.     

Development of a simple high-throughput screening protocol based on biosynthetic activity of Mycobacterium tuberculosis glutamine synthetase for the identification of novel Inhibitors

A high-throughput screening protocol has been developed for Mycobacterium tuberculosis glutamine synthetase by quantitative estimation of inorganic phosphate. The K(m) values determined at pH 6.8 are 22 mM for L-glutamic acid, 0.75 mM for NH(4)Cl, 3.25 mM for MgCl(2), and 2.5 mM for adenosine triphosphate. The K(m) value for glutamine is affected significantly by the increase in pH of assay buffer. At the saturating level of the substrate, the enzyme activity at pH 6.8 and 25 degrees C is found to be linear up to 3 h. The reduction of enzyme activity is negligible even in presence of 10% DMSO. The Z' factor and signal-to-noise ratio are found to be 0.75 and 6.18, respectively, when the enzyme is used at 62.5 microg/ml concentration. The IC(50) values obtained at pH 6.8 for both L-methionine S-sulfoximine and DL-phosphothriacin are 500 microM and 30 microM, respectively, which is lowest compared to the values obtained at other pH levels. The Beckman Coulter high-throughput screening platform was found to take 5 h 9 min to complete the screening of 60 plates. For each assay plate, a replica plate is used to normalize the data. Screening of 1164 natural product fractions/extracts and synthetic molecules from an in-house library was able to identify 12 samples as confirmed hits. Altogether, the validation data from screening of a small set of an in-house library coupled with Z' and signal-to-noise values indicate that the protocol is robust for high-throughput screening of a diverse chemical library.