Kinetic Constants (kinetic + constant)

Distribution by Scientific Domains
Distribution within Chemistry

Selected Abstracts

Characterization of phycoviolobilin phycoerythrocyanin-,84-cystein-lyase-(isomerizing) from Mastigocladus laminosus

FEBS JOURNAL, Issue 18 2002
Kai-Hong Zhao
Cofactor requirements and enzyme kinetics have been studied of the novel, dual-action enzyme, the isomerizing phycoviolobilin phycoerythrocyanin-,84-cystein-lyase(PVB-PEC-lyase) from Mastigocladus laminosus, which catalyses both the covalent attachment of phycocyanobilin to PecA, the apo-,-subunit of phycoerythrocyanin, and its isomerization to phycoviolobilin. Thiols and the divalent metals, Mg2+ or Mn2+, were required, and the reaction was aided by the detergent, Triton X-100. Phosphate buffer inhibits precipitation of the proteins present in the reconstitution mixture, but at the same time binds the required metal. Kinetic constants were obtained for both substrates, the chromophore (Km = 12,16 m, depending on [PecA], kcat , 1.2 10,4s,1) and the apoprotein (Km = 2.4 m at 14 m PCB, kcat = 0.8 10,4s,1). The kinetic analysis indicated that the reconstitution reaction proceeds by a sequential mechanism. By a combination of untagged and His-tagged subunits, evidence was obtained for a complex formation between PecE and PecF (subunits of PVB-PEC-lyase), and by experiments with single subunits for the prevalent function of PecE in binding and PecF in isomerizing the chromophore. [source]

Modeling of Slurry Polymerization of Ethylene Using a Soluble Cp2ZrCl2/MAO Catalytic System

Mostafa Ahmadi
Abstract The slurry homopolymerization of ethylene catalyzed by a Cp2ZrCl2/MAO catalytic system was studied. A simple kinetic model including initiation, propagation, transfer to monomer and cocatalyst, spontaneous transfer and spontaneous deactivation was developed to predict dynamic yield of polymerization and molecular weight of final products. Kinetic constants were estimated by numerical solution of polymerization kinetic model, combined with Nelder-Mead simplex method. The model predicts that the propagation reaction has the lower activation energy in relation to chain transfer reactions which leads to decrease of molecular weight at elevated temperatures. The initiation reaction has however, the highest activation energy that results in raising the peak of reaction rate at higher temperatures. [source]

Atmospheric pressure desorption/ionization on silicon ion trap mass spectrometry applied to the quantitation of midazolam in rat plasma and determination of midazolam 1,-hydroxylation kinetics in human liver microsomes

Rick C. Steenwyk
The application of atmospheric pressure desorption/ionization on silicon (AP-DIOS) coupled with ion trap mass spectrometry (ITMS) was investigated for the quantification of midazolam in rat plasma, and determination of midazolam 1,-hydroxylation kinetics in pooled human liver microsomes. Results indicate good sensitivity with absolute detection limits for midazolam in rat plasma of approximately 300 femtograms. A linear dynamic range from approximately 10,5000,ng/mL was obtained in rat plasma with analysis times of 1,min per sample. Kinetic constants for midazolam 1,-hydroxylation in human liver microsomes yielded an apparent Km of 10.0,M and Vmax of 6.4,nmol/min/mg. Studies investigating the inhibition of 1,-hydroxymidazolam formation by the cytochrome P450 3A4 model inhibitor ketoconazole yielded an IC50 of 0.03,M. Quantitative precision for replicate analysis of rat plasma and human liver microsomal samples was variable with relative standard deviation (RSD) values ranging from a low of approximately 3% to over 50%, with the highest variability observed in data from human liver microsomal incubations. While preliminary studies investigating the application of AP-DIOS-ITMS suggested feasibility of this technique to typical pharmacokinetic applications, further work is required to understand the underlying causes for the high variability observed in these investigations. Copyright 2006 John Wiley & Sons, Ltd. [source]

Biosynthesis reaction mechanism and kinetics of deoxynucleoside triphosphates, dATP and dGTP

Jie Bao
Abstract The enzyme reaction mechanism and kinetics for biosyntheses of deoxyadenosine triphosphate (dATP) and deoxyguanosine triphosphate (dGTP) from the corresponding deoxyadenosine diphosphate (dADP) and deoxyguanosine diphosphate (dGDP) catalyzed by pyruvate kinase were studied. A kinetic model for this synthetic reaction was developed based on a Bi-Bi random rapid equilibrium mechanism. Kinetic constants involved in this pyruvate kinase catalyzed phosphorylation reactions of deoxynucleoside diphosphates including the maximum reaction velocity, Michaelis-Menten constants, and inhibition constants for dATP and dGTP biosyntheses were experimentally determined. These kinetic constants for dATP and dGTP biosyntheses are of the same order of magnitude but significantly different between the two reactions. Kinetic constants involved in ATP and GTP biosyntheses as reported in literature are about one order of magnitude different from those involved in dATP and dGTP biosyntheses. This enzyme reaction requires Mg2+ ion and the optimal Mg2+ concentration was also determined. The experimental results showed a very good agreement with the simulation results obtained from the kinetic model developed. This kinetic model can be applied to the practical application of a pyruvate kinase reaction system for production of dATP and dGTP. There is a significant advantage of using enzymatic biosyntheses of dATP and dGTP as compared to the chemical method that has been in commercial use. 2005 Wiley Periodicals, Inc. [source]

