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1.
Hepatitis B surface antibody (HBsAb) was immobilized to the surface of a gold electrode modified with cysteamine and colloidal gold as matrices to detect hepatitis B surface antigen (HBsAg). Differential pulse voltammetry (DPV) method was used for the investigation of the specific interaction between the immobilized HBsAb and HBsAg in solution, which was followed as a change of peak current in DPV with time. With the modified gold electrode, the differences in affinity of HBsAb with HBsAg at the temperatures of 37 and 40 °C were easily distinguished and the kinetic rate constants (kass and kdiss) and kinetic affinity constant K were determined from the curves of current versus time. In addition, the thermodynamic constants, ΔG, ΔH and ΔS, of the interaction at 37 °C were calculated, which were −56.65, −64.54 and −25.45 kJ mol−1, respectively. 相似文献
2.
Toshiaki Mori Momoko Toyoda Tatsuro Ohtsuka Yoshio Okahata 《Analytical biochemistry》2009,395(2):211-216
A biotinylated mannotriose (Man3-bio) was dispersively immobilized in the matrix of biotinylated lactose (Gal-Glc-bio) on a streptavidin-covered, 27-MHz quartz crystal microbalance (QCM), and binding kinetics of concanavalin A (Con A) to Man3-bio in the Gal-Glc-bio matrix could be obtained from frequency decreases (mass increases) of the QCM. Association constants (Ka) and binding and dissociation rate constants (kon and koff) could be determined separately as the 1:1 and 1:2 bindings of Con A to Man3-bio on the surface. When Man3-bio was immobilized with content of 1 to 5 mol% in the matrix, the 1:1 binding of Con A to Man3-bio was obtained as Ka = (4 ± 1) × 106 M−1, kon = (4 ± 1) × 104 M−1 s−1, and koff = (12 ± 2) × 10–3 s−1. On the contrary, when Man3-bio was immobilized with content of 20 to 100 mol% in the matrix, the 1:2 binding of Con A to Man3-bio was obtained as Ka = (14 ± 2) × 106 M−1, kon = (14 ± 2) × 104 M−1 s−1, and koff = (7 ± 2) × 10–3 s−1. Thus, Ka for the 1:2 binding was 10 times larger than that for the 1:1 binding, with a three times larger binding rate constant (kon) and a three times smaller dissociation rate constant (koff). This is the first example to obtain separate kinetic parameters for the 1:1 and 1:2 bindings of lectins to carbohydrates on the surface. 相似文献
3.
Elicitins are low-molecular-weight proteins representing the elicitor family secreted by many species of the oomycete Phytophthora. Elicitins induce a hypersensitive reaction in tobacco, a process that is triggered by binding of elicitin to the high-affinity site on the plasma membrane. Specific interaction of cryptogein with the binding sites on tobacco plasma membranes was studied using the piezoelectric biosensor in real time in a flow-through mode. Cryptogeins (wild-type and mutant forms) were covalently immobilized on the sensing surface, and membrane vesicles containing receptors were in solution. Kinetic characterization of the interaction provided values of kinetic rate association (ka) = 5.74 · 106 M1 s−1 and kinetic rate dissociation (kd) = 6.87 10−4 s−1 constants, respectively. The kinetic equilibrium dissociation constant was calculated as KD = 12.0 nM. The piezoelectric biosensor appeared to be a convenient tool for studying interactions of receptors embedded in membrane vesicles. 相似文献
4.
