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RX-series References
The last 25 references for the RX-series stopped-flow rapid reaction analyzers are listed below. A complete searchable database with all known stopped-flow references can be accessed by logging into the APL members area.
| Authors | Title | Year | Keywords | Journal/Proceedings | |
|---|---|---|---|---|---|
| Ruth E. Silversmith, Matthew D. Levin, Elmar Schilling, and Robert B. Bourret | Kinetic Characterization of Catalysis by the Chemotaxis Phosphatase CheZ: MODULATION OF ACTIVITY BY THE PHOSPHORYLATED CheY SUBSTRATE | 2008 | dephosphorylation, CheZ, chemotaxis, phosphatase | J BIOL CHEM, 2008, Vol 283, Iss 2, pp 756-765 | |
| Abstract: CheZ catalyzes the dephosphorylation of the response regulator CheY in the two-component regulatory system that mediates chemotaxis in Escherichia coli. CheZ is a homodimer with two active sites for dephosphorylation. To gain insight into cellular mechanisms for the precise regulation of intracellular phosphorylated CheY (CheYp) levels, we evaluated the kinetic properties of CheZ. The steady state rate of CheZ-mediated dephosphorylation of CheYp displayed marked sigmoidicity with respect to CheYp concentration and a kcat of 4.9 s–1. In contrast, the gain of function mutant CheZ-I21T with an amino acid substitution far from the active site gave hyperbolic kinetics and required far lower CheYp for half-saturation but had a similar kcat value as the wild type enzyme. Stopped flow fluorescence measurements demonstrated a 6-fold faster CheZ/CheYp association rate for CheZ-I21T (kassoc = 3.4 x 107 M –1 s–1) relative to wild type CheZ (kassoc = 5.6 x 106 M–1 s–1). Dissociation of the CheZ·CheYBeF3 complex was slow for both wild type CheZ (kdissoc = 0.040 s–1) and CheZ-I21T (kdissoc = 0.023 s–1) and, when taken with the kassoc values, implied Kd values of 7.1 and 0.68 nM, respectively. However, comparison of the kdissoc and kcat values implied that CheZ and CheYp are not at binding equilibrium during catalysis and that once CheYp binds, it is almost always dephosphorylated. The rate constants were collated to formulate a kinetic model for CheZ-mediated dephosphorylation that includes autoregulation by CheYp and allowed prediction of CheZ activities at CheZ and CheYp concentrations likely to be present in cells. | |||||
| Michael RUSSWURM, Florian MULLERSHAUSEN1, Andreas FRIEBE, Ronald JÄGER, Corina RUSSWURM and Doris KOESLING | Design of fluorescence resonance energy transfer (FRET)-based cGMP indicators: a systematic approach | 2007 | cGMP biosensor, cGMP-dependent protein kinase (cGK), fluorescence resonance energy transfer (FRET), phosphodiesterase (PDE) | BIOCHEM J, 2007, Vol 407, pp 69.77 | |
| Abstract: The intracellular signalling molecule cGMP regulates a variety of physiological processes, and so the ability to monitor cGMP dynamics in living cells is highly desirable. Here, we report a systematic approach to create FRET (fluorescence resonance energy transfer)-based cGMP indicators from two known types of cGMP-binding domains which are found in cGMP-dependent protein kinase and phosphodiesterase 5, cNMP-BD [cyclic nucleotide monophosphate-binding domain and GAF [cGMP-specific and -stimulated phosphodiesterases, Anabaena adenylate cyclases and Escherichia coli FhlA] respectively. Interestingly, only cGMP-binding domains arranged in tandem configuration as in their parent proteins were cGMP-responsive. However, the GAF-derived sensors were unable to be used to study cGMP dynamics because of slow response kinetics to cGMP. Out of 24 cGMP-responsive constructs derived from cNMP-BDs, three were selected to cover a range of cGMP affinities with an EC50 between 500 nM and 6 µM. These indicators possess excellent specifity for cGMP, fast binding kinetics and twice the dynamic range of existing cGMP sensors. The in vivo performance of these new indicators is demonstrated in living cells and validated by comparison with cGMP dynamics as measured by radioimmunoassays. | |||||
| Kathrin Lang, Renate Rieder, and Ronald Micura | Ligand-induced folding of the thiM TPP riboswitch investigated by a structure-based fluorescence spectroscopic approach | 2007 | riboswitches, thiamine pyrophosphate, RNA, | NUCL ACID RES, 2007, Vol 35, Iss 16, pp 5370-5378 | |
| Abstract: Riboswitches are genetic control elements within non-coding regions of mRNA. They consist of a metabolite-sensitive aptamer and an adjoining expression platform. Here, we describe ligand-induced folding of a thiamine pyrophosphate (TPP) responsive riboswitch from Escherichia coli thiM mRNA, using chemically labeled variants. Referring to a recent structure determination of the TPP/aptamer complex, each variant was synthesized with a single 2-aminopurine (AP) nucleobase replacement that was selected to monitor formation of tertiary interactions of a particular region during ligand binding in real time by fluorescence experiments. We have determined the rate constants for conformational adjustment of the individual AP sensors. From the 7-fold differentiation of these constants, it can be deduced that tertiary contacts between the two parallel helical domains (P2/J3-2/P3/L3 and P4/P5/L5) that grip the ligand's ends in two separate pockets, form significantly faster than the function-critical three-way junction with stem P1 fully developed. Based on these data, we characterize the process of ligand binding by an induced fit of the RNA and propose a folding model of the TPP riboswitch aptamer. For the full-length riboswitch domain and for shorter constructs that represent transcriptional intermediates, we have additionally evaluated ligand-induced folding via AP-modified variants and provide insights into the sequential folding pathway that involves a finely balanced equilibrium of secondary structures. | |||||