Thermally induced conformational changes in horseradish peroxidase

FEBS JOURNAL, Issue 1 2001
David G. Pina
Detailed differential scanning calorimetry (DSC), steady-state tryptophan fluorescence and far-UV and visible CD studies, together with enzymatic assays, were carried out to monitor the thermal denaturation of horseradish peroxidase isoenzyme c (HRPc) at pH 3.0. The spectral parameters were complementary to the highly sensitive but integral method of DSC. Thus, changes in far-UV CD corresponded to changes in the overall secondary structure of the enzyme, while that in the Soret region, as well as changes in intrinsic tryptophan fluorescence emission, corresponded to changes in the tertiary structure of the enzyme. The results, supported by data about changes in enzymatic activity with temperature, show that thermally induced transitions for peroxidase are irreversible and strongly dependent upon the scan rate, suggesting that denaturation is under kinetic control. It is shown that the process of HRPc denaturation can be interpreted with sufficient accuracy in terms of the simple kinetic scheme where k is a first-order kinetic constant that changes with temperature, as given by the Arrhenius equation; N is the native state, and D is the denatured state. On the basis of this model, the parameters of the Arrhenius equation were calculated. [source]

A kinetic evaluation of the anaerobic digestion of two-phase olive mill effluent in batch reactors

Francisco Raposo
Abstract A comparative kinetic study was carried out on the anaerobic digestion of two-phase olive mill effluent (TPOME) using three 1-dm3 volume stirred tank reactors, one with freely suspended biomass (control), and the other two with biomass supported on polyvinyl chloride (PVC) and bentonite (aluminium silicate), respectively. The reactors were batch fed at mesophilic temperature (35 C) using volumes of TPOME of between 50 and 600 cm3, corresponding to chemical oxygen demand (COD) loadings in the range of 1.02,14.22 g, respectively. The process followed first-order kinetics and the specific rate constants, K0, were calculated. The K0 values decreased considerably from 2.59 to 0.14 d,1, from 1.93 to 0.23 d,1 and from 1.52 to 0.17 d,1 for the reactors with suspended biomass (control) and biomass immobilized on PVC and bentonite, respectively, when the COD loadings increased from 1.02 to 14.22 g; this showed an inhibition phenomenon in the three reactors studied. The values of the critical inhibitory substrate concentration (S*), theoretical kinetic constant without inhibition (KA) and the inhibition coefficient or inhibitory parameter for each reactor (n) were determined using the Levenspiel model. Copyright 2004 Society of Chemical Industry [source]

Aggregation kinetics of recombinant human FVIII (rFVIII)

Karthik Ramani
Abstract The physical phenomenon of aggregation can have profound impact on the stability of therapeutic proteins. This study focuses on the aggregation behavior of recombinant human FVIII (rFVIII), a multi-domain protein used as the first line of therapy for hemophilia A, a bleeding disorder caused by the deficiency or dysfunction of factor VIII (FVIII). Thermal denaturation of rFVIII was investigated using circular dichroism (CD) spectroscopy and size exclusion chromatography (SEC). The dependence of unfolding on heating rate indicated that the thermal denaturation of the protein was at least partly under kinetic control. The data was interpreted in terms of a simple two-state kinetic model, , where k is a first-order kinetic constant that changes with temperature, as given by the Arrhenius equation. Analysis of the data in terms of the above scheme suggested that under the experimental conditions used in this study, the rate-controlling step in the aggregation of rFVIII may be a unimolecular reaction involving conformational changes. 2005 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 94:2023,2029, 2005 [source]

Incubation period in the 2,2,4,4-tetramethyl-1-piperidinyloxy-mediated thermal autopolymerization of styrene: Kinetics and simulations

Enrique Saldvar-Guerra
Abstract Mechanisms and simulations of the induction period and the initial polymerization stages in the nitroxide-mediated autopolymerization of styrene are discussed. At 120,125 C and moderate 2,2,4,4-tetramethyl-1-piperidinyloxy (TEMPO) concentrations (0.02,0.08 M), the main source of radicals is the hydrogen abstraction of the Mayo dimer by TEMPO [with the kinetic constant of hydrogen abstraction (kh)]. At higher TEMPO concentrations ([N,] > 0.1 M), this reaction is still dominant, but radical generation by the direct attack against styrene by TEMPO, with kinetic constant of addition kad, also becomes relevant. From previous experimental data and simulations, initial estimates of kh , 1 and kad , 6 10,7 L mol,1 s,1 are obtained at 125 C. From the induction period to the polymerization regime, there is an abrupt change in the dominant mechanism generating radicals because of the sudden decrease in the nitroxide radicals. Under induction-period conditions, the simulations confirm the validity of the quasi-steady-state assumption (QSSA) for the Mayo dimer in this regime; however, after the induction period, the QSSA for the dimer is not valid, and this brings into question the scientific basis of the well-known expression kth[M]3 (where [M] is the monomer concentration and kth is the kinetic constant of autoinitiation) for the autoinitiation rate in styrene polymerization. 2006 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 44: 6962-6979, 2006 [source]