The aggregation of proteins is believed to be intimately connected to many neurodegenerative disorders. We recently reported an “Ockham's razor”/minimalistic approach to analyze the kinetic data of protein aggregation using the Finke–Watzky (F–W) 2-step model of nucleation (A → B, rate constant k1) and autocatalytic growth (A + B → 2B, rate constant k2). With that kinetic model we have analyzed 41 representative protein aggregation data sets in two recent publications, including amyloid β, α-synuclein, polyglutamine, and prion proteins (Morris, A. M., et al. (2008) Biochemistry 47, 2413-2427; Watzky, M. A., et al. (2008) Biochemistry 47, 10790–10800). Herein we use the F–W model to reanalyze protein aggregation kinetic data obtained under the experimental conditions of variable temperature or pH 2.0 to 8.5. We provide the average nucleation (k1) and growth (k2) rate constants and correlations with variable temperature or varying pH for the protein α-synuclein. From the variable temperature data, activation parameters ΔG‡, ΔH‡, and ΔS‡ are provided for nucleation and growth, and those values are compared to the available parameters reported in the previous literature determined using an empirical method. Our activation parameters suggest that nucleation and growth are energetically similar for α-synuclein aggregation (ΔG‡nucleation = 23(3) kcal/mol; ΔG‡growth = 22(1) kcal/mol at 37 °C). From the variable pH data, the F–W analyses show a maximal k1 value at pH ~ 3, as well as minimal k1 near the isoelectric point (pI) of α-synuclein. Since solubility and net charge are minimized at the pI, either or both of these factors may be important in determining the kinetics of the nucleation step. On the other hand, the k2 values increase with decreasing pH (i.e., do not appear to have a minimum or maximum near the pI) which, when combined with the k1 vs. pH (and pI) data, suggest that solubility and charge are less important factors for growth, and that charge is important in the k1, nucleation step of α-synuclein. The chemically well-defined nucleation (k1) rate constants obtained from the F–W analysis are, as expected, different than the 1/lag-time empirical constants previously obtained. However, k2 × [A]0 (where k2 is the rate constant for autocatalytic growth and [A]0 is the initial protein concentration) is related to the empirical constant, kapp obtained previously. Overall, the average nucleation and average growth rate constants for α-synuclein aggregation as a function of pH and variable temperature have been quantitated. Those values support the previously suggested formation of a partially folded intermediate that promotes aggregation under high temperature or acidic conditions. 相似文献
5.
zlem Alptekin S. Seyhan Tükel Deniz Yldrm Dilek Alagz 《Journal of Molecular Catalysis .B, Enzymatic》2009,58(1-4):124-131
Bovine liver catalase was covalently immobilized onto controlled pore glass (CPG) beads modified with 3-aminopropyltriethoxysilane (3-APTES) followed by treatment with glutaraldehyde. Coupling of catalase onto CPG was optimized to improve the efficiency of the overall immobilization procedure. The optimum coupling conditions: pore diameter of CPG, pH, buffer concentration, temperature, coupling time and initial catalase amount per grams of carrier were determined as 70 nm, 6.0, 75 mM, 5 °C, 7 h and 6 mg catalase, respectively. Catalytic efficiencies (kcat/Km) and thermal inactivation rate constants (ki) of ICPG1 were determined and compared with that of free catalase. Suitability of ICPG1 was also investigated by using it in batch and plug-flow type reactors. When the remaining activity of ICPG1 retained was about 50% of its initial activity the highest total productivity of ICPG1 was determined as 7.6 × 106 U g immobilized catalase−1 in plug-flow type reactor. However, the highest total productivity of ICPG1 was 6.2 × 105 U g immobilized catalase−1 in batch type reactor. ICPG1 may have great potentials as biocatalyst for the application in decomposition of hydrogen peroxide in plug-flow type reactor. 相似文献
6.
Shveta Chaudhary J. David Van Horn 《Mutation Research - Genetic Toxicology and Environmental Mutagenesis》2006,610(1-2):56
Kinetics for the breakdown of the trinuclear chromium acetate cluster with a series of monoprotic and diprotic amino acid ligands and with glutathione in aqueous media have been investigated spectrophotometrically at pH 3.5–5.5 and in a temperature range of 45–60 °C. Under pseudo-first-order conditions, reactions with these ligands exhibited biphasic kinetic behavior that can be accounted for by a consecutive two-step reaction, A → B → C, where A is assumed to be a forced ion pair, B an intermediate and C is the product; experimental data fit to a biexponential equation for the transformation. Rates for kshort, klong, and kobs were determined by manual extrapolation of absorbance data or curve-fitting routines; associated activation parameters for each step of the reaction were calculated using the Eyring equation. Rates for the first and second steps of the reaction are on the order of 10−4 and 10−5 s−1, respectively. The large negative values of ΔS‡ and smaller ΔH‡ in the first step indicate an associative step, while high positive values of ΔS‡ in the second step indicate dissociation. To account for the results mechanistically, the results are interpreted to be a first step of ligand exchange with a pseudo-axial aqua ligand, followed by a dissociative step involving acetate or oxo ligand displacement. The dissociative step is the rate determining step, with kobs ≈ klong.The results demonstrate reaction pathways that are available to the Cr(III) metal centers that may be physiologically relevant in the ligand-rich environment of biological systems. Under general conditions Cr(III) clusters may be expected to be broken down, unless some unique biological environment stabilizes the cluster. The present study has application to the processes related to Cr(III) transport and excretion, to potential mechanisms of Cr(III) action in a biological setting, and to the pharmacokinetics of Cr(III) supplements for animal and human consumption. 相似文献
7.
