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1.
An amperometric glucose biosensor was fabricated by the electrochemical polymerization of pyrrole onto a platinum electrode in the presence of the enzyme glucose oxidase in a KCl solution at a potential of + 0·65 V versus SCE. The enzyme was entrapped into the polypyrrole film during the electropolymerization process. Glucose responses were measured by potentio-statting the enzyme electrode at a potential of + 0·7 V versus SCE in order to oxidize the hydrogen generated by the oxidation of glucose by the enzyme in the presence of oxygen. Experiments were performed to determined the optimal conditions of the polypyrrole glucose oxidase film preparation (pyrrole and glucose oxidase concentrations in the plating solution) and the response to glucose from such electrodes was evaluated as a function of film thickness, pH and temperature. It was found that a concentration of 0·3 M pyrrole in the presence of 65 U/ml of glucose oxidase in 0·01 M KCl were the optimal parameters for the fabrication of the biosensor. The optimal response was obtained for a film thickness of 0·17 μm (75 mC/cm 2) at pH 6 and at a temperature of 313 K. The temperature dependence of the amperometric response indicated an activation energy of 41 kJ/mole. The linearity of the enzyme electrode response ranged from 1·0 mM to 7·5 mM glucose and kinetic parameters determined for the optimized biosensors were 33·4 mM for the K m and 7·2 μA for the I max. It was demonstrated that the internal diffusion of hydrogen peroxide through the polypyrrole layer to the platinum surface was the main limiting factor controlling the magnitude of the response of the biosensor to glucose. The response was directly related to the enzyme loading in the polypyrrole film. The shelf life and the operational stability of the optimized biosensor exceed 500 days and 175 assays, respectively. The substrate specificity of the entrapped glucose oxidase was not altered by the immobilization procedure. 相似文献
2.
The growth of the freshwater microalga Scenedesmus obliquus was studied at 30°C in a mineral culture medium with phosphorus concentrations of between 0 and 372 μ
. The values for the specific growth rates, between
and
, fitted a semistructured substrate-limitation model with μ m1 = 0·0466 h −1, μ m2 = 0·0256 h −1 and
. The specific uptake rate of phosphorus reached a maximum value of qSm1 = 658·01 × 10 −4 μmol P mg −1 biomass h −1. 相似文献
3.
The binding of [ 3H]proctolin to oviduct membranes has been analyzed to investigate the nature of proctolin binding sites in the oviduct. Proctolin binding was found to be time dependent, proportional to concentration of membrane protein, saturable, specific and reversible. Two apparent proctolin binding sites were recognized. The first had a Kd of 400 ± 82 nM and a Bmax of 23.7 ± 6.7 pmol/mg protein. The second had a Kd of 2.4 ± 0.2 μM and a Bmax of 96.3 ± 16.7 pmo/mg protein. Binding was specific in thatcompetition experiments with a wide range of peptides showed that only Arg-Tyr-Leu-Pro-Ala was an effective competitor at μM concentrations. All other peptides examined weekly reduced proctolin binding at concentrations above 50 μM. Certain peptides were found to potentiate [3]pproctolin binding at low μM concentrations (1–10 μM) and to inhibit proctolin binding at higher concentrations. The significance of these findings is discussed. 相似文献
4.
Previous research has shown that lactate dehydrogenase (LDH) was competitively inhibited by pentachlorophenol (PCP) and a modified assay produced a detection limit of 1 μM (270 μg l −1). This work used spectrophotometric rate-determination but in order to move towards biosensor development the selected detection method was electrochemical. The linkage of LDH to lactate oxidase (LOD) provided the electroactive species, hydrogen peroxide. This could be monitored using a screen-printed carbon electrode (SPCE) incorporating the mediator, cobalt phthalocyanine, at a potential of +300 mV (vs. Ag/AgCl). A linked LDH/LOD system was optimised with respect to inhibition by PCP. It was found that the SPCE support material, PVC, acted to reduce inhibition, possibly by combining with PCP. A cellulose acetate membrane removed this effect. Inhibition of the system was greatest at enzyme activities of 5 U ml −1 LDH and 0.8 U ml −1 LOD in reactions containing 246 μM pyruvate and 7.5 μM NADPH. PCP detection limits were an EC10 of 800 nM (213 μg l −1) and a minimum inhibition detectable ( MID) limit of 650 nM (173 μg l −1). The inclusion of a third enzyme, glucose dehydrogenase (GDH), provided cofactor recycling to enable low concentrations of NADPH to be incorporated within the assay. NADPH was reduced from 7.5 to 2 μM. PCP detection limits were obtained for an assay containing 5 U ml −1 LDH, 0.8 U ml −1 LOD and 0.1 U ml −1 GDH with 246 μM pyruvate, 400 mM glucose and 2 μM NADPH. The EC10 limit was 150 nM (39.9 μg l −1) and the MID was 100 nM (26.6 μg l −1). The design of the inhibition assays discussed has significance as a model for other enzymes and moves forward the possibility of an electrochemical biosensor array for pollution monitoring. 相似文献
5.