Photoinitiated polymerization of methacrylic monomers in a polystyrene matrix: Kinetic, mechanistic, and structural aspects

J. L. Mateo
Abstract The kinetics and mechanism of the photoinitiated polymerization of tetrafunctional and difunctional methacrylic monomers [1,6-hexanediol dimethacrylate (HDDMA) and 2-ethylhexyl methacrylate (EHMA)] in a polystyrene (PS) matrix were studied. The aggregation state, vitreous or rubbery, of the monomer/matrix system and the intermolecular strength of attraction in the monomer/matrix and growing macroradical/matrix systems are the principal factors influencing the kinetics and mechanism. For the PS/HDDMA system, where a relatively high intermolecular force of attraction between monomer and matrix and between growing macroradical and matrix occurs, a reaction-diffusion mechanism takes place at low monomer concentrations (<30,40%) from the beginning of the polymerization. For the PS/EHMA system, which presents low intermolecular attraction between monomer and matrix and between growing macroradical and matrix, the reaction-diffusion termination is not clear, and a combination of reaction-diffusion and diffusion-controlled mechanisms explains better the polymerization for monomer concentrations below 30,40%. For both systems, for which a change from a vitreous state to a rubbery state occurs when the monomer concentration changes from 10 to 20%, the intrinsic reactivity and kp/kt1/2 ratio (where kp is the propagation kinetic constant and kt is the termination kinetic constant) increase as a result of a greater mobility of the monomer in the matrix (a greater kp value). The PS matrix participates in the polymerization process through the formation of benzylic radical, which is bonded to some extent by radical,radical coupling with the growing methacrylic radica, producing grafting on the PS matrix. 2001 John Wiley & Sons, Inc. J Polym Sci Part A: Polym Chem 39: 2049,2057, 2001 [source]

Kinetics of Core-Shell Nanoparticle Formation by Two-Dimensional Nuclear Magnetic Resonance

Alaitz Ruiz de Luzuriaga
Abstract We have studied the kinetics of polymeric nanoparticle formation for poly(styrene- block -4-vinylpyridine) [P(S- b -4-VPy)], chains in a non-selective solvent using 1,4-dibromobutane (DBB) as a cross-linker by means of different nuclear magnetic resonance (NMR) spectroscopy techniques. The kinetic process was followed using 1H, 13C, and 2-D Heteronuclear Single Quantum Correlation (HSQC) NMR experiments. The kinetic data obtained from 2-D HSQC and 1H NMR experiments were in good agreement between them, proving the reliability of the 2-D HSQC NMR technique for the in situ study of the kinetics of core-shell nanoparticle formation. A value of 1.5,,10,5 s,1 was determined for the apparent kinetic constant of the P(S- b -4-VPy)-DBB core-shell nanoparticle formation process. [source]

Engineered Pyranose 2-Oxidase: Efficiently Turning Sugars into Electrical Energy

Oliver Spadiut
Abstract Due to the recent interest in enzymatic biofuel cells (BFCs), sugar oxidizing enzymes other than the commonly used glucose oxidase are gaining more importance as possible bioelements of implantable microscale-devices, which can, for example, be used in biosensors and pacemakers. In this study we used rational and semi-rational protein design to improve the catalytic activity of the enzyme pyranose 2-oxidase (P2Ox) with its alternative soluble electron acceptors 1,4-benzoquinone and ferricenium ion, which can serve as electron mediators, to possibly boost the power output of enzymatic BFCs. Using a screening assay based on 96-well plates, we identified the variant H450G, which showed lower KM and higher kcat values for both 1,4-benzoquinone and ferricenium ion compared to the wild-type enzyme, when either D -glucose or D -galactose were used as saturating electron donors. Besides this variant, we analyzed the variants V546C and T169G/V546C for their possible application in enzymatic BFCs. The results obtained in homogeneous solution were compared with those obtained when P2Ox was immobilized on the surface of graphite electrodes and either "wired" to an osmium redox polymer or using soluble 1,4-benzoquinone as mediator. According to the spectrophotometrically determined kinetic constants, the possible energy output, measured in flow injection analysis experiments with these variants, increased up to 4-fold compared to systems employing the wild-type enzyme. [source]