Adam Sikora Jacek Zielonka Marcos Lopez Joy Joseph B. Kalyanaraman 《Free radical biology & medicine》2009,47(10):1401-1407
In this study, we show that boronates, a class of synthetic organic compounds, react rapidly and stoichiometrically with peroxynitrite (ONOO−) to form stable hydroxy derivatives as major products. Using a stopped-flow kinetic technique, we measured the second-order rate constants for the reaction with ONOO−, hypochlorous acid (HOCl), and hydrogen peroxide (H2O2) and found that ONOO− reacts with 4-acetylphenylboronic acid nearly a million times (k = 1.6 × 106 M− 1 s− 1) faster than does H2O2 (k = 2.2 M− 1 s− 1) and over 200 times faster than does HOCl (k = 6.2 × 103 M− 1 s− 1). Nitric oxide and superoxide together, but not alone, oxidized boronates to the same phenolic products. Similar reaction profiles were obtained with other boronates. Results from this study may be helpful in developing a novel class of fluorescent probes for the detection and imaging of ONOO− formed in cellular and cell-free systems. 相似文献
8.
Decarboxylation kinetics for the carbonatotris(pyridine)cobalt(III) ion in perchloric acid solutions
The kinetics of the decomposition reactions of the CO(py)3(CO3)(H2O)+ ion have been investigated in aqueous perchloric acid solutions over a range of hydrogen ion concentrations (0.10 to 5.0 M) and at two ionic strengths (I = 1.0 and 5.0 M). At the lower ionic strength, plots of ln (At–A∞ versus time show a nonlinearity that is consistent with that expected for consecutive first-order reactions. The rates of the faster reaction are similar to those reported for the spontaneous reduction of aquopyridine-cobalt(III) cations. At the higher ionic strength, the above noted curvature is not apparent and the decarboxylation kinetics of the title complex may be described by a pseudo-first-order rate constant: kobs = k[H3O+]. At 20°C, k = (1.75−+0.09) s−1 M−1 with activation parameters ofΔH‡ = (97 −+ 4) kJ mol−1 and ΔS‡ = −(54 −+ 32) J deg−1 mol−1. These kinetic parameters are compared with those previously reported for the similar complexes, Co(py)4CO3+ and Co(py)2(CO3)(H2O)2+. 相似文献
9.
The kinetics of formation of the complex ion, μ-carbonato-di-μ-hydroxo-bis((1,5-diamino-3-aza-pentane) cobalt(III), from the tri-μ-hydroxo-bis((1,5-diamino-3-aza-pentane(III)cobalt(III)) ion in aqueous buffered carbonate solution have been studied spectrophotometrically at 295 nm over the ranges 20.0θ°C34.8, 8.03pH9.44, 5 mM [CO32−35 mM and at an ionic strength of 0.1 M (LiClO4). On the basis of the kinetic results a mechanism, involving rapid cleavage of an hydroxo bridge followed by carbon dioxide uptake with subsequent bridge formation, has been proposed. At 25 °C, the rate of the carbon dioxide uptake is 0.58 M−1 s−1 with ΔH≠ = (13.2±0.7) kcal mol−1 and ΔS≠ = (−15.1 ± 0.7) cal deg−1 mol−1. The results are composed with those obtained for several mononuclear cobalt(III) and one dinuclear cobalt(III) complexes. 相似文献
10.