| Luyben, W. L. | Effect of Kinetic and Design Parameters on Ternary Reactive Distillation Columns | 2007 | IND ENG CHEM RES, 2007, Vol 46, Iss 21, pp 6944-6952 | ||
| Abstract: Many types of reactive distillation columns have been studied in the literature. Ideal hypothetical and real chemical systems have been investigated from the standpoint of steady-state design, bifurcation phenomena, and dynamic control. Most papers dealing with ideal systems consider the quaternary ideal two-reactant, two-product chemistry A + B C + D. This paper explores the ideal ternary system with the chemistry A + B C. Two cases are considered. In the first, there are only three components. In the second, the feed contains another inert component that does not participate in the reaction but has a major impact on the structure of the column and the vapor-liquid phase equilibrium. The effects of a number of kinetic and design parameters are explored. Significant differences are observed between the system without inerts and that with inerts. | |||||
| Moller, M. N.; Li, Q.; Vitturi, D. A.; Robinson, J. M.; Lancaster, J. R., Jr.; Denicola, A. | Membrane `Lens` Effect: Focusing the Formation of Reactive Nitrogen Oxides from the NO/O2 Reaction | 2007 | membrane lens effect, NO, nitric oxide, | CHEM RES TOXICOL, 2007, Vol 20, Iss 4, pp 709-714 | |
| Abstract: It was previously observed that lipid membranes accelerate NO disappearance (Liu et al. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 2175), and here, we demonstrate that this translates into increased rates of NO2 production and nitrosative chemistry. Not only the phospholipid membranes but also the atherosclerosis-related low-density lipoprotein (LDL) were able to accelerate the formation of NO2, studied by stopped-flow spectrophotometry using ABTS as a probe. In addition, membranes, LDL, and Triton X-100 micelles significantly accelerated S-nitrosation of glutathione and captopril. It is shown here that autoxidation of NO occurs 30 times more rapidly within the hydrophobic interior of these particles than in an equal volume of water, approximately 1 order of magnitude less than previous reports. This acceleration can be explained by the ~3 times higher solubility of NO and O2 into these hydrophobic phases relative to water, which results in a higher local concentration of reactants (`lens effect`) and, therefore, a higher rate of reaction. It is predicted that 50% of the oxidizing and nitrosating species derived from NO autoxidation in cells will be formed in the small volume comprising cellular membranes (3% of the total); thus, biomolecules near the membranes will be exposed to fluxes of reactive nitrogen species 30-fold higher than their cytosolic counterparts. | |||||
| Lisa Ghezzi, Brian H. Robinson, Fernando Secco, Maria Rosaria Tinéa and Marcella Venturinia | Binding of Pd(II) to Pada in water/micellar system: Complex formation, kinetics in water and DTAC solution | 2007 | Palladium; PADA; DTAC; Micellar catalysis; Kinetics | COLLOID SURFACE A, 2007, Vol 292, Iss 2, pp 139-147 | |
| Abstract: The kinetics of binding of Pd(II), initially present as PdCl42-, to the azo-dye ligand pyridine-2-azo-p-dimethylaniline (PADA) in dodecyltrimethylammonium chloride (DTAC) micellar solution has been investigated as a preliminary study on palladium micellar extraction and recovery. For comparison, the same investigation has been performed in water. Palladium reacts with PADA forming a strong 1:1 complex both in water and in DTAC micellar solution. Addition of DTAC, at concentrations just above the critical micellar concentration, results in a large enhancement of the rate of complex formation. The analysis of the kinetic behaviour reveals that several reaction paths are present. In water, under the experimental hydrogen ion concentration range investigated, reaction steps involving the protonated ligand forms PADAH+ and PADAH2++ are important and the reacting palladium species are PdCl42- and Pd(H2O)Cl3-. In DTAC micellar solution, PADA reacts in its unprotonated form and a new reaction path appears involving the hydroxo species PdCl3OH2- which is about 10-times faster than the other reaction paths. The large catalytic effect produced by DTAC micellar solution provides a promising basis for the extraction of palladium from water using the PADA/DTAC system. | |||||
| Ruben G.M. Moreno, María V. Alipázaga, Osmar F. Gomes, Edlaine Linares, Marisa H.G. Medeiros and Nina Coichev | DNA damage and 2'-deoxyguanosine oxidation induced by S(IV) autoxidation catalyzed by copper(II) tetraglycine complexes: Synergistic effect of a second metal ion | 2007 | DNA damage; Tetraglycine; Copper; Synergistic effect; Sulfite | J INORG BIOCHEM, 2007, Vol 101, Iss 5, pp 866-875 | |