Polyethylene glycol-modified glucose oxidase (PEG-GOD) was prepared. Carbon paste (CP) containing PEG-GOD retained enzyme activity of 0·02 U cm −2. Anodic and cathodic peak currents of modified GOD in CP matrix were observed on the differential pulse voltammograms at the potential of −0·36 and −0·36 V vs. Ag/AgCl, respectively. The addition of glucose to a test solution brought about an increase in the anodic current on the PEG-GOD-based electrode at the potential as low as 0·0 V vs. Ag/AgCl. The current increase was proportional to the concentration of glucose up to 50 mM. 相似文献
6.
The aim of our study was to determine whether a meal modifies the antisecretory response induced by PYY and the structural requirements to elicit antisecretory effects of analogue PYY(22–36) for potential antidiarrhea therapy. The variations in short-circuit current (Isc) due to the modification of ionic transport across the rat intestine were assessed in vitro, using Ussing chambers. In fasted rats, PYY induced a dose- and time-dependent reduction in Isc, with a sensitivity threshold at 5 × 10 −11 M (ΔIsc −2 ± 0.5 μA/cm 2). The reduction was maximal at 10 −7 M (Isc −23 ± 2 μA/cm 2), and the concentration producing half-maximal inhibition was 10 −9 M. At 10 −7 M, reduction of Isc by PYY reached 90% of response to 5 × 10 −5 M bumetanide. The PYY effect was partly reversed by 10 −5 M forskolin (Isc +13.43 ± 2.91 μA/h·cm 2, p < 0.05) or 10 −3 M dibutyryl adenosine 3′,5′ cyclic monophosphate (Isc +12 ± 1.69 μA/cm 2, p < 0.05). Naloxone and tetrodotoxin did not alter the effect of PYY. In addition, PYY and its analogue P915 reduced net chloride ion secretion to 2.85 and 2.29 μEq/cm 2 ( p < 0.05), respectively. The antisecretory effect of PYY was accompanied by dose- and time-dependent desensitization when jejunum was prestimulated by a lower dose of peptide. The antisecretory potencies exhibited by PYY analogues required both a C-terminal fragment (22–36) and an aromatic amino acid residue (Trp or Phe) at position 27. At 10 −7 M the biological activity of PYY was lower in fed than fasted rats ( p < 0.001). Our results confirm the antisecretory effect of PYY, but show that the fed period is accompanied by desensitization, similar to the transient desensitization observed in the fasted period with cumulative doses. This suggests that PYY may act as a physiological mediator that reduces intestinal secretion. 相似文献
7.
A novel chitosan/PVI-Os(polyvinylimidazole-Os)/CNT(carbon nanotube)/LOD (lactate oxidase) network nanocomposite was constructed on gold electrode for detection of lactate. The composite was nanoengineered by selected matched material components and optimized composition ratio to produce a superior lactate sensor. Positively charged chitosan and PVI-Os were used as the matrix and the mediator to immobilize the negatively charged LOD and to enhance the electron transfer, respectively. CNTs were introduced as the essential component in the composite for the network nanostructure. FESEM (field emission scan electron microscopy) and electrochemical characterization demonstrated that CNT behaved as a cross-linker to network PVI and chitosan due to its nanoscaled and negative charged nature. This significantly improved the conductivity, stability and electroactivity for detection of lactate. The standard deviation of the sensor without CNT in the composite was greatly reduced from 19.6 to 4.9% by addition of CNTs. With optimized conditions the sensitivity and detection limit of the lactate sensor was 19.7 μA mM −1 cm −2 and 5 μM, respectively. The sensitivity was remarkably improved in comparison to the newly reported values of 0.15–3.85 μA mM −1 cm −2. This novel nanoengineering approach for selecting matched components to form a network nanostructure could be extended to other enzyme biosensors, and to have broad potential applications in diagnostics, life science and food analysis. 相似文献
8.