Ty1 is a retrotransposon of the yeast Saccharomyces cerevisiae whose transposition at new locations in the host genome is activated by stress conditions, such as exposure to UV light, X-rays, nitrogen starvation. In this communication, we supply evidence that cooling for 2 h at +4 °C followed by freezing for 1 h at −10 °C and 16 h at −20 °C also increased Ty1 transposition. The mobility of Ty1 was induced by cooling at slow rates (3 °C/min) and the accumulation of trehalose inside cells or the cooling at high rates (100 °C/min) inhibited significantly the induction of the transposition. The freeze-induced Ty1 transposition did not occur in mitochondrial mutants (rho−) and in cells with disrupted SCO1 gene (Δsco1 cells) evidencing that the Ty1 transposition induced by cooling depends on the mitochondrial oxidative phosphorylation. We also found that the freeze induced Ty1 transposition is associated with increased synthesis and accumulation of superoxide anions (O−2) into the cells. Accumulation of O−2 and activation of Ty1 transposition were not observed after cooling of cells with compromised mitochondrial functions (rho−, Δsco1), or in cells pretreated with O−2 scavengers. It is concluded that (i) elevated levels of reactive oxygen species (ROS) have a key role in activation the transposition of Ty1 retrotransposon in yeast cells undergoing freezing and (ii) given the deleterious effect of increased ROS levels on cells, special precautions should be taken to avoid ROS production and accumulation during cryopreservation procedures. 相似文献
11.
N.N. Mendona D.C. Masui J.C. McNamara F.A. Leone R. P.M. Furriel 《Comparative biochemistry and physiology. Part A, Molecular & integrative physiology》2007,146(4):534
The kinetic properties of a microsomal gill (Na+,K+)-ATPase from the freshwater shrimp, Macrobrachium olfersii, acclimated to 21‰ salinity for 10 days were investigated using the substrate p-nitrophenylphosphate. The enzyme hydrolyzed this substrate obeying cooperative kinetics at a rate of 123.6 ± 4.9 U mg− 1 and K0.5 = 1.31 ± 0.05 mmol L− 1. Stimulation of K+-phosphatase activity by magnesium (Vmax = 125.3 ± 7.5 U mg− 1; K0.5 = 2.09 ± 0.06 mmol L− 1), potassium (Vmax = 134.2 ± 6.7 U mg− 1; K0.5 = 1.33 ± 0.06 mmol L− 1) and ammonium ions (Vmax = 130.1 ± 5.9 U mg− 1; K0.5 = 11.4 ± 0.5 mmol L− 1) was also cooperative. While orthovanadate abolished p-nitrophenylphosphatase activity, ouabain inhibition reached 80% (KI = 304.9 ± 18.3 μmol L− 1). The kinetic parameters estimated differ significantly from those for freshwater-acclimated shrimps, suggesting expression of different isoenzymes during salinity adaptation. Despite the ≈2-fold reduction in K+-phosphatase specific activity, Western blotting analysis revealed similar α-subunit expression in gill tissue from shrimps acclimated to 21‰ salinity or fresh water, although expression of phosphate-hydrolyzing enzymes other than (Na+,K+)-ATPase was stimulated by high salinity acclimation. 相似文献
12.
The kinetics of substitution reactions of [η-CpFe(CO)3]PF6 with PPh3 in the presence of R-PyOs have been studied. For all the R-PyOs (R = 4-OMe, 4-Me, 3,4-(CH)4, 4-Ph, 3-Me, 2,3-(CH)4, 2,6-Me2, 2-Me), the reactions yeild the same product [η5-CpFe(CO)2PPh3]PF6, according to a second-order rate law that is first order in concentrations of [η5-CpFe(CO)3]PF6 and of R-PyO but zero order in PPh3 concentration. These results, along with the dependence of the reaction rate on the nature of R-PyO, are consistent with an associative mechanism. Activation parameters further support the bimmolecular nature of the reactions: ΔH≠ = 13.4 ± 0.4 kcal mol−1, ΔS≠ = −19.1 ± 1.3 cal k−1 mol−1 for 4-PhPyO; ΔH≠ = 12.3 ± 0.3 kcal mol−1, ΔS≠ = 24.7 ±1.0 cal K−1 mol−1 for 2-MePyO. For the various substituted pyridine N-oxides studied in this paper, the rates of reaction increase with the increasing electron-donating abilities of the substituents on the pyridine ring or N-oxide basicities, but decrease with increasing 17O chemical shifts of the N-oxides. Electronic and steric factors contributing to the reactivity of pyridine N-oxides have been quantitatively assessed. 相似文献
13.