| Abstract: S(IV) Click to view the (SO2, HSO-3, SO2-)autoxidation catalyzed by Cu(II)/tetraglycine complexes in the presence of DNA or 2'-deoxyguanosine (dGuo) resulted in DNA strand breaks and formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), respectively. Ni(II), Co(II) or Mn(II) (1.0 × 10-4 M) complexes had much smaller effects. Cu(II)/tetraglycine (1.0 × 10-4 M) in the presence of Ni(II) or Mn(II) (10-7–10-6 M) and S(IV) showed remarkable synergistic effect with these metal ions producing a higher yield of 8-oxodGuo. Oxidation of dGuo and DNA damage were attributed to oxysulfur radicals formed as intermediates in S(IV) autoxidation catalyzed by transition metal ions. Click to view the SO3- and HOradical dot radicals were detected by EPR-spin trapping experiments with DMPO (5,5-dimethyl-1-pyrroline-N-oxide). | |||||
| Tanya S. Freedman, Holger Sondermann, Gregory D. Friedland, Tanja Kortemme, Dafna Bar-Sagi, Susan Marqusee, and John Kuriyan | A Ras-induced conformational switch in the Ras activator Son of sevenless | 2006 | Cdc25, nucleotide-exchange factor, crystal structure, Ras exchanger motif (Rem) domain, Ras guanine nucleotide-releasing factor 1 | PROC NAT ACAD SCI USA, 2006, Vol 103, Iss 45, pp 16692-16697 | |
| Abstract: The Ras-specific guanine nucleotide-exchange factors Son of sevenless (Sos) and Ras guanine nucleotide-releasing factor 1 (RasGRF1) transduce extracellular stimuli into Ras activation by catalyzing the exchange of Ras-bound GDP for GTP. A truncated form of RasGRF1 containing only the core catalytic Cdc25 domain is sufficient for stimulating Ras nucleotide exchange, whereas the isolated Cdc25 domain of Sos is inactive. At a site distal to the catalytic site, nucleotide-bound Ras binds to Sos, making contacts with the Cdc25 domain and with a Ras exchanger motif (Rem) domain. This allosteric Ras binding stimulates nucleotide exchange by Sos, but the mechanism by which this stimulation occurs has not been defined. We present a crystal structure of the Rem and Cdc25 domains of Sos determined at 2.0-Ĺ resolution in the absence of Ras. Differences between this structure and that of Sos bound to two Ras molecules show that allosteric activation of Sos by Ras occurs through a rotation of the Rem domain that is coupled to a rotation of a helical hairpin at the Sos catalytic site. This motion relieves steric occlusion of the catalytic site, allowing substrate Ras binding and nucleotide exchange. A structure of the isolated RasGRF1 Cdc25 domain determined at 2.2-Ĺ resolution, combined with computational analyses, suggests that the Cdc25 domain of RasGRF1 is able to maintain an active conformation in isolation because the helical hairpin has strengthened interactions with the Cdc25 domain core. These results indicate that RasGRF1 lacks the allosteric activation switch that is crucial for Sos activity. | |||||
| Wu, F.; Gaffney, B. J. | Dynamic Behavior of Fatty Acid Spin Labels within a Binding Site of Soybean Lipoxygenase-1 | 2006 | soybean lipoxygenase-1, fatty acids, spin labels | BIOCHEMISTRY-USA, 2006, Vol 45, Iss 41, pp 12510-12518 | |
| Abstract: The putative substrate-binding site in lipoxygenases is long and internal. There is little direct evidence about how the unsaturated fatty acid substrates enter and move within the cavity to position themselves correctly for electron transfer reactions with the catalytic non-heme iron. An EPR spectroscopy approach, with spin-labeled fatty acids, is taken here to investigate dynamic behavior of fatty acids bound to soybean lipoxygenase-1. The probes are labeled on C5, C8, C10, C12, and C16 of stearic acid. The EPR-determined affinity for the enzyme increases as the length of the alkyl end of the probe increases, with a ΔΔG of -190 cal/methylene. The probes in the series exhibit similar enhanced paramagnetic relaxation by the iron center. These results indicate that the members of the series have a common binding site. All of the bound probes undergo considerable local mobility. The stearate spin-labeled at C5 has the highest affinity for the lipoxygenase, and it is a competitive inhibitor, with a Ki of 9 µM. Surprisingly, this stearate labeled near the carboxyl end undergoes more local motion than those labeled in the middle of the chain, when it is bound. This shows that the carboxyl end of the fatty-acid spin label is not rigidly docked on the protein. During catalysis, repositioning of the substrate carboxyl on the protein surface may be coupled to motion of portions of the chain undergoing reaction. | |||||
| da Silva, G.; Kennedy, E. M.; Dlugogorski, B. Z. | Ab Initio Procedure for Aqueous-Phase pKa Calculation: The Acidity of Nitrous Acid | 2006 | pKa, nitrous acide, ab initio calculations, stopped-flow | J PHYS CHEM A, 2006, Vol 110, Iss 39, pp 11371-11376 | |
| Abstract: We present an ab initio procedure for accurately calculating aqueous-phase pKa values and apply it to study the acidity of nitrous acid (HNO2, or HONO). The aqueous-phase pKa of nitrous acid was obtained from calculated gas-phase acidities and solvation free energies via a thermodynamic cycle and the solvation model chemistry of Barone et al. (J. Chem. Phys. 1997, 107, 3210). Solvation free energies were calculated at the HF/6-31G(d) level using the dielectric-polarizable continuum and the integral equation formalism-polarizable continuum solvent models (D-PCM and IEF-PCM, respectively), with the D-PCM model yielding the most accurate pKa values. For HF free energies of solvation, significant improvements in accuracy could be made by moving to the larger 6-311++G(3df,3pd) and aug-cc-pVQZ basis sets. Solvation free energies were also calculated using the density functional theory (DFT) methods B3LYP, TPSS, PBE0, B1B95, VSXC, B98 and O3LYP, with the most accurate methods being TPSS and VSXC, which provided average errors of less than 0.11 pKa units. Solvation free energies calculated with the different DFT methods were relatively insensitive to the basis set used. Our theoretical calculations are compared with experimental results obtained using stopped flow spectrophotometry. The pKa of nitrous acid was measured as 3.16 at 25 C, and the enthalpy and entropy of nitrous acid dissociation were calculated from measurements as 6.7 kJ mol-1 and -38.4 J mol-1 K-1, respectively, between 25 and 45 C. The UV/visible absorption spectra of the nitrite ion and nitrous acid were also examined, and molar extinction coefficients were obtained for each. | |||||