Subsite specificity of trypanosomal cathepsin L-like cysteine proteases

FEBS JOURNAL, Issue 9 2001
Probing the S2 pocket with phenylalanine-derived amino acids
The S2 subsite of mammalian cysteine proteinases of the papain family is essential for specificity. Among natural amino acids, all these enzymes prefer bulky hydrophobic residues such as phenylalanine at P2. This holds true for their trypanosomal counterparts: cruzain from Trypanosoma cruzi and congopain from T. congolense. A detailed analysis of the S2 specificity of parasitic proteases was performed to gain information that might be of interest for the design of more selective pseudopeptidyl inhibitors. Nonproteogenic phenylalanyl analogs (Xaa) have been introduced into position P2 of fluorogenic substrates dansyl-Xaa-Arg-Ala-Pro-Trp, and their kinetic constants (Km, kcat/Km) have been determined with congopain and cruzain, and related host cathepsins B and L. Trypanosomal cysteine proteases are poorly stereoselective towards d/l -Phe, the inversion of chirality modifying the efficiency of the reaction but not the Km. Congopain binds cyclohexylalanine better than aromatic Phe derivatives. Another characteristic feature of congopain compared to cruzain and cathepsins B and L was that it could accomodate a phenylglycyl residue (kcat/Km = 1300 mm,1s,1), while lengthening of the side chain by a methylene group only slightly impaired the specificity constant towards trypanosomal cysteine proteases. Mono- and di-halogenation or nitration of Phe did not affect Km for cathepsin L-like enzymes, but the presence of constrained Phe derivatives prevented a correct fitting into the S2 subsite. A model of congopain has been built to study the fit of Phe analogs within the S2 pocket. Phe analogs adopted a positioning within the S2 pocket similar to that of the Tyr of the cruzain/Z-Tyr-Ala-fluoromethylketone complex. However, cyclohexylalanine has an energetically favorable chair-like conformation and can penetrate deeper into the subsite. Fitting of modeled Phe analogs were in good agreement with kinetic parameters. Furthermore, a linear relationship could be established with logP, supporting the suggestion that fitting into the S2 pocket of trypanosomal cysteine proteases depends on the hydrophobicity of Phe analogs. [source]

Inorganic reactions of iodine(+1) in acidic solutions

Guy Schmitz
We present a thorough analysis of the former works concerning the hydrolysis of iodine and its mechanism in acidic or neutral solutions and recommend values of equilibrium and kinetic constants. Since the literature value for the reaction H2OI+ , HOI + H+ appeared questionable, we have measured it by titration of acidic iodine solutions with AgNO3. Our new value, K(H2OI+ , HOI + H+) , 2 M at 25C, is much larger than accepted before. It decreases slowly with the temperature. We have also measured the rate of the reaction 3HOI , IO3, + 2I, + 3H+ in perchloric acid solutions from 5 10,2 M to 0.5 M. It is a second order reaction with a rate constant nearly independent on the acidity. Its value is 25 M,1 s,1 at 25C and decreases slightly when the temperature increases, indicating that the disproportionation mechanism is more complicated than believed before. An analysis of the studies of this disproportionation in acidic and slightly basic solutions strongly supports the importance of a dimeric intermediate 2HOI , I2OH2O in the mechanism. 2004 Wiley Periodicals, Inc. Int J Chem Kinet 36:480,493, 2004 [source]

Urease immobilization on an ion-exchange textile for urea hydrolysis

Kyeong-Ho Yeon
Abstract Ion-exchange textiles are used as organic supports for urease immobilization with the aim of developing reactive fibrous materials able to promote urea removal. A non-woven, polypropylene-based cation-exchange textile was prepared using UV-induced graft polymerization. Urease was covalently immobilized onto the cation-exchange textile using three different coupling agents: N -(3-dimethylaminopropyl)- N,-ethylcarbodiimide hydrochloride (EDC), N -cyclohexyl- N,-(b -[N -methylmorpholino]ethyl)carbodiimide p -toluenesulfonate (CMC), and glutaraldehyde (GA). The immobilized biocatalyst was characterized by means of FT-IR spectrometry, SEM micrographs, dependence of the enzyme activity on pH and temperature, and according to the kinetic constants of the free and immobilized ureases. The biotextile prepared with EDC in the presence of N -hydroxysuccinimide performs best. The optimum pH was 7.2 for the free urease and 7.6 for the immobilized ureases. The reactivity was maximal at 45 C for free urease, 50 C for biotextiles prepared using EDC or CMC, and 55 C for biotextiles prepared with GA. The activation energy for the immobilized ureases was 4.73,5.67 kcal mol,1, which is somewhat higher than 4.3 kcal mol,1 for free urease. The urea conversion for a continuous-flow immobilized urease reactor is nearly as good as a continuously stirred tank reactor having a much longer residence time, suggesting that the packed bed reactor had sufficient diffusive mixing and residence time to reach nearly optimal results. Urease immobilized on a biotextile using EDC has good storage and operational stability. Copyright 2006 Society of Chemical Industry [source]

Effect of xylanase on ozone bleaching kinetics and properties of Eucalyptus kraft pulp

M Blanca Roncero
Abstract Environmental pressure has led the pulp and paper industry to develop new technologies in order to reduce or suppress the presence of various pollutants in effluents from bleaching plants. One of the choices for this purpose is enzyme-based biotechnology. This study deals with the effect of using a xylanase-based enzymatic pretreatment, in a TCF (Totally Chlorine Free) sequence, on the properties of the resulting paper pulps. The hexenuronic acid content in the pulp and the physical properties of the paper were also studied. The performance of the xylanase was analysed through kinetic studies on ozone bleaching. The enzymatic pretreatment results in easier bleaching and delignification of the pulp, causing a bleach-boosting effect. The decreased consumption of reagent is related to a decreased content of hexenuronic groups. The physical properties of the treated pulp are similar to those of untreated pulps. Cellulose degradation, delignification and chromophores' removal show first-order kinetics. Enzyme pretreatment leads to differences between the kinetic constants of cellulose degradation and chromophores' removal, due to an increased accessibility to bleaching agents. The xylanase treatment leads to a lower floor kappa number (IK,) during the ozone stage. Copyright 2003 Society of Chemical Industry [source]

