Rapid scanning stopped-flow scan rates

The aquated iron(III) ion is an oxidant. Reaction with reducing ligands probably proceeds through complexing. Rapid scan spectrophotometry of the Fe(III)-cysteine system shows a transient blue Fe(lII)-cysteine complex and formation of Fe(II) and cystine. The reduction of Fe(lII) by hydroquinone, in concentrated solution has been probed by stopped-flow linked to x-ray absorption spectrometry. The changing charge on the iron is thereby assessed. In the reaction of Fe(III) with a number of reducing transition metal ions M in acid, the rate law... 

Later, Smith and coworkers succeeded in measuring rate constants of the reaction of MeLi with a carbonyl compound at various reagent concentrations with a stopped-flow/rapid scan spectroscopic method, and demonstrated that the reaction also exhibited a fractional kinetic order . Thus, the reaction of 2,4-dimethyl-4 -methylmercaptobenzophenone with MeLi in diethyl ether at 25 °C showed one-fourth order in MeLi in the concentration range of MeLi between 3.9 mM and 480 mM (Figure 1). The rate constant was 200 7 M s . Under these conditions, the monomer was considered the reactive species that exists in equilibrium with the tetramer. Addition of LiBr or Lil depressed the reaction rate but did not change the kinetic order. The same... 

The chromophoric pyridoxal phosphate coenzyme provides a useful spectrophotometric probe of catalytic events and of conformational changes that occur at the pyridoxal phosphate site of the P subunit and of the aiPi complex. Tryptophan synthase belongs to a class of pyridoxal phosphate enzymes that catalyze /3-replacement and / -elimination reactions.3 The reactions proceed through a series of pyridoxal phosphate-substrate intermediates (Fig. 7.6) that have characteristic spectral properties. Steady-state and rapid kinetic studies of the P subunit and of the aiPi complex in solution have demonstrated the formation and disappearance of these intermediates.73-90 Fig. 7.7 illustrates the use of rapid-scanning stopped-flow UV-visible spectroscopy to investigate the effects of single amino acid substitutions in the a subunit on the rate of reactions of L-serine at the active site of the P subunit.89 Formation of enzyme-substrate intermediates has also been observed with the 012P2 complex in the crystalline state.91 ... 

The stopped-flow mixing can be combined with a variety of detection methods in addition to spectrophotometry. A nice example is provided by a rapid-scanning EPR spectrometer in which the reagents are delivered from a stopped-flow mixer into an EPR cell. This action triggers the generation of a wave form to drive a rapid-scan unit. The rate constants are obtained from the digitized spectra [14]. 

Silverman s studies on mechanism based MAO inactivation have provided overwhelming support for the role of electron transfer in the MAO catalyzed dealkylation of amines. It must be mentioned however that spectroscopic attempts for detecting the radical ion intermediates have hitherto been unsuccessful. Yasanobu and coworkers could not find EPR spectral evidence for radical intermediates in MAO-catalyzed oxidation of benzylamine [205]. Miller et al. failed to observe the flavin semiquinone or an amine-flavin adduct in rapid-scan-stopped flow spectroscopy [206]. The only time-dependent absorption change observed in this study was the bleaching of the oxidized flavin. Furthermore, no influence of a magnetic field up to 6500 G was observed on the rate of MAO B reduction. The reaction rates of systems with kinetically significant radical pair intermediates are known to be altered... 

Fig. 11. Rapid-scanning, stopped-flow data comparing the accumulation of transient intermediates during the reaction of 8 mM z>i-[a- H]-serine (A) and 8 mM o/.-[a-2H]-serine (B) with 13.3 M a232 from E. coli. The trace designated 0 is the reconstructed spectrum of the reactants before mixing. The insets to both (A) and (B) are 460-nm reaction time courses reconstructed from the RSSF data. Each experiment was conducted using 0.1 M potassium phosphate and 1 mM EDTA buffer at pH 7.80 and25°C. All conditions refer to concentrations immediately after mixing. The initiation of scanning in both (A) and (B) occurred 2 ms after flow stopped. Scans 2 through 19 were collected at 4.7, 9.3, 14.0, 18.7,23.4,28.0,32.7,42.0,51.4,60.7,70.1,107, 154,247,387, 761, 1135, and 1980 ms after the first scan, respectively, with a repetitive scan rate of 4.7 ms/scan. [Taken from Drewe and Dunn (85) with permission.]...

Fig. 21. Rapid-scanning, stopped-flow spectra for the reaction of / -cysteine with CGS pre-incubated with i-allylglycine. Concentrations refer to conditions immediately after mixing [CGS] = 7.08 im, [i-allylglycine] = 28mM, [i-cysteine] = 10 mM, 0.1 M potassium phosphate, 5 mM DTE, 1 mM EDTA, pH 7.2 and 25°C. Initiation of scanning occurred 2.3 ms after flow stopped. Data shown are representative scans from a sequential 79-scan data set collected with a repetitive scan rate of 8.9 ms/scan. The spectra shown were acquired at 2.3, 11.2,20.1,29.0, 37.9, 55.7, 73.5,109.1,162.5, 289.6, and 696.5 ms after flow stopped. The inset to (A) represents single-wavelengths time courses taken from the entire set of 79 scans at (a) 300 nm and (b) 420 nm. The left and right ordinates correspond to the absorbance values at 300 and 420 nm, respectively. [Taken from Brzovic et al. (107) with permission.]...

The optical absorption spectra of the stable Fe, Fe, and Fe NO species were determined, and stopped-flow rapid scan, flash photolysis, and low-temperature spectroscopic methods have been applied to study the reactions of Equations (7)-(9). The species P450nor(Fe Njqo) formed in reaction (7) was characterized by stopped-flow as having a diminished Soret absorbance at 434 nm and a single broad band at 558 nm. Formation of this species was complete within the dead time of the stopped-flow apparatus, even at 10 °C and [NO] of 5 pM, allowing a limit for k, the rate constant for formation of this species, to be estimated as larger than 10 s ... 

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