Stopped-flow Time Research Articles

Redox responsive bi/tri-nuclear complexes incorporating ferrocenyl unit: Synthesis, characterization, physicochemical studies and X-ray structure of [C 5H 5FeC 5H 4CH  CHC 5H 4NCH 3]PF 6 with a mirror creating disorder

( Trans-1(4-pyridyl)-ethylene) ferrocene, L reacts with K[Ru III(edtaH)]Cl to form binuclear complex. K[Ru III(edtaH)]Cl exist as Ru III(edtaH(H 2O) in aqueous solutions and substitution of the aqua molecule by L occurs within the stopped flow time scale. Rate constants for the reaction are 1620 ± 20, 2080 ± 35, 2690 ± 50 M −1 s −1 at 28, 34 and 39.9°C, respectively [ ΔH # is 30.3 ± 1.1 kJ mol −1 and ΔS # is − 124 ± 4 J K −1 mol −1]. R II(2,2′-bipy) 2Cl 2 reacts with L to form bi/trinuclear complexes. [Ru II(2,2′-bipy) 2LCl]PF 6 and [Ru II(2,2′-bipy) 2L 2](PF 6) 2, depending on the reaction conditions. Luminescence of the Ru II(2,2′-bipy) 2(py) 2 2+ center in the trinuclear complexes is completely quenched presumably through energy transfer pathway. There exist a moderate to weak electrochemical interaction between the two redox centers either in N-methylated form of L or in the bi/trinuclear complexes derived from Ru(II) or Ru(III). All these new bi/trinuclear complexes are characterized by physicochemical methods. Single crystal X-ray structure of [LCH 3]PF 0 is reported. A very low value of powder SHG efficiency observed earlier for this salt can now be well understood in terms of the centrosymmetric molecular arrangement created due to the disorder in the crystal.

Determination of Amyloglucosidase Activity Using Flow Injection Analysis With Fourier Transform Infrared Spectrometric detection

An automated method for the determination of amyloglucosidase (glucan 1,4-α-glucosidase) activity in aqueous solution is proposed. The method is based on the direct FTIR spectrometric monitoring of starch hydrolysis catalyzed by amyloglucosidase. It is shown that spectral changes caused by the enzyme action can be directly related to the enzyme activity, hence eliminating the need for additional consecutive reaction steps which are normally required to derive a reaction product detectable by conventional techniques such as UV/VIS spectrometry or electrochemistry. Unspecific background absorption was eliminated by calculation of a difference spectrum of the FTIR spectrum of unreacted starch and the FTIR spectrum of the reaction mixture. To guarantee optimum handling of the viscous starch solution and to obtain high sensitivity, a flow injection analysis system combining the merging zone approach and the stopped flow technique was developed. Using a stopped flow time of 5 min and a 55 g l -1 starch substrate solution, linear calibration curves from 50 to 2000 U -1 (830 - 33 300 nkat l -1 ) were obtained. Recoveries for spiked fermentation broth samples ranged from 102 to 98% and were determined by the standard addition method.

Spectrophotometric determination of mercury with 5,10,15,20-tetrakis(3-chloro-4-sulfophenyl)porphine by flow injection analysis

5,10,15,20-tetrakis(3-chloro-4-sulfophenyl)porphine (m-Cl-TPPS4) was synthesized and used for the Spectrophotometric determination of mercury by flow injection analysis. A pseudo-first-order reaction kinetic mechanism was proposed with a rate constant of 0.8 min−1 for Hg(II) withm-Cl-TPPS4 in the presence of 8-hydroxyquinoline in a medium of 1.0M acetic acid and sodium acetate buffer solution (pH 6.22). In the optimum conditions of reaction temperature (85 ° C), stopped-flow time (60 s) and sampling volume (100 μl), the method's relative standard deviation was 0.82% (n = 12) at 5.0 μg ml−1 mercury, with a linear range of 0–12.0 μg ml−1 and an analytical frequency of 60h−1. The detection limit (3σ) was 0.025 μg ml−1. Interference studies showed that most metal ions co-existing with Hg2+ could be tolerated at 100-fold excess levels, but Zn2+, Cu2+ and Mn2+ needed to be masked. The method has been applied to the analysis of water samples with satisfactory results.