| José Manuel Amigo, Anna de Juan, Jordi Coello and Santiago Maspoch | A mixed hard- and soft-modelling approach for the quantitative determination of oxipurines and uric acid in human urine | 2006 | Enzymatic data analysis; Oxipurines; Human urine; Hard-modelling; Soft-modelling; Multivariate curve resolution | ANAL CHIM ACTA, 2006, Vol 567, Iss 2, pp 236-244 | |
| Abstract: A new treatment of kinetic-enzymatic data, joining the benefits of hard- and soft-modelling methods, is proposed for the kinetic resolution and simultaneous quantification of xanthine oxidase induced oxipurines (hypoxanthine and xanthine) and uric acid in human urine without any further separation.nnThese three analytes show highly overlapped spectral bands in the range of 232–320 nm and urine contains absorbing species in this wavelength range that behave as a constant interference throughout the monitoring of the kinetic experiment. The proposed method allows the determination of the analytes in human urine by simultaneous analysis of the urine sample and a few (even only one) standards of pure analyte mixtures of hypoxanthine and xanthine in aqueous solution.nnAn iterative mixed hard- and soft-modelling multivariate curve resolution (HS-MCR) algorithm, which includes a hard-modelling constraint based on the enzymatic model in the parent multivariate curve resolution-alternating least squares method (MCR-ALS), is applied to a data set formed by the standards and the urine samples. Hard-modelling is applied to the concentration profiles of the three analytes in the standards, whereas soft-modelling on the analytes and the urine in the samples gives the quantitative information. As a result, quantitative information similar in quality to that obtained by separation techniques (HPLC and HPCE) and other chemometric approaches (PLS and N-PLS) is obtained with less experimental effort and a much smaller number of standards in aqueous solution that do not need to contain the interferences present in the samples. | |||||
| Gialih Lin, Hsin-Chang Tseng, Ai-Chi Chio, Tsao-Ming Tseng and Bo-Yi Tsai | A rate determining step change in the pre-steady state of acetylcholinesterase inhibitions by 1,n-alkane-di-N-butylcarbamates | 2005 | acetylcholinesterase, enyme inhibition, enyme kinetics | BIOORG MED CHEM LETT, 2005, Vol 15, Iss 4, pp 951-955 | |
| Abstract: Alkane-1-N-butylcarbamate-n-ols (1–7) and 1,n-alkane-di-N-butylcarbamates (8–14) are potent pseudo-substrate inhibitors of acetylcholinesterase. For inhibitors 1–7, the pre-steady state -log Ks values and steady state -log Ki, values are linearly correlated with the tether length (N). However, for inhibitors 8–14, correlation of the -log Ks or -log Ki values against N deviates from linearity. A discontinuity of the -log Ks versus N plot, concave downwards, is indicative of a rate determining step change in the pre-steady state of acetylcholinesterase inhibitions by inhibitors | |||||
| Rajesh Mishra, Robert Seckler, and Rajiv Bhat | Efficient Refolding of Aggregation-prone Citrate Synthase by Polyol Osmolytes: HOW WELL ARE PROTEIN FOLDING AND STABILITY ASPECTS COUPLED? | 2005 | protein folding, citrate synthase, aggregation | J BIOL CHEM, 2005, Vol 280, Iss 16, pp 15553-15560 | |
| Abstract: Efficient refolding of proteins and prevention of their aggregation during folding are of vital importance in recombinant protein production and in finding cures for several diseases. We have used citrate synthase (CS) as a model to understand the mechanism of aggregation during refolding and its prevention using several known structure-stabilizing cosolvent additives of the polyol series. Interestingly, no parallel correlation between the folding effect and the general stabilizing effect exerted by polyols was observed. Although increasing concentrations of polyols increased protein stability in general, the refolding yields for CS decreased at higher polyol concentrations, with erythritol reducing the folding yields at all concentrations tested. Among the various polyols used, glycerol was the most effective in enhancing the CS refolding yield, and a complete recovery of enzymatic activity was obtained at 7 M glycerol and 10 µg/ml protein, a result superior to the action of the molecular chaperones GroEL and GroES in vitro. A good correlation between the refolding yields and the suppression of protein aggregation by glycerol was observed, with no aggregation detected at 7 M. The polyols prevented the aggregation of CS depending on the number of hydroxyl groups in them. Stopped-flow fluorescence kinetics experiments suggested that polyols, including glycerol, act very early in the refolding process, as no fast and slow phases were detectable. The results conclusively demonstrate that both the thermodynamic and kinetic aspects are critical in the folding process and that all structure-stabilizing molecules need not always help in productive folding to the native state. These findings are important for the rational design of small molecules for efficient refolding of various aggregation-prone proteins of commercial and medical relevance. | |||||