Sharon A. Hibdon Charolette A. Coleman Jiabing Wang Christopher J. Murray 《Bioorganic chemistry》1992,20(4)
Association constants for N---H+…−O hydrogen bond formation between substituted ammonium dications and phenolate ion were measured in water and deuterium oxide at 25°C and 2.0
ionic strength. In combination with isotopic fractionation factors for phenol and the conjugate diacid of 1,2-ethanediamine determined by 13C NMR spectroscopy, these yield isotopic fractionation factors for amine dication-phenolate ion hydrogen bonds in water: φAB = 0.69 for 1,2-propanediamine dication with a pK difference between donor and acceptor, ΔpKa = −0.45, φAB = 0.88 for 1,2-ethanediamine dication (ΔpKa = −2.1), and φAB = 1.1 for piperizine dication (ΔpKa = −3.5). The hydrogen bond association constants follow Brønsted correlations α = 0.19 in water and α = 0.27 in deuterium oxide. The results are consistent with a double-minimum potential with a significant barrier for motion across the hydrogen bond. 相似文献
14.
Jeffrey A. Seminoff T. Todd Jones Tomoharu Eguchi Mervin Hastings David R. Jones 《Journal of experimental marine biology and ecology》2009,381(1):33-41
The trophic ecology of marine vertebrates has been increasingly studied via stable isotope analysis of body tissues. However, the theoretical basis for using stable isotopes to elucidate consumer–prey relationships remains poorly validated for most taxa despite numerous studies using this technique in natural systems. In this study, we measured stable carbon and stable nitrogen diet-tissue discrimination (Δdt) in whole blood, red blood cells, blood plasma solutes, and skin of leatherback sea turtles (Dermochelys coriacea; N = 7) maintained in captivity for up to 424 days and fed an isotopically consistent control diet with a mean C:N ratio of 2.94:1.00 and an energetic content of 20.16 ± 0.39 kJ g− 1 Dry Mass. We used a random-effect repeated measure model to evaluate isotopic consistency among tissue samples collected on days 276, 348, and 424. Both δ13C and δ15N remained consistent among sampling events in all tissues (all 95% posterior intervals for the slopes of a linear model included zero), indicating that all tissues had fully integrated diet-derived stable isotope compositions. Mean tissue-specific δ13C ranged from − 18.30 ± 0.16‰ (plasma solutes) to − 15.54 ± 0.14‰ (skin), whereas mean δ15N was from 10.06 ± 0.22‰ (whole blood) to 11.46 ± 0.10‰ (plasma solutes). The computed Δdt factors for carbon ranged from − 0.58‰ (plasma solutes) to + 2.25‰ (skin), whereas Δdt for nitrogen was from + 1.49 (red blood cells) to + 2.85 (plasma solutes). As the only discrimination factors available for leatherback turtles, our data will be useful for future interpretations of field-derived stable isotope data for this species. The inherent variability in Δdt values among individuals was low, which supports the value of these data for dietary reconstructions. However, it is important to note that tissue-specific discrimination factors for leatherbacks contrast with the widely accepted values for endothermic species (0–1‰ for C, 3–5‰ for N), and are also different from values established for hard-shelled turtles. This underscores the need for species- and tissue-specific discrimination factors before interpreting trophic studies of wild animals, including marine turtles. 相似文献
15.