Kinetics and mechanism of the oxidation of hydroxylamine by aqueous chlorine

The kinetics and mechanism of the reaction between hydroxylamine and chlorine in 0.1-1 M hydrochloric acid have been determined. In 10-fold or greater excess of NH 3 OH + , on the stopped-flow time scale, three distinct steps are observed: the initial rapid formation of an intermediate, which reacts with further hydroxylamine to form nitrous acid, HONO, followed by a slower reduction of HONO with encess NH 3 OH + to form nitrous oxide

Equilibria and kinetics of the reactions between hydrogen peroxide and methyltrioxorhenium in aqueous perchloric acid solutions

In aqueous solutions the colorless compounds CH[sub 3]ReO[sub 3] (=MTO) and H[sub 2]O[sub 2] form 1:1 and 1:2 adducts. The latter is yellow, with [epsilon][sub 360] = 1.1 x 10[sup 3] L mol[sup [minus]1] cm[sup [minus]1]. Peroxide binding to MTO shows cooperativity, as shown by the inversion of the usual order of binding constants. The stepwise equilibrium constants are K[sub 1] = 7.7 L mol[sup [minus]1] and K[sub 2] = 145 L mol[sup [minus]1] at 25[degrees]C. The buildup of product, which occurs on the stopped-flow time scale at 9-680 mM H[sub 2]O[sub 2], is fit by biexponential kinetics. The equilibria and rates are independent of [H[sub 3]O[sup +]] in the range 10[sup [minus]1]-10[sup [minus]3] M. The peroxide complexes decompose more rapidly at lower [H[sub 3]O[sup +]], particularly at pH > 3. Possible structures for 1:1 and 1:2 MOT-H[sub 2]O[sub 2] adducts are presented and discussed.

Transient spectroscopy of the reaction of cyanide with ferrous myoglobin. Effect of distal side residues.

The reaction of cyanide metmyoglobin with dithionite conforms to a two-step sequential mechanism with formation of an unstable intermediate, identified as cyanide bound ferrous myoglobin. This reaction was investigated by stopped-flow time resolved spectroscopy using different myoglobins, i.e. those from horse heart, Aplysia limacina buccal muscle, and three recombinant derivatives of sperm whale skeletal muscle myoglobin (Mb) (the wild type and two mutants). The myoglobins from horse and sperm whale (wild type) have in the distal position (E7) a histidyl residue, which is missing in A. limacina Mb as well as the two sperm whale mutants (E7 His----Gly and E7 His----Val). All these proteins in the reduced form display an extremely low affinity for cyanide at pH less than 10. The differences in spectroscopy and kinetics of the ferrous cyanide complex of these myoglobins indicate a role of the distal pocket on the properties of the complex. The two mutants of sperm whale Mb are characterized by a rate constant for the decay of the unstable intermediate much faster than that of the wild type, at all pH values explored. Therefore, we envisage a specific role of the distal His (E7) in controlling the rate of cyanide dissociation and also find that this effect depends on the protonation of a single ionizable group, with pK = 7.2, attributed to the E7 imidazole ring. The results on A. limacina Mb, which displays the slowest rate of cyanide dissociation, suggests that a considerable stabilizing effect can be exerted by Arg E10 which, according to Bolognesi et al. (Bolognesi, M., Coda, A., Frigerio, F., Gatti, C., Ascenzi, P., and Brunori, M. (1990) J. Mol. Biol. 213, 621-625), interacts inside the pocket with fluoride bound to the ferric heme iron. A mechanism of control for the rate of dissociation of cyanide from ferrous myoglobin, involving protonation of the bound anion, is discussed.