Production of L -methionine by immobilized pellets of Aspergillus oryzae in a packed bed reactor

Ying-Jin Yuan
Abstract Production of L -methionine by immobilized pellets of Aspergillus oryzae in a packed bed reactor was investigated. Based on the determination of relative enzymatic activity in the immobilized pellets, the optimum pH and temperature for the resolution reaction were 8.0 and 60,C, respectively. The effects of substrate concentration on the resolution reaction were also investigated and the kinetic constants (Km and Vm) of immobilized pellets were found to be 7.99,mmol,dm,3 and 1.38,mmol,dm,3 h,1, respectively. The maximum substrate concentration for the resolution reaction without inhibition was 0.2,mol,dm,3. The L -methionine conversion rate reached 94% and 78% when substrate concentrations were 0.2 and 0.4,mol,dm,3, respectively, at a flow rate of 7.5,cm3,h,1 using the small-scale packed bed reactor developed. The half-life of the L -aminoacylase in immobilized pellets was 70 days in continuous operation. All the results obtained in this paper exhibit a practical potential of using immobilized pellets of Aspergillus oryzae in the production of L -methionine. 2002 Society of Chemical Industry [source]

Monitoring of a second-order reaction by electronic absorption spectroscopy using combined chemometric and kinetic models

Tom J. Thurston
Abstract This paper reports the application of 11 methods for obtaining kinetic constants from a second-order reaction, that between phenylhydrazine and benzophenone. In this type of reaction the number of absorbing species is lower than the number of steps in the reaction minus one, resulting in a rank-deficient response matrix. The methods used include traditional univariate curve fitting, classical least squares using previously recorded pure spectra, alternating least squares methods with both kinetic and non-negativity constraints, and target-testing methods using principal component scores. An additional recently proposed method based on difference spectra is also examined, suitable for any single-step closed reaction. The methods that performed best were difference spectra, kinetically constrained alternating least squares, and target-testing approaches. Limitations of the traditional methods are described. Copyright 2003 John Wiley & Sons, Ltd. [source]

Comparison of Deamidation Activity of Transglutaminases

T. Ohtsuka
ABSTRACT: A comparison was made of the deamidation activity of transglutaminases fro m guinea pig liver (GTGase), fish red sea bream liver (FTGase) and microorganisms (MTGase). Against the Z-Gln-Gly, kinetic constants of the deamidation and incorporation of primary amine were measured. GTGase and FTGase showed similar deamidation activity, however, that of MTGase was less than 1/7 of the other two TGases. Against the proteins, N, N-dimethylated casein and native gliadin, FTGase was the most active and deamidated respectively 45.5% and 38.2% of Gln residues. The deamidation rate of these proteins by GTGase was less than 1/2 and these results were expected to be caused by the difference of substrate specificity of the TGases. [source]

Bubble size distribution modeling in stirred gas,liquid reactors with QMOM augmented by a new correction algorithm

AICHE JOURNAL, Issue 1 2010
Miriam Petitti
Abstract Local gas hold-up and bubbles size distributions have been modeled and validated against experimental data in a stirred gas,liquid reactor, considering two different spargers. An Eulerian multifluid approach coupled with a population balance model (PBM) has been employed to describe the evolution of the bubble size distribution due to break-up and coalescence. The PBM has been solved by resorting to the quadrature method of moments, implemented through user defined functions in the commercial computational fluid dynamics code Fluent v. 6.2. To overcome divergence issues caused by moments corruption, due to numerical problems, a correction scheme for the moments has been implemented; simulation results prove that it plays a crucial role for the stability and the accuracy of the overall approach. Very good agreements between experimental data and simulations predictions are obtained, for a unique set of break-up and coalescence kinetic constants, in a wide range of operating conditions. 2009 American Institute of Chemical Engineers AIChE J, 2010 [source]

The turnover of the H3 deuterons from (2- 13C) glutamate and (2- 13C) glutamine reveals subcellular trafficking in the brain of partially deuterated rats

Sebastin Cerdn
Abstract We investigated by 13C NMR the turnover of the H3 deuterons of (2- 13C) glutamate and (2- 13C) glutamine in the brain of partially deuterated rats. Adult animals (150,200 g) fed ad libitum received 50%2H2O or tap water 9 days before infusing (1- 13C) glucose or (2- 13C) acetate for 5, 10, 15, 30, 60, or 90 min. The brains were then funnel-frozen and acid extracts were prepared and analyzed by high-resolution 13C NMR. The deuteration of one or the two H3 hydrogens of (2- 13C) glutamate or glutamine resulted in single (,0.07 ppm) or double (,0.14 ppm) isotopic shifts upfield of the corresponding C2 perprotonated resonance, demonstrating two sequential deuteration steps. The faster monodeuteration generated 3R or 3S (2- 13C, 3- 2H) glutamate or glutamine through the alternate activities of cerebral aconitase or isocitrate dehydrogenase, respectively. The slower process produced bideuterated (2- 13C, 3,3,- 2H2) glutamate or glutamine through the consecutive activity of both enzymes. The kinetics of deuteration was fitted to a Michaelis,Menten model including the apparent Km, and Vmax, values for the observed deuterations. Our results revealed different kinetic constants for the alternate and consecutive deuterations, suggesting that these processes were caused by the different cytosolic or mitochondrial isoforms of aconitase and isocitrate dehydrogenase, respectively. The deuterations of (2- 13C) glutamate or glutamine followed also different kinetics from (1- 13C) glucose or (2- 13C) acetate, revealing distinct deuteration environments in the neuronal or glial compartments. [source]