Sandeep Kumar Alka Dwevedi Arvind M. Kayastha 《Journal of Molecular Catalysis .B, Enzymatic》2009,58(1-4):138-145
The soybean (Glycine max) urease was immobilized on alginate and chitosan beads and various parameters were optimized and compared. The best immobilization obtained were 77% and 54% for chitosan and alginate, respectively. A 2% chitosan solution (w/v) was used to form beads in 1N KOH. The beads were activated with 1% glutaraldehyde and 0.5 mg protein was immobilized per ml of chitosan gel for optimum results. The activation and coupling time were 6 h and 12 h, respectively. Further, alginate and soluble urease were mixed to form beads and final concentrations of alginate and protein in beads were 3.5% (w/v) and 0.5 mg/5 ml gel. From steady-state kinetics, the optimum temperature for urease was 65 °C (soluble), 75 °C (chitosan) and 80 °C (alginate). The activation energies were found to be 3.68 kcal mol−1, 5.02 kcal mol−1, 6.45 kcal mol−1 for the soluble, chitosan- and alginate-immobilized ureases, respectively. With time-dependent thermal inactivation studies, the immobilized urease showed improved stability at 75 °C and the t1/2 of decay in urease activity was 12 min, 43 min and 58 min for soluble, alginate and chitosan, respectively. The optimum pH of urease was 7, 6.2 and 7.9 for soluble, alginate and chitosan, respectively. A significant change in Km value was noticed for alginate-immobilized urease (5.88 mM), almost twice that of soluble urease (2.70 mM), while chitosan showed little change (3.92 mM). The values of Vmax for alginate-, chitosan-immobilized ureases and soluble urease were 2.82 × 102 μmol NH3 min−1 mg−1 protein, 2.65 × 102 μmol NH3 min−1 mg−1 protein and 2.85 × 102 μmol NH3 min−1 mg−1 protein, respectively. By contrast, reusability studies showed that chitosan–urease beads can be used almost 14 times with only 20% loss in original activity while alginate–urease beads lost 45% of activity after same number of uses. Immobilized urease showed improved stability when stored at 4 °C and t1/2 of urease was found to be 19 days, 80 days and 121 days, respectively for soluble, alginate and chitosan ureases. The immobilized urease was used to estimate the blood urea in clinical samples. The results obtained with the immobilized urease were quite similar to those obtained with the autoanalyzer®. The immobilization studies have a potential role in haemodialysis machines. 相似文献
16.
A new electrochemical method to monitor biotin–streptavidin interaction on carbon paste electrode, based on silver electrodeposition catalyzed by colloidal gold, was investigated. Silver reduction potential changed when colloidal gold was attached to an electrode surface through the biotin–streptavidin interaction. Thus, the direct reduction of silver ions on the electrode surface could be avoided and therefore, they were only reduced to metallic silver on the colloidal gold particle surface, forming a shell around these particles. When an anodic scan was performed, this shell of silver was oxidized and an oxidation process at +0.08 V was recorded in NH3 1.0 M. Biotinylated albumin was adsorbed on the pretreated electrode surface. This modified electrode was immersed in colloidal gold-streptavidin labeled solutions. The carbon paste electrode was then activated in adequate medium (NaOH 0.1 M and H2SO4 0.1 M) to remove proteins from the electrode surface while colloidal gold particles remained adsorbed on it. Then, a silver electrodeposition at −0.18 V for 2 min and anodic stripping voltammetry were carried out in NH3 1.0 M containing 2.0×10−5 M of silver lactate. An electrode surface preparation was carried out to obtain a good reproducibility of the analytical signal (5.3%), using a new electrode for each experiment. In addition, a sequential competitive assay was carried out to determine streptavidin. A linear relationship between peak current and logarithm of streptavidin concentration from 2.25×10−15 to 2.24×10−12 M and a limit of detection of 2.0×10−15 M were obtained. 相似文献
17.
Silanized palygorskite for lipase immobilization 总被引:2,自引:0,他引:2
Jianhua Huang Yuanfa Liu Xingguo Wang 《Journal of Molecular Catalysis .B, Enzymatic》2009,57(1-4):10-15
Lipase from Candida lipolytica has been immobilized on 3-aminopropyltriethoxysilane-modified palygorskite support. Scanning electron micrographs proved the covalently immobilization of C. lipolytica lipase on the palygorskite support through glutaraldehyde. Using an optimized immobilization protocol, a high activity of 3300 U/g immobilized lipase was obtained. Immobilized lipase retained activity over wider ranges of temperature and pH than those of the free enzyme. The optimum pH of the immobilized lipase was at pH 7.0–8.0, while the optimum pH of free lipase was at 7.0. The retained activity of the immobilized enzyme was improved both at lower and higher pH in comparison to the free enzyme. The immobilized enzyme retained more than 70% activity at 40 °C, while the free enzyme retained only 30% activity. The immobilization stabilized the enzyme with 81% retention of activity after 10 weeks at 30 °C whereas most of the free enzyme was inactive after a week. The immobilized enzyme retains high activity after eight cycles. The kinetic constants of the immobilized and free lipase were also determined. The Km and Vmax values of immobilized lipase were 0.0117 mg/ml and 4.51 μmol/(mg min), respectively. 相似文献
18.