Spectrophotometric determination of protein by the stopped-flow time difference analysis technique to enhance the sensitivity of the Coomassie brilliant blue-binding method.

Spectrophotometric determination of protein by the stopped-flow time difference analysis technique to enhance the sensitivity of the Coomassie brilliant blue-binding method.

Application of the "micro-stopped-flow time difference analysis" technique to a dehydrogenase-catalyzed reaction for rapid and sensitive determination of pyruvate.

The great usefulness of the previously reported “stopped-flow time difference analysis” technique for improving enzymatic analyses based on dehydrogenase-catalyzed reactions is demonstrated by combining the technique with the conventional enzymatic method for pyruvate determination. Reagent economy was accomplished by using a micro-stopped-flow apparatus designed by one of the authors. The running speed and sensitivity were improved by about 100 and 7 times the figures for the conventional enzymatic method, respectively. Furthermore, as procedural blanks were unnecessary, a simpler method could be developed. The interference by several other keto acids was easily eliminated by analyzing the reaction curve, and the apparent first-order rate constant proved to be useful for qualitative analysis.

Solvent Extraction-Spectrophotometric Determination of Trace Orthophosphate in Water by Stopped-Flow Time Difference Analysis Technique

For the determination of phosphate from μg l-1 to sub-μg l-1 level in water, the time difference analysis technique (a sort of end-point assay using the stopped-flow method) was used by combining the Molybdenum Blue method and the pre-concentrating solvent extraction of 12-molybdophosphate. Isobutyl alcohol and stannous chloride were used as the solvent and the reductant, respectively. The reducing reaction was fast and the colored complex formed by the reaction was unstable. Phosphate concentration was easily determined by analyzing the reaction curves recorded for only 0.5s. The apparent molar absorptivity of phosphate was about 2×105M-1 cm-1 at 730nm, and the detection limit was 0.3μg l-1. The calibration curve was linear from zero to 5μg l-1. The method was applied to the determination of phosphate in tap water samples; the relative standard deviation was less than 3% and the recovery of 20μg l-1 phosphate was 100±6%.

Determination of Low Concentrations of Protein by the Biuret Method Using the “Stopped-flow Time Difference Analysis” Technique

The “stopped-flow time difference analysis” technique (a novel and sensitive technique, involving a sort of end-point assay using the stopped-flow method) was applied to the modified biuret method (micro-biuret method) for protein determination. Protein concentration was easily determined from a reaction curve recorded for only 2 sec. The calibration curve for bovine serum albumin was linear from zero to 1000μg/ml. The detection limit for bovine serum albumin was 4 μg/ml and is comparable to that of the widely used Lowry method. The important advantage of the present method, compared with the conventional spectrophotometric methods, is that it was much less affected by the variation of protein species, and was almost free from interference by coexisting materials, including widely used tris(hydroxymethyl)aminomethane and peptides. The method is thus useful for a sensitive and accurate determination of either an unknown individual protein or total proteins in an aqueous solution.

Determination of low concentrations of protein by the biuret method using the "stopped-flow time difference analysis" technique.

The “stopped-flow time difference analysis” technique (a novel and sensitive technique, involving a sort of end-point assay using the stopped-flow method) was applied to the modified biuret method (micro-biuret method) for protein determination. Protein concentration was easily determined from a reaction curve recorded for only 2 sec. The calibration curve for bovine serum albumin was linear from zero to 1000μg/ml. The detection limit for bovine serum albumin was 4 μg/ml and is comparable to that of the widely used Lowry method. The important advantage of the present method, compared with the conventional spectrophotometric methods, is that it was much less affected by the variation of protein species, and was almost free from interference by coexisting materials, including widely used tris(hydroxymethyl)aminomethane and peptides. The method is thus useful for a sensitive and accurate determination of either an unknown individual protein or total proteins in an aqueous solution.