Redox enzyme mediated biocatalysis has the potential to regio- and stereo-specifically oxidize hydrocarbons producing valuable products with minimal by-product formation. In vitro reactions of the camphor (cytochrome P-450) 5-monooxygenase enzyme system with naphthalene-like substrates yield stereospecifically hydroxylated products from nonactivated hydrocarbons. Specifically, the enzyme system catalyzes the essentially stereospecific conversion of the cycloarene, tetralin (1,2,3,4-tetrahydronaphthalene) to ( R)-1-tetralol (( R)-(−)-1,2,3,4-tetrahydro-1-naphthol). It is shown that this reaction obeys Michaelis–Menten kinetics and that interactions between the enzyme subunits are not affected by the identity of the substrate. This subunit independence extends to the efficiency of NADH usage by the enzyme system—subunit ratios do not effect efficiency, but substrate identity does. Tetralin is converted at an efficiency of 13±3%, whereas ( R)-1-tetralol is converted at 7.8±0.7%. A model of this system based on Michaelis–Menten parameters for one subunit (Pdx: KM=10.2±2 μM) and both substrates (tetralin: KM=66±26 μM, νmax=0.11±0.04 s −1, and ( R)-1-tetralol: KM=2800±1300 μM, νmax=0.83±0.22 s −1) is presented and used to predict the consumption and production of all substrates, products and cofactors. 相似文献
9.
1. 1. Cyanide inhibits the catalytic activity of cytochrome aa3 in both polarographic and spectrophotometric assay systems with an apparent velocity constant of 4·103 M−1·s−1 and a Ki that varies from 0.1 to 1.0 μM at 22 °C, pH 7·3. 2. 2. When cyanide is added to the ascorbate-cytochrome c-cytochromeaa3−O2 system a biphasic reduction of cytochrome c occurs corresponding to an initial Ki of 0.8 μM and a final Ki of about 0.1 μM for the cytochrome aa3−cyanide reaction. 3. 3. The inhibited species (a2+a33+HCN) is formed when a2+a33+ reacts with HCN, when a2+a32+HCN reacts with oxygen, or when a3+a33+HCN (cyano-cytochrome aa3) is reduced. Cyanide dissociates from a2+a33+HCN at a rate of 2·10−3 s−1 at 22 °C, pH 7.3. 4. 4. The results are interpreted in terms of a scheme in which one mole of cyanide binds more tightly and more rapidly to a2+a33+ than to a3+a33+.
Abbreviations: TMPD, N,N,N′,N′-tetramethyl-p-phenylenediamine 相似文献
10.
Androgen aromatase was found to also be estrogen 2-hydroxylase. The substrate specificity among androgens and estrogens and multiplicity of aromatase reactions were further studied. Through purification of human placental microsomal cytochrome P-450 by monoclonal antibody-based immunoaffinity chromatography and gradient elution on hydroxyapatite, aromatase and estradiol 2-hydroxylase activities were co-purified into a single band cytochrome P-450 with approx. 600-fold increase of both specific activities, while other cytochrome P-450 enzyme activities found in the microsomes were completely eliminated. The purified P-450 showed M r of 55 kDa, specific heme content of 12.9 ± 2.6 nmol·mg −1 (±SD, N = 4), reconstituted aromatase activity of 111 ± 19 nmol·min −1·mmg −1 and estradiol 2-hydroxylase activity of 5.85 ± 1.23 nmol·min −1·mg −1. We found no evidence for the existence of catechol estrogen synthetase without concomitant aromatase activity. The identity of the P-450 for the two different hormone synthetases was further confirmed by analysis of the two activities in the stable expression system in Chinese hamster ovarian cells transfected with human placental aromatase cDNA, pH β-Aro. Kinetic analysis of estradiol 2-hydroxylation by the purified and reconstituted aromatase P-450 in 0.1 M phosphate buffer (pH 7.6) showed Km of 1.58 μM and Vmax of 8.9 nmol·min −1·mg −1. A significant shift of the optimum pH and Vmax, but not the Km, for placental estrogen 2-hydroxylase was observed between microsomal and purified preparations. Testosterone and androstenedione competitively inhibited estradiol 2-hydroxylation, and estrone and estradiol competitively inhibited aromatization of both testosterone and androstenedione. Estrone and estradiol showed Ki of 4.8 and 7.3 μM, respectively, for testosterone aromatization, and 5.0 and 8.1 μM, respectively, for androstenedione aromatization. Androstenedione and testosterone showed Ki of 0.32 and 0.61 μM, respectively, for estradiol 2-hydroxylation. Our studies showed that aromatase P-450 functions as estrogen 2-hydroxylase as well as androgen 19-, 1β-,and 2β-hydroxylase and aromatase. The results indicate that placental aromatase is responsible for the highly elevated levels of the catechol estrogen and 19-hydroxyandrogen during pregnancy. These results also indicate that the active site structure holds the steroid ssubstrates to face their β-side of the A-ring to the heme, tilted in such a way as to make the 2-position of estrogens and 19-, 1-, and 2-positions of androgens available for monooxygenation. 相似文献
11.