Wei Choon Alvin Koh Eun Sang Choe Dong Kun Lee Seung-Cheol Chang Yoon-Bo Shim 《Biosensors & bioelectronics》2009,25(1):211-217
An all solid state potentiometric immunosensor (ASPI) has been developed to study the activation process of neuronal nitric oxide synthase (nNOS), the enzyme involved in the synthesis of nitric oxide generated under physiological conditions. At first, an all solid state H+-selective ISE was fabricated with the carboxylated poly(vinyl chloride) (PVC-COOH) film containing H+ ionophore, antibody was then immobilized on the polymer layer. The immunocomplex formation was detected by monitoring pH change due to interaction between urease labeled secondary antibody and antigen. Experimental parameters such as the amount of phosphorylated nNOS immobilized on the electrode surface and pH responses due to the antibody–antigen reaction were studied in detail. The calibration plot of the potentiometric potential vs. phosphorylated nNOS concentration exhibited a linear relationship in the range of 3.4–340.0 μg/ml. The calibration sensitivity of the phosphorylated nNOS immunosensor was −0.073 ± 0.002 mV/μg ml−1. The detection limit of nNOS was determined to be 0.2 μg/ml based on five-time measurements (95% confidence level, k = 3, n = 5). The reliability of the immunosensor was examined with rat brain tissues as well as neuronal cells, and the results shown were good, implying a promising approach for a novel electrochemical immunosensor platform with potential applications to clinical diagnosis. 相似文献
19.
A hydrogen peroxide permselective membrane with asymmetric structure was prepared and
-glucose oxidase (EC 1.1.3.4) was immobilized onto the porous layer. The activity of the immobilized
-glucose oxidase membrane was 0.34 units cm−2 and the activity yield was 6.8% of that of the native enzyme. Optimum pH, optimum temperature, pH stability and temperature stability were found to be pH 5.0, 30–40°C, pH 4.0–7.0 and below 55°C, respectively. The apparent Michaelis constant of the immobilized
-glucose oxidase membrane was 1.6 × 10−3 mol l−1 and that of free enzyme was 4.8 × 10−2 mol l−1. An enzyme electrode was constructed by combination of a hydrogen peroxide electrode with the immobilized
-glucose oxidase membrane. The enzyme electrode responded linearly to
-glucose over the concentration 0–1000 mg dl−1 within 10 s. When the enzyme electrode was applied to the determination of
-glucose in human serum, within day precision (CV) was 1.29% for
-glucose concentration with a mean value of 106.8 mg dl−1. The correlation coefficient between the enzyme electrode method and the conventional colorimetric method using a free enzyme was 0.984. The immobilized
-glucose oxidase membrane was sufficiently stable to perform 1000 assays (2 to 4 weeks operation) for the determination of
-glucose in human whole blood. The dried membrane retained 77% of its initial activity after storage at 4°C for 16 months. 相似文献
20.
Om Prakash Mahe Talat Syed Hadi Hasan 《Journal of Molecular Catalysis .B, Enzymatic》2009,56(4):265-271
Soluble and alginate immobilized urease was utilized for detection and quantitation of mercury in aqueous samples. Urease from the seeds of pumpkin, being a vegetable waste, was extracted and purified to apparent homogeneity (sp. activity 353 U/mg protein; A280/A260 = 1.12) by heat treatment at 48 ± 0.1 °C and gel filtration through Sephadex G-200. Homogeneous enzyme preparation was immobilized in 3.5% alginate leading to 86% immobilization, no leaching of enzyme was found over a period of 15 days at 4 °C. Urease catalyzed urea hydrolysis by soluble and immobilized enzyme revealed a clear dependence on the concentration of Hg2+. Inhibition caused by Hg2+ was non-competitive (Ki = 1.2 × 10−1 μM for soluble and 1.46 × 10−1 μM for alginate immobilized urease.). Time-dependent inhibition both in presence and in absence of Hg2+ ion revealed a biphasic inhibition in activity. For optimization of this process response surface methodology (RSM) was utilized where two-level-two-full factorial (22) central composite design (CCD) has been employed. The regression equation and analysis of variance (ANOVA) were obtained using MINITAB® 15 software. Predicted values thus obtained were closed to experimental value indicating suitability of the model. 3D response surface plot, iso-response contour plot and process optimization curve were helpful to predict the results by performing only limited set of experiments. 相似文献