Carboxy terminus of secreted phosphoprotein-24 kDa (spp24) is essential for full inhibition of BMP-2 activity

Elsa J. Brochmann
Abstract Secreted phosphoprotein-24,kDa (spp24) is a bone morphogenetic protein (BMP)-binding protein isolated from bone. It exists in a number of size forms and is hypothesized to function as a BMP latency protein and/or a "slow release" mechanism for BMPs involved in bone turnover and repair. We have examined the hypothesis that proteolytic modification of the C-terminus of spp24 affects its BMP-2,binding properties and bioactivity in the BMP-2,stimulated ectopic bone forming bioassay. Three different size forms of recombinant spp24 that correspond to predicted 18.1,kDa, 16.0,kDa, and 14.5,kDa proteolytic products were compared to full-length (fl) spp24. One of these forms (spp18.1) we hypothesize to be the protein which Urist initially, but apparently inaccurately, called "BMP." Only full-length spp24 completely inhibited BMP-2,induced bone formation. The 18.1,kDa truncated isoform of spp24 which we hypothesize to be Urist's protein did not. The inhibitory capacity of the proteins was correlated with their kinetic constants, assessed by surface plasmon resonance. At the highest, inhibitory, dose of spp24 and its derivatives, kd ("stability") best predicted the extent of ectopic bone formation whereas at the lowest dose, which was not inhibitory, ka ("recognition") best predicted the extent of ectopic bone formation. We conclude that proteolytic processing of spp24 affects the interaction of this protein with BMP-2 and this affects the function of the protein. 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:1200,1207, 2010 [source]

Photoinitiated polymerization of methacrylic monomers in a polybutadiene matrix (PB): Kinetic, mechanistic, and structural aspects

J. L. Mateo
Abstract The kinetics and mechanism of the photoinitiated polymerization of tetrafunctional and difunctional methacrylic monomers [1,6-hexanediol dimethacylate (HDDMA) and 2-ethylhexyl methacrylate (EHMA)] in a polybutadiene matrix (PB) have been studied. The maximum double-bond conversion, the maximum polymerization rate, the intrinsic reactivity, and the kinetic constants for propagation and termination have been calculated. Unlike the behavior followed by the SBS-HDDMA and PS-HDDMA systems, where a reaction-diffusion mechanism occurs from the start of the polymerization at low monomer concentrations (<30,40%), in the PB-HDDMA system the reaction diffusion controls the termination process only after approximately 10% conversion is reached, as for the bulk polymerization of polyfunctional methacrylic monomers. Before reaching 10% conversion the behavior observed can be better explained by a combination of segmental diffusion-controlled (autoaccelerated) and reaction-diffusion mechanisms. This is probably a consequence of the lower force of attraction between the monomer and the matrix and between the growing macroradical and the matrix than those corresponding to the other systems mentioned. For the PB-EHMA system, the termination mechanism is principally diffusion-controlled from the beginning of the polymerization for monomer concentrations below 30,40%, and for higher monomer concentrations, a standard termination mechanism takes place (kt , 106) at low double-bond conversions, which is diffusion-controlled for high conversions (>40%). For PB-HDDMA and PB-EHMA systems, crosslinked polymerized products are obtained as a result of the participation of the double bonds of the matrix in the polymerization process. 2001 John Wiley & Sons, Inc. J Polym Sci Part A: Polym Chem 39: 2444,2453, 2001 [source]

Purification and some properties of a cysteine proteinase from sorghum malt variety SK5912

Augustine C Ogbonna
Abstract A cysteine proteinase from sorghum malt variety SK5912 was purified by a combination of 4 M sucrose fractionation, ion-exchange chromatography on Q- and S-Sepharose (fast flow), gel filtration chromatography on Sephadex G-100 and hydrophobic interaction chromatography on Phenyl Sepharose CL-4B. The enzyme was purified 8.4-fold to give a 13.4% yield relative to the total activity in the crude extract and a final specific activity of 2057.1 U mg,1 protein. SDS,PAGE revealed two migrating protein bands corresponding to apparent relative molecular masses of 55 and 62 kDa, respectively. The enzyme was optimally active at pH 6.0 and 50 C, not influenced across a relatively broad pH range of 5.0,8.0 and retained over 60% activity at 70 C after 30-min incubation. It was highly significantly (P < 0.001) inhibited by Hg2+, appreciably (P < 0.01) inhibited by Ag+, Ba2+ and Pb2+ but highly significantly (P < 0.001) activated by Co2+, Mn2+ and Sr2+. The proteinase was equally highly significantly (P < 0.001) inhibited by both iodoacetate and p -chloromercuribenzoate and hydrolysed casein to give the following kinetic constants: Km = 0.33 mg ml,1; Vmax = 0.08 mol ml,1 min,1. Copyright 2004 Society of Chemical Industry [source]

Kinetic Study of the Thermopolymerization of Furfuryl Methacrylate in Bulk by Mathematical Modeling.