The thymidylate synthase (TS)-encoding gene from Cryptococcus neoformans ( Cn) has been isolated from cDNA and genomic libraries. The 1127-bp gene contains three introns and a 951-bp open reading frame encoding a 35844-Da protein. The cDNA clones lack 324 bp of the 5' coding region of the gene. The complete coding sequence was assembled as an expression cassette in pUC19 using parts of the coding sequence from the cDNA and genomic DNA and completing the sequence using synthetic DNA. Production of active TS from Cn (CnTS) was first demonstrated by complementation of a thymine(Thy)-requiring Escherichia coli strain. The expression cassette was subsequently subcloned into the T7 polymerase vector pET15-b. In this construct, CnTS is produced as approximately 10% of the total soluble protein in E. coli. Homogeneous enzyme was obtained at a 36% yield after consecutive chromatography on DEAE-cellulose, Q-Sepharose, phenyl-Sepharose and Affi-Gel Blue. Steady-state kinetic analysis showed that the Km values for dUMP and CH 2H 4-folate were 2.7 ± 0.5 μM and 38.2 ± 2.5 μM, respectively, and the Kcat was 5.1 s −1. The enzyme was stable upon storage at −80°C in Tris-HCl pH 7.4 and thiol. 相似文献
12.
Purified RNase Rs, from Rhizopus stolonifer, when covalently coupled to aminoethyl (AE) Bio-Gel P-2, via its carbohydrate moiety, retained 35–40% activity of the soluble enzyme. Optimization of coupling conditions showed that the most active immobilized preparations are obtained when 400 units of 100 μM periodate oxidized enzyme are allowed to react with 1 ml (packed volume) of AE-Bio-Gel P-2 at 6±1°C for 15 h. Immobilization did not change the pH and temperature optima of the enzyme but it increased the temperature stability. Immobilization did not bring about a change in the K m but resulted in a 2·5-fold decrease in the V max. Substrate concentrations as high as 25 mg of RNA could be converted to more than 80% 2′,3′ cyclic nucleotides in 14 h, at pH 5·5 and 37°C. On repeated use, the bound enzyme retained 70% of its initial activity after six cycles of use. The bound enzyme could be stored in wet state for 60 days without any significant loss in its initial activity. 相似文献
13.
Estimation of the ammonia production of the shrimp C. crangon in two littoral ecosystems (oligotrophic sand and eutrophic mud) was determined in winter and summer conditions from laboratory observations in experimental microcosms. The ammonia excretion rate of C. crangon was not influenced by either the sediment type or the ammonia concentration of the overlying water; on the other hand, the mean excretion rate and the response to initial handling stress increased markedly as shrimp were deprived of soft substratum. The daily ammonia production of C. crangon was 16 μmol NH3 · g −1 wet wt · day −1 in winter and 40 μmol in summer. A gross production of 12 μmol NH3 · m−2 · day −1 and 300–700 μmol μ m−2 · day−1, respectively, could be expected in the two ecosystems studied. This would account for 5% (winter) and 2–4% (summer) of the total NH+4 flux at the sediment-water interface. The contribution of the excretion of all macrofauna to the NH+4 flux from the sediment is discussed. 相似文献
14.