SPECTROPHOTOMETRIC DETERMINATION OF PARTS PER BILLION LEVEL PHOSPHATE BY “STOPPED-FLOW TIME DIFFERENCE ANALYSIS” USING MALACHITE GREEN AND MOLYBDATE

Abstract The “stopped-flow time difference analysis” method [K. Hiromi, C. Kuwamoto, and M. Ohnishi, Anal. Biochem., 101, 421 (1980)] was applied to the rapid and simple determination of trace amounts of phosphate, using Malachite Green and molybdate. The specific determination of phosphate down to the ppb level was successfully carried out even in the presence of 20 fold-excess (w/w) of AsO43−. The detection limit was as low as 2 ppb.

Kinetics and thermodynamics of the reversible reaction between carbon monoxide and palladium(I) dimers containing bis(diphenylphosphino)methane

The kinetic and thermodynamic parameters for the reversible insertion of carbon monoxide into the palladium-palladium bond of Pd/sub 2/ (dpm)/sub 2/X/sub 2/ complexes (dpm = Ph/sub 2/PCH/sub 2/PPh/sub 2/; X = Cl, Br, I, NCO) in N,N'-dimethylacetamide solution have been determined by spectrophotometric methods. The forward reaction is first order in both Pd/sub 2/ and CO and occurs on the stopped-flow time scale at around ambient temperatures, the rate constants being similar for the four systems (3.15-6.60) x 10/sup 3/ M/sup -1/ s/sup -1/ at 24/sup 0/C. The equilibrium constants, estimated by kinetic and/or static methods, decrease in the order X = NCO > Cl > Br > I (from 3.7 x 10/sup 5/ to 1.3 x 10/sup 3/ M/sup -1/ at 24/sup 0/C) and are governed largely by the first-order off-rates, which probably depend more on a weakening of the Pd-CO bonds throughout the series than changes in the metal-metal bond strength. A correlation between ln k/sub -1/ for the off-rates and the energy of a high intensity UV band of the Pd/sub 2/(dpm)/sub 2/X/sub 2/ complexes is noted. The CO addition to give Pd/sub 2/(dpm)/sub 2/(..mu..-CO)X/sub 2/ occurs because of the exothermicity of the reaction that is unfavorablemore » in terms of ..delta..S; an approximate value of 90-120 kJ mol/sup -1/ is estimated for the Pd-Pd bond strength. The systems show high selectivity for binding of CO in the presence of H/sub 2/, C/sub 2/H/sub 2/, C/sub 2/H/sub 4/, CO/sub 2/, O/sub 2/, or air. 39 references, 5 figures, 1 table.« less

Kinetics of Tryptophanase Inactivation/Activation by Sudden Removal/Addition of Potassium Ions with the Aid of a Crown Ether or Cryptand

This chapter discusses kinetics of tryptophanase inactivation/activation by sudden removal/addition of potassium ions with the aid of a crown ether or cryptand. Tryptophanase from Escherichia coli B/lt7-A is a member of a group of monovalent cation-activated pyridoxal phosphate-dependent enzymes that catalyze α - β elimination reactions of amino acid substrates. Two moles of thallium are bound per mole of subunit. Thallium substitutes for K + . The spectrum of the active enzyme contains absorption maxima centered at 337 nm and 420 nm. Their relative amplitudes are sensitive to the nature and concentration of the monovalent cation and to pH. The absorption band at 420 nm dominates the absorption spectrum of the inactive K + -free enzyme. This chapter explores the kinetics of deactivation of the enzyme by mixing, on a stopped-flow time scale, the holoenzyme K + complex with an excess of 18-crown-6 or cryptand, both of which are well-known complexants for K + . This deactivation process was followed spectrophotometrically by noting the changes in absorbance at 420 and 337 nm as a function of time. To circumvent the instability of the enzyme in the absence of monovalent cations, the activation process was studied by mixing a solution of the enzyme, freshly deactivated by the addition of complexant, with an excess of K + . This technique proved to be a useful new tool for the study of enzyme activation/deactivation by monovalent cations. The chapter gives a description of the method and a preliminary account of the results.