Part A: Simulation of Experimental Data, Sensitivity Analysis of Kinetic Parameters
Abstract Mathematical modeling of the thermopolymerization of FM and CMFMA was carried out using a cross-linked kinetic model proposed for the photo-initiated polymerization of acryl-furanic compounds. In this model, the photochemical initiation step was substituted by a thermal one and it was assumed that the constant of radical termination was time-dependent, which allowed the gel effect (Trommsdorff) at high monomer conversion to be simulated. Optimization of all kinetic constants was achieved and the results of simulation suitably fitted the experimental data of the monomer conversion. The contribution of each step in the mechanism and its dependence on the experimental conditions were estimated by a sensitivity analysis technique. [source]

Mechanistic Studies of Catechol Generation from Secondary Quinone Amines Relevant to Indole Formation and Tyrosinase Activation

Edward J. Land
The biological significance of the spontaneous cyclization and redox reactions of ortho -quinone amines is that these appear to be the mechanism of formation of the indolic components of melanin and are also involved in the autoactivation of tyrosinase. We have previously shown that activation of tyrosinase is prevented by the formation of a cyclic betaine from a tertiary amine analogue. Evidence is presented to show that cyclization of ortho -quinones by Michael addition also occurs in the oxidation of secondary catecholamines. Three varieties of cyclic product have been detected and their formation is influenced by the nature of the N -substituent. Five-membered betaine rings form directly and, although six- and seven-membered rings also form, a transient spiro isomer of the ortho -quinone was in some cases detected as an intermediate. The heterocyclic products formed as betaines undergo redox exchange with residual quinone to form the corresponding aminochromes. We have established the kinetic constants of these reactions, either directly by pulse radiolysis measurements or by inference using a computer model of the reaction pathway to fit the observed data. To investigate the potential biological applications of this chemistry the system was also examined by tyrosinase-catalysed oxidation of the catecholamine substrates in which there is re-oxidation of the catechol formed by the redox exchange reaction and enables measurement of oxygen utilization stoichiometry. We show that the redox exchange reaction is unaffected by side-chain modification whereas cyclization is dependent on both electronic and steric factors. In the light of these studies we conclude that the failure of tertiary amine-derived betaines to undergo redox exchange, and thus block in vitro activation of tyrosinase, is due to the absence of a second exchangeable proton. [source]

On the need to incorporate sensitivity to CO2 transfer conductance into the Farquhar,von Caemmerer,Berry leaf photosynthesis model

ABSTRACT Virtually all current estimates of the maximum carboxylation rate (Vcmax) of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and the maximum electron transport rate (Jmax) for C3 species implicitly assume an infinite CO2 transfer conductance (gi). And yet, most measurements in perennial plant species or in ageing or stressed leaves show that gi imposes a significant limitation on photosynthesis. Herein, we demonstrate that many current parameterizations of the photosynthesis model of Farquhar, von Caemmerer & Berry (Planta 149, 78,90, 1980) based on the leaf intercellular CO2 concentration (Ci) are incorrect for leaves where gi limits photosynthesis. We show how conventional A,Ci curve (net CO2 assimilation rate of a leaf ,An, as a function of Ci) fitting methods which rely on a rectangular hyperbola model under the assumption of infinite gi can significantly underestimate Vcmax for such leaves. Alternative parameterizations of the conventional method based on a single, apparent Michaelis,Menten constant for CO2 evaluated at Ci[Km(CO2)i] used for all C3 plants are also not acceptable since the relationship between Vcmax and gi is not conserved among species. We present an alternative A,Ci curve fitting method that accounts for gi through a non-rectangular hyperbola version of the model of Farquhar et al. (1980). Simulated and real examples are used to demonstrate how this new approach eliminates the errors of the conventional A,Ci curve fitting method and provides Vcmax estimates that are virtually insensitive to gi. Finally, we show how the new A,Ci curve fitting method can be used to estimate the value of the kinetic constants of Rubisco in vivo is presented [source]

Would transformation of C3 crop plants with foreign Rubisco increase productivity?