| Roncaroli, F.; Olabe, J. A. | The Reactions of Nitrosyl Complexes with Cysteine | 2005 | cysteine, ruthenium nitrosyl complex, | INORG CHEM, 2005, Vol 44, Iss 13, pp 4719-4727 | |
| Abstract: The reaction kinetics of a set of ruthenium nitrosyl complexes, {(X)5MNO}n, containing different coligands X (polypyridines, NH3, EDTA, pz, and py) with cysteine (excess conditions), were studied by UV-vis spectrophotometry, using stopped-flow techniques, at an appropriate pH, in the range 3-10, and T = 25 C. The selection of coligands afforded a redox-potential range from -0.3 to +0.5 V (vs Ag/AgCl) for the NO+/NO bound couples. Two intermediates were detected. The first one, I1, appears in the range 410-470 nm for the different complexes and is proposed to be a 1:1 adduct, with the S atom of the cysteinate nucleophile bound to the N atom of nitrosyl. The adduct formation step of I1 is an equilibrium, and the kinetic rate constants for the formation and dissociation of the corresponding adducts were determined by studying the cysteine-concentration dependence of the formation rates. The second intermediate, I2, was detected through the decay of I1, with a maximum absorbance at ca. 380 nm. From similar kinetic results and analyses, we propose that a second cysteinate adds to I1 to form I2. By plotting ln k1(RS-) and ln k2(RS-) for the first and second adduct formation steps, respectively, against the redox potentials of the NO+/NO couples, linear free energy plots are obtained, as previously observed with OH- as a nucleophile. The addition rates for both processes increase with the nitrosyl redox potentials, and this reflects a more positive charge at the electrophilic N atom. In a third step, the I2 adducts decay to form the corresponding Ru-aqua complexes, with the release of N2O and formation of cystine, implying a two-electron process for the overall nitrosyl reduction. This is in contrast with the behavior of nitroprusside ([Fe(CN)5NO]2-; NP), which always yields the one-electron reduction product, [Fe(CN)5NO]3-, either under substoichiometric or in excess-cysteine conditions. | |||||
| da Silva, G.; Kennedy, E. M.; Dlugogorski, B. Z. | Effect of Added Nucleophilic Species on the Rate of Primary Amino Acid Nitrosation | 2005 | J AM CHEM SOC, 2005, Vol 127, Iss 11, pp 3664-3665 | ||
| Abstract: The rate of primary amino acid nitrosation, both with and without the addition of nucleophilic species, has been studied using stopped-flow spectrophotometry. The rate of nitrosation in the presence of strong nucleophilic species such as thiocyanate and thiourea was shown to be much faster than nitrosation without the addition of a nucleophile. Rate constants were determined at 25 C for reaction of the amino acids alanine, glycine, and valine with five common nitrosating agents. For the nitrosating agents nitrosyl chloride, nitrosyl bromide, and dinitrogen trioxide the rate of reaction was observed to approach the predicted encounter-controlled limit. However, for nitrosyl thiocyanate and S-nitrosothiourea nitrosation was found to be reaction-controlled. In the reaction-controlled regime, rate constants were found to increase with increasing electrophilic strength of the nitrosating agent, as measured by the parameter En, with a slope indicative of a product-like transition state. Activation energies were also measured, being around 10-30 kJ mol-1 for encounter-controlled rate constants, and 30-50 kJ mol-1 for reaction-controlled rate constants. Our results are discussed in the context of in vivo amino acid nitrosation, where it is proposed that the rate of nitrosation may be considerably greater than currently thought, due to the presence of nucleophilic species. | |||||
| Zhang, R.; Horner, J. H.; Newcomb, M. | Laser Flash Photolysis Generation and Kinetic Studies of Porphyrin-Manganese-Oxo Intermediates. Rate Constants for Oxidations Effected by Porphyrin-MnV-Oxo Species and Apparent Disproportionation Equilibrium Constants for Porphyrin-MnIV-Oxo Species | 2005 | kinetic isotope effects, porphyrin-manganese, oxidation | J AM CHEM SOC, 2005, Vol 127, Iss 18, pp 6573-6582 | |
| Abstract: Porphyrin-manganese(V)-oxo and porphyrin-manganese(IV)-oxo species were produced in organic solvents by laser flash photolysis (LFP) of the corresponding porphyrin-manganese(III) perchlorate and chlorate complexes, respectively, permitting direct kinetic studies. The porphyrin systems studied were 5,10,15,20-tetraphenylporphyrin (TPP), 5,10,15,20-tetrakis(pentafluorophenyl)porphyrin (TPFPP), and 5,10,15,20-tetrakis(4-methylpyridinium)porphyrin (TMPyP). The order of reactivity for (porphyrin)MnV(O) derivatives in self-decay reactions in acetonitrile and in oxidations of substrates was (TPFPP) > (TMPyP) > (TPP). Representative rate constants for reaction of (TPFPP)MnV(O) in acetonitrile are k = 6.1 × 105 M-1 s-1 for cis-stilbene and k = 1.4 × 105 M-1 s-1 for diphenylmethane, and the kinetic isotope effect