The electrocatalytic reduction of H 2O 2 was studied for carbonaceous electrodes modified with horse-radish peroxidase (HRP), microperoxidase (MP), and lactoperoxidase (LP). The carbonaceous electrodes were of three different graphites, carbon and glassy carbon. The peroxidase modified electrode was inserted as the working electrode in a flow through amperometric cell of the wall jet type and connected to a flow injection system. The effect of different pretreatments of the electrode surface prior to adsorption of the enzyme was investigated. Heating the electrodes in a muffle furnace at 700°C for 1.5 min was found to yield the highest currents. The electrocatalytic current for HRP-modified electrodes starts at about +600 mV vs. Ag/AgCl (pH 7.0) and reaches a maximum value at about −200 mV. For MP- and LP-modified electrodes the currents start at a lower potential (≈ 300 mV). For the best electrode material for HRP, straight calibration curves were obtained between 1 and 500 μM H 2O 2 at 0 mV. The mechanism for the electron transfer from the electrode to the adsorbed peroxidase is discussed. Deliberate modification of the electrode surface with quinoid type electroactive species was found to mediate the reaction. It is proposed that spontaneously occurring electrochemically active surface groups mediate the electron transfer to the adsorbed enzyme. However, a contribution to the observed current from a direct electron transfer cannot be ruled out. 相似文献
15.
The response characteristics of a new enzyme electrode for determining choline are reported. The enzyme electrode consists of a polyvinylferrocenium perchlorate coated Pt surface onto which the enzyme, choline oxidase, is attached. Choline oxidase catalyzes the oxidation of choline to betaine, producing H 2O 2. Current due to H 2O 2 oxidation catalyzed by polyvinylferrocenium centers was measured. The effects of choline concentration, the amount of enzyme immobilized and the operating pH and temperature on the response of the enzyme electrode were studied. The effects of interferents were also investigated. The response time was found to be 60–70 s and the upper limit of the linear working portion was found to be 1.2 mM choline concentration. The minimum substrate concentration that produced detectable current was 4.0×10 −6 M choline concentration. The steady-state current of this enzyme electrode was reproducible within ±4.6% of relative error. The apparent Michaelis–Menten constant ( KMapp) and the activation energy, Ea, of this immobilized enzyme system were found to be 2.32 mM and 38.91 kJ/mol, respectively. 相似文献
16.
A reversed-phase high-performance liquid chromatographic method has been developed and validated for the quantification of the novel anticancer drug Ecteinascidin 743 in human plasma. The sample pretreatment of the plasma samples involved a solid-phase extraction (SPE) on cyano columns. Propyl- p-hydroxybenzoate was added after the sample pretreatment to correct for variability in injection volumes. The separation was performed on a Zorbax SB-C 18 column (75×4.6 mm I.D., particle size 3.5 μm) with acetonitrile–25 m M phosphate buffer, pH 5.0 (70:30, v/v) as the mobile phase. The flow-rate was 1.0 ml/min and the eluent was monitored at 210 nm. The accuracies and precisions of the assay fall within ±15% for all quality control samples and within ±20% for the lower limit of quantitation, which was 1.0 ng/ml using 500 μl of plasma. The overall recovery of the sample pretreatment procedure for Ecteinascidin 743 was 87.0±5.9%. The drug was found to be stable in human plasma at −30°C for at least 2 months. At room temperature Ecteinascidin 743 was stable in human plasma for 5 h at most. 相似文献
17.