A computational analysis extrapolating from kinetic properties to canopy photosynthesis
ABSTRACT Genetic modification of Rubisco to increase the specificity for CO2 relative to O2 (,) would decrease photorespiration and in principle should increase crop productivity. When the kinetic properties of Rubisco from different photosynthetic organisms are compared, it appears that forms with high , have low maximum catalytic rates of carboxylation per active site (kcc). If it is assumed that an inverse relationship between kcc and , exists, as implied from measurements, and that an increased concentration of Rubisco per unit leaf area is not possible, will increasing , result in increased leaf and canopy photosynthesis? A steady-state biochemical model for leaf photosynthesis was coupled to a canopy biophysical microclimate model and used to explore this question. C3 photosynthetic CO2 uptake rate (A) is either limited by the maximum rate of Rubisco activity (Vcmax) or by the rate of regeneration of ribulose-1,5-bisphosphate, in turn determined by the rate of whole chain electron transport (J). Thus, if J is limiting, an increase in , will increase net CO2 uptake because more products of the electron transport chain will be partitioned away from photorespiration into photosynthesis. The effect of an increase in , on Rubisco-limited photosynthesis depends on both kcc and the concentration of CO2 ([CO2]). Assuming a strict inverse relationship between kcc and ,, the simulations showed that a decrease, not an increase, in , increases Rubisco-limited photosynthesis at the current atmospheric [CO2], but the increase is observed only in high light. In crop canopies, significant amounts of both light-limited and light-saturated photosynthesis contribute to total crop carbon gain. For canopies, the present average , found in C3 terrestrial plants is supra-optimal for the present atmospheric [CO2] of 370 mol mol,1, but would be optimal for a CO2 concentration of around 200 mol mol,1, a value close to the average of the last 400 000 years. Replacing the average Rubisco of terrestrial C3 plants with one having a lower and optimal , would increase canopy carbon gain by 3%. Because there are significant deviations from the strict inverse relationship between kcc and ,, the canopy model was also used to compare the rates of canopy photosynthesis for several Rubiscos with well-defined kinetic constants. These simulations suggest that very substantial increases (> 25%) in crop carbon gain could result if specific Rubiscos having either a higher , or higher kcc were successfully expressed in C3 plants. [source]

Studies on a Tyr residue critical for the binding of coenzyme and substrate in mouse 3(17),-hydroxysteroid dehydrogenase (AKR1C21): structure of the Y224D mutant enzyme

Urmi Dhagat
Mouse 3(17),-hydroxysteroid dehydrogenase (AKR1C21) is the only aldo,keto reductase that catalyzes the stereospecific reduction of 3- and 17-ketosteroids to the corresponding 3(17),-hydroxysteroids. The Y224D mutation of AKR1C21 reduced the Km value for NADP(H) by up to 80-fold and completely reversed the 17, stereospecificity of the enzyme. The crystal structure of the Y224D mutant at 2.3, resolution revealed that the mutation resulted in a change in the conformation of the flexible loop B, including the V-shaped groove, which is a unique feature of the active-site architecture of wild-type AKR1C21 and is formed by the side chains of Tyr224 and Trp227. Furthermore, mutations (Y224F and Q222N) of residues involved in forming the safety belt for binding of the coenzyme showed similar alterations in kinetic constants for 3,-hydroxy/3-ketosteroids and 17-hydroxy/ketosteroids compared with the wild type. [source]

Kinetics and modeling of charge transfer polymerization of methyl methacrylate

P. G. Rao
Abstract The charge transfer polymerization of methyl methacrylate (MMA) in presence of n -butyl amine (nBA) and carbon tetrachloride (CCl4) with dimethyl sulfoxide (DMSO) as solvent was experimentally investigated. The variables include concentrations of MMA, nBA, CCl4, DMSO and reaction temperature. The effect of these variables on monomer conversion, number and weight average molecular weights is discussed. A reaction mechanism is proposed and overall kinetics, propagation, chain transfer and termination constants are evaluated. A mathematical model is proposed for estimating monomer conversion, number and weight average molecular weights. The proposed model is validated using the kinetic constants and compared satisfactorily with experimental data of the present study. Copyright 2009 Curtin University of Technology and John Wiley & Sons, Ltd. [source]

Metabolism, oral bioavailability and pharmacokinetics of chemopreventive kaempferol in rats

Avantika Barve
Abstract The purpose of this study was to compare the hepatic and small intestinal metabolism, and to examine bioavailability and gastro-intestinal first-pass effects, of kaempferol in rats. Liver and small intestinal microsomes fortified with either NADPH or UDPGA were incubated with varying concentrations of kaempferol for up to 120,min. Based on the values of the kinetic constants (Km and Vmax), the propensity for UDPGA-dependent conjugation compared with NADPH-dependent oxidative metabolism was higher for both hepatic and small intestinal microsomes. Male Sprague-Dawley rats were administered kaempferol intravenously (i.v.) (10, 25,mg/kg) or orally (100, 250,mg/kg). Gastro-intestinal first-pass effects were observed by collecting portal blood after oral administration of 100,mg/kg kaempferol. Pharmacokinetic parameters were obtained by non-compartmental analysis using WinNonlin. After i.v. administration, the plasma concentration,time profiles for 10 and 25,mg/kg were consistent with high clearance (,3,L/hr/kg) and large volumes of distribution (8,12,L/hr/kg). The disposition was characterized by a terminal half-life value of 3,4,h. After oral administration the plasma concentration,time profiles demonstrated fairly rapid absorption (tmax,1,2,h). The area under the curve (AUC) values after i.v. and oral doses increased approximately proportional to the dose. The bioavailability (F) was poor at ,2%. Analysis of portal plasma after oral administration revealed low to moderate absorption. Taken together, the low F of kaempferol is attributed in part to extensive first-pass metabolism by glucuronidation and other metabolic pathways in the gut and in the liver. Copyright 2009 John Wiley & Sons, Ltd. [source]