in oxidation of ethylbenzene and ethylbenzene-d10 is kH/kD = 2.3. Competitive oxidation reactions conducted under catalytic conditions display approximately the same relative rate constants as were found in the LFP studies of (porphyrin)MnV(O) derivatives. The apparent rate constants for reactions of (porphyrin)MnIV(O) species show inverted reactivity order with (TPFPP) < (TMPyP) < (TPP) in reactions with cis-stilbene, triphenylamine, and triphenylphosphine. The inverted reactivity results because (porphyrin)MnIV(O) disproportionates to (porphyrin)MnIIIX and (porphyrin)MnV(O), which is the primary oxidant, and the equilibrium constants for disproportionation of (porphyrin)MnIV(O) are in the order (TPFPP) < (TMPyP) < (TPP). The fast comproportionation reaction of (TPFPP)MnV(O) with (TPFPP)MnIIICl to give (TPFPP)MnIV(O) (k = 5 × 108 M-1 s-1) and disproportionation reaction of (TPP)MnIV(O) to give (TPP)MnV(O) and (TPP)MnIIIX (k 2.5 × 109 M-1 s-1) were observed. The relative populations of (porphyrin)MnV(O) and (porphyrin)MnIV(O) were determined from the ratios of observed rate constants for self-decay reactions in acetonitrile and oxidation reactions of cis-stilbene by the two oxo derivatives, and apparent disproportionation equilibrium constants for the three systems in acetonitrile were estimated. A model for oxidations under catalytic conditions is presented. | |||||
| Pokorny, A.; Almeida, P. F. F. | Permeabilization of Raft-Containing Lipid Vesicles by d-Lysin: A Mechanism for Cell Sensitivity to Cytotoxic Peptides | 2005 | lysin, phosphatidylcholine, membrane binding, | BIOCHEMISTRY-USA, 2005, Vol 44, Iss 27, pp 9538-9544 | |
| Abstract: delta-Lysin is a linear, 26-residue peptide that adopts an a-helical, amphipathic structure upon binding to membranes. delta-Lysin preferentially binds to mammalian cell membranes, the outer leaflets of which are enriched in sphingomyelin, cholesterol, and unsaturated phosphatidylcholine. Mixtures including these lipids have been shown to exhibit separation between liquid-disordered (ld) and liquid-ordered (lo) domains. When rich in sphingomyelin and cholesterol, these ordered domains have been called lipid `rafts`. We found that delta-lysin binds poorly to the lo (raft) domains; therefore, in mixed-phase lipid vesicles, -lysin preferentially binds to the ld domains. This leads to the concentration of -lysin in ld domains, enhancing peptide aggregation and, consequently, the rate of peptide-induced dye efflux from lipid vesicles. The efficient lysis of eukaryotic cells by delta-lysin can thus be attributed not to specific -lysin-cholesterol or delta-lysin-sphingomyelin interactions but, rather, to the exclusion of delta-lysin from ordered rafts. The degree to which the kinetics of dye efflux are enhanced in mixed-phase vesicles over those observed in pure, unsaturated phosphatidylcholine vesicles directly reflects the amount of ld phase present in mixed-phase systems. This effect of lipid domains has broader consequences, beyond the hemolytic efficiency of delta-lysin. We discuss the hypothesis that bacterial sensitivity to antimicrobial peptides may be determined by a similar mechanism. | |||||
| Ribacka, C.; Verkhovsky, M. I.; Belevich, I.; Bloch, D. A.; Puustinen, A.; Wikstrom, M. | An Elementary Reaction Step of the Proton Pump Is Revealed by Mutation of Tryptophan-164 to Phenylalanine in Cytochrome c Oxidase from Paracoccus denitrificans | 2005 | cytochrome c oxidase, proton pumps, proton translocation, mitochondrial membrane | BIOCHEMISTRY-USA, 2005, Vol 44, Iss 50, pp 16502-16512 | |
| Abstract: Cytochrome c oxidase couples reduction of dioxygen to water to translocation of protons over the inner mitochondrial or bacterial membrane. A likely proton acceptor for pumped protons is the -propionate of heme a3, which may receive the proton via water molecules from a conserved glutamic acid (E278 in subunit I of the Paracoccus denitrificans enzyme) and which receives a hydrogen bond from a conserved tryptophan, W164. Here, W164 was mutated to phenylalanine (W164F) to further explore the role of the heme a3 -propionate in proton translocation. FTIR spectroscopy showed changes in vibrations possibly attributable to heme propionates, and the midpoint redox potential of heme a3 decreased by ~50 mV. The reaction of the oxidized W164F enzyme with hydrogen peroxide yielded substantial amounts of the intermediate F' even at high pH, which suggests that the mutation rearranges the local electric field in the binuclear center that controls the peroxide reaction. The steady-state proton translocation stoichiometry of the W164F enzyme dropped to ~0.5 H+/e- in cells and reconstituted proteoliposomes. Time-resolved electrometric measurements showed that when the fully reduced W164F enzyme reacted with O2, the membrane potential generated in the fast phase of this reaction was far too small to account either for full proton pumping or uptake of a substrate proton from the inside of the proteoliposomes. Time-resolved optical spectroscopy showed that this fast electrometric phase occurred with kinetics corresponding to the transition from state A to PR, whereas the subsequent transition to the F state was strongly delayed. This is due to a delay of reprotonation of E278 via the D-pathway, which was confirmed by observation of a slowed rate of CuA oxidation and which explains the small amplitude of the fast charge transfer phase. Surprisingly, the W164F mutation thus mimics a weak block of the D-pathway, which is interpreted as an effect on the side chain isomerization of E278. The fast charge translocation may be due to transfer of a proton from E278 to a `pump site` above the heme groups and is likely to occur also in wild-type enzyme, though not distinguished earlier due to the high-amplitude membrane potential formation during the PR F transition. | |||||