The in vitro metabolism of cortisol in human liver fractions is highly complex and variable. Cytosolic metabolism proceeds predominantly via A-ring reduction (to give 3,5β-tetrahydrocortisol; 3,5β-THF), while microsomal incubations generate upto 7 metabolites, including 6β-hydroxycortisol (6β-OHF), and 6β-hydroxycortisone (6β-OHE), products of the cytochrome P450 (CYP) 3A subfamily. The aim of the present study was, therefore, to examine two of the main enzymes involved in cortisol metabolism, namely, microsomal 6β-hydroxylase and cytosolic 4-ene-reductase. In particular, we wished to assess the substrate specificity of these enzymes and identify compounds with inhibitory potential. Incubations for 30 min containing [ 3H]cortisol, potential inhibitors, microsomal or cytosolic protein (3 mg), and co-factors were followed by radiometric HPLC analysis. The Km value for 6β-OHF and 6β-OHE formation was 15.2 ± 2.1 μM (mean ± SD; n = 4) and the Vmax value 6.43 ± 0.45 pmol/min/mg microsomal protein. The most potent inhibitor of cortisol 6β-hydroxylase was ketoconazole ( Ki = 0.9 ± 0.4 μM; N = 4), followed by gestodene ( Ki = 5.6 ± 0.6 μM) and cyclosporine ( Ki = 6.8 ± 1.4 μM). Both betamethasone and dexamethasone produced some inhibition ( Ki = 31.3 and 54.5 μ, respectively). However, substrates for CYP2C (tolbutamide), CYP2D (quinidine), and CYP1A (theophylline) were essentially non-inhibitory. The Km value for cortisol 4-ene-reductase was 26.5 ± 11.2 μM ( n = 4) and the Vmax value 107.7 ± 46.0 pmol/min/mg cytosolic protein. The most potent inhibitors were androstendione ( Ki = 17.8 ± 3.3 μM) and gestodene ( Ki = 23.8 ± 3.8 μM). Although both compounds have identical A-rings to cortisol, and undergo reduction, inhibition was non-competitive. 相似文献
18.
Chalcones xanthohumol (X) and desmethylxanthohumol (DMX), present in hops ( Humulus lupulus L.), and the corresponding flavanones isoxanthohumol (IX, from X), 8-prenylnaringenin (8-PN, from DMX), and 6-prenylnaringenin (6-PN, from DMX), have been examined in vitro for their anti-proliferative activity on human prostate cancer cells PC-3 and DU145. X proved to be the most active compound in inhibiting the growth of the cell lines with IC 50 values of 12.3±1.1 μM for DU145 and 13.2±1.1 μM for PC-3. 6-PN was the second most active growth inhibitor, particularly in PC-3 cells (IC 50 of 18.4±1.2 μM). 8-PN, a highly potent phytoestrogen, exhibited pronounced anti-proliferative effects on PC-3 and DU145 (IC 50 of 33.5±1.0 and 43.1±1.2 μM, respectively), and IX gave comparable activities (IC 50 of 45.2±1.1 μM for PC-3 and 47.4±1.1 μM for DU145). DMX was the least active compound. It was evidenced for the first time that this family of prenylated flavonoids from hops effectively inhibits proliferation of prostate cancer cells in vitro. 相似文献
19.
Butylmalonate (butyl propanedioic acid) is a slow-binding inhibitor of porcine renal aminoacylase I (EC 3.5.1.14), causing transients of activity with half-times of more than 10 min. At 25°C and pH 7.0, the dissociation rate of the complex is approximately 6 × 10 −4 s −1, while the rate constant of complex formation is in the order of 20 M −1·s −1. In good agreement with these data, steady-state kinetics yield an estimated inhibition constant around 100 μM. Molecular mechanics calculations showed that conformation and charge distribution of butylmalonate are strikingly similar to those of the putative transition state of aminoacylase catalysis. 相似文献
20.
Live, intact third-stage larvae (L3s) of Strongyloides ratti in the absence of exogenous substrates consumed oxygen at a rate (E-QO 2) of 181.8 ± 12.4 ng atoms min −1 mg dry weight −1 at 35°C. Respiratory electron transport (RET) Complex I inhibitor rotenone (2 μ
) produced 33 ± 6.5% inhibition of the E-QO 2. Unusually the rotenone-induced inhibition was not relieved by 5 μ
-succinate. The E-QO 2 of intact L3s was refractory to RET Complex III inhibitor antimycin A at 2 μ
; 4 μ
-antimycin inhibited ≤ 10% of the E-QO 2. The electron donor couple ascorbate/TMPD augmented the E-QO 2 in the presence of rotenone (2 μ
) and antimycin A (4 μ
) by 110%. Azide (1 m
) stimulated the antimycin A refractory QO 2 by 36.6 ± 7.2% which was only partially inhibited by 1.0 m
-KCN (
). The data suggest the presence of classical (CPW) and alternate (APW) electron transport pathways in S. ratti L3s. 相似文献
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