| Yan, X.; Habbersett, R. C.; Yoshida, T. M.; Nolan, J. P.; Jett, J. H.; Marrone, B. L. | Probing the Kinetics of SYTOX Orange Stain Binding to Double-Stranded DNA with Implications for DNA Analysis | 2005 | DNA, sytox orange stain, dna binding, DNA stain | ANAL CHEM, 2003, Vol 77, Iss 11, pp 3554-3562 | |
| Abstract: Rapid binding kinetics of SYTOX Orange stain with double-stranded DNA (dsDNA) was revealed on the DNA fragment sizing flow cytometer. We demonstrated for the first time that the dye molecules could be adsorbed onto the capillary surface and native DNA fragments can be dynamically stained while passing through the capillary. High-quality burst size distribution histograms were obtained for DNA samples analyzed immediately after staining, dilution, or mixing. These observations indicated that rapid interactions exist between SYTOX Orange dye molecules and dsDNA. A stopped-flow fluorescence apparatus was set up to capture the fast association traces of intercalating dyes binding to dsDNA. Kinetic equations were derived to fit the association curves for determination of association rates and to model the dynamic staining, dilution, and mixing processes of DNA samples stained with intercalating dyes. The measured association rates for both SYTOX Orange and PicoGreen stains intercalating into dsDNA were on the order of 108 M-1 s-1, suggesting a diffusion-controlled process. Simulations indicate that reequilibration can be reached in seconds upon staining, dilution, or mixing. Insight into the kinetics of DNA binding dyes will help implement efficient sample-handling practices in DNA analysis, including DNA fragment sizing flow cytometry. | |||||
| Heim-N Griesbeck-O | Genetically Encoded Indicators of Cellular Calcium Dynamics Based on Troponin C and Green Fluorescent Protein | 2004 | green fluorescent protein, calmodulin, calcium | J BIOL CHEM, 2003, Vol 279, Iss 14, pp 14280-14286 | |
| Abstract: Genetic calcium probes offer tremendous potential in the fields of neuroscience, cell biology, and pharmaceutical screening. Previously, ratiometric and non-ratiometric indicators of cellular calcium dynamics have been described that consist of mutants of the green fluorescent protein (GFP) as fluorophores and calmodulin as calcium-binding moiety in several configurations. However, these calmodulin-based types of probes have a series of deficiencies, such as reduced dynamic ranges, when expressed within transgenic organisms and lack of calcium sensitivity in certain targetings. We developed novel types of calcium probes based on troponin C variants from skeletal and cardiac muscle. These indicators have ratio changes up to 140%, Kds ranging from 470 nM to 29 µM, and improved subcellular targeting properties. We targeted the indicators to the plasma membrane of HEK293 cells and primary hippocampal neurons. Upon long lasting depolarization, submembrane calcium levels in hippocampal neurons were found to be in equilibrium with bulk cytosolic calcium levels, suggesting no standing gradient persists from the membrane toward the cytosol. We expect that such novel indicators using specialized calcium sensing proteins will be minimally interacting with the cellular biochemical machinery. | |||||
| Chen-IA Szostak-JW | Membrane growth can generate a transmembrane pH gradient in fatty acid vesicles | 2004 | electrochemical proton gradients, membrane gradient, | PROC NAT ACAD SCI USA, 2004, Vol 101, Iss 21, pp 7965-7970 | |
| Abstract: Electrochemical proton gradients are the basis of energy transduction in modern cells, and may have played important roles in even the earliest cell-like structures. We have investigated the conditions under which pH gradients are maintained across the membranes of fatty acid vesicles, a model of early cell membranes. We show that pH gradients across such membranes decay rapidly in the presence of alkali-metal cations, but can be maintained in the absence of permeable cations. Under such conditions, when fatty acid vesicles grow through the incorporation of additional fatty acid, a transmembrane pH gradient is spontaneously generated. The formation of this pH gradient captures some of the energy released during membrane growth, but also opposes and limits further membrane area increase. The coupling of membrane growth to energy storage could have provided a growth advant | |||||
| Warren-DB Grieser-F Perera-JM Stevens-GW | Kinetics and the effect of electrostatic surface potential on nickel(II) extraction by 2-hydroxy-5-nonylacetophenone oxime (LIX 84) in a micellar phase | 2004 | HNAPO, micelles, | COLLOID SURFACE A, 2004, Vol 243, Iss 1-3, pp 127-132 | |
| Abstract: The kinetics of extraction and the effect of electrostatic surface potential on the nickel(II)/2-hydroxy-5-nonylacetophenone oxime (HNAPO) extraction system were characterised in a non-ionic micellar system. A two step reaction mechanism between nickel(II) and HNAPO was found to satisfactorily explain the observed kinetics. Simulation of the kinetics of the complexation reaction over a wide range of concentrations provided the rate constants for the proposed mechanism. The addition of a cationic surfactant, dodecyl trimethyl ammonium chloride (DTAC), slowed the reaction kinetics, while the inclusion of an anionic surfactant, sodium dodecyl sulphate (SDS), enhanced the reaction kinetics relative to the non-ionic micellar system. The charge effects on the reaction rate could be successfully modelled based on the electrostatic surface potential generated by the anionic and cationic surfactants. | |||||
| Lowinsohn-D Alipazaga-MV Coichev-N Bertotti-M | Studies on the stability of the electrochemically generated Cu(III)–triglycine complex | 2004 | Copper(III); Triglycine; Rotating ring-disc voltammetry; EC mechanism; Rate constant | ELECTROCHIM ACTA, 2004, Vol 49, Iss 11, pp1761-1766 | |
| Abstract: The electrochemistry of Cu(II) in aqueous solutions containing triglycine has been investigated at glassy carbon surfaces. Experiments were carried out at the pH range comprising 7 and 10 and a correlation between the electrochemical behaviour and the nature of the Cu(II) species was performed. Spectrophotometric and coulometric studies were also done both to characterise Cu(II) and Cu(III) species in solution and to prove the existence of a degradation step related to Cu(III) complexes. Rotating ring-disc voltammetry was employed to follow this chemical step coupled to the electrode reaction and rate constant values were calculated at three different temperatures. Data show that Cu(III)–triglycine species degrades with faster kinetics compared to tetraglycine and pentaglycine Cu(II) complexes. | |||||
| Alluisetti-GE Almaraz-AE Amorebieta-VT Doctorovich-F Olabe-JA | Metal-Catalyzed Anaerobic Disproportionation of Hydroxylamine. Role of Diazene and Nitroxyl Intermediates in the Formation of N2, N2O, NO+, and NH3 | 2004 | Nitric oxide, disproportionation of NH2OH | J AM CHEM SOC, 2004, Vol 126, Iss 41, pp 13432-13442 | |
| Abstract: The catalytic disproportionation of NH2OH has been studied in anaerobic aqueous solution, pH 6-9.3, at 25.0 C, with Na3[Fe(CN)5NH3]·3H2O as a precursor of the catalyst, [FeII(CN)5H2O]3-. The oxidation products are N2, N2O, and NO+ (bound in the nitroprusside ion, NP), and NH3 is the reduction product. The yields of N2/N2O increase with pH and with the concentration of NH2OH. Fast regime conditions involve a chain process initiated by the NH2 radical, generated upon coordination of NH2OH to [FeII(CN)5H2O]3-. NH3 and nitroxyl, HNO, are formed in this fast process, and HNO leads to the production of N2, N2O, and NP. An intermediate absorbing at 440 nm is always observed, whose formation and decay depend on the medium conditions. It was identified by UV-vis, RR, and 15NMR spectroscopies as the diazene-bound [FeII(CN)5N2H2]3- ion and is formed in a competitive process with the radical path, still under the fast regime. At high pH's or NH2OH concentrations, an inhibited regime is reached, with slow production of only N2 and NH3. The stable red diazene-bridged [(NC)5FeHN=NHFe(CN)5]6- ion is formed at an advanced degree of NH2OH consumption. | |||||
| Pokorny-A Almeida-PFF | Kinetics of Dye Efflux and Lipid Flip-Flop Induced by delta-Lysin in Phosphatidylcholine Vesicles and the Mechanism of Graded Release by Amphipathic, -Helical Peptides | 2004 | delta-lysin, vesicles, amphipathic peptides | BIOCHEMISTRY-USA, 2004, Vol 43, Iss 27, pp 8846-8857 | |
| Abstract: delta-Lysin is a 26-residue, amphipathic, -helical peptide of bacterial origin. Its specificity is to some extent complementary to that of antimicrobial peptides. Therefore, understanding its mechanism is important for the more general goal of understanding the interaction of amphipathic peptides with membranes. In this article, we show that -lysin induces graded efflux of the contents of phosphatidylcholine vesicles. In view of this finding, carboxyfluorescein efflux kinetics were re-examined. In addition, peptide-induced lipid flip-flop was directly measured using fluorescence energy transfer between two lipid fluorophores initially placed on opposite leaflets of the bilayer. Carboxyfluorescein efflux and lipid flip-flop occur with essentially identical rate constants. On the basis of a detailed, quantitative analysis of the kinetics of peptide-vesicle interactions, we conclude that the peptide translocates across the bilayer as a small, transient aggregate, most likely a trimer. Dye efflux and lipid flip-flop occur concomitantly with the transient peptide-induced perturbation of the membrane. The experimental data are interpreted by comparing the predictions of the available models for the mechanism of action of amphipathic -helical peptides. We demonstrate how the combination of the quantitative kinetic analysis, graded efflux, and reversibility of the peptide-vesicle interaction can be used to reject several models for this particular peptide. Two models are compatible with the data, the toroidal pore model and the sinking raft model. On the basis of the small aggregate size, a trimer, the latter appears to be more plausible. Some significant modifications are introduced in the sinking raft model to take into account the new finding of graded dye release. Furthermore, we present an explanation for the phenomenon of graded release in general, which, contrary to all-or-none efflux, has not been well-understood. | |||||