Rapid scanning stopped-flow

Similarly, the spectrum of a mixture of Fe(tpps)H20 and Fe(tpps)(OH) can be measured by rapid scan/stopped-flow at various pH s within a few milliseconds after generation (Fig. 3.9). In this short time, dimerization is unimportant so that the spectrum of Fe(tpps)OH can be measured and the pAi of Fe(tpps)H20 estimated. 

Me had a number of goals in mind when we set out to design a new vidicon rapid scanning stopped-flow spectrophotometer. 

Before any instrument can be used with confidence its operating characteristics must be well understood. The exceptional versatility of the vidicon rapid scanning stopped-flow system allowed a large variety of characterization experiments to be easily performed. A few of the more important experiments designed to help characterize the vidicon detector will be presented before the actual application of the instrument is discussed. 

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 ... 

Fig. 7.7 Rapid-scanning stopped-flow spectra for the reaction of 10 pM wild and mutant aifii complexes with 40 mM L-serine at pH 7.8. A) Wild type, B) o E49F, C), aG51L and D) aD60Y. Sprectra were collected at various intervals (0-640 msec) after mixing.

General aspects of the stereochemistry and mechanism of PLP-catalyzed reactions have been reviewed by Floss and Vederas75 and Miles.77 In this section we briefly describe the catalytic cycle of tryptophanase. New transient intermediates have recently been detected in this cycle by Phillips et a/.,41-42-78 using rapid-scanning stopped-flow spectrophotometry, and they are included in the reaction mechanism depicted in Fig. 9.13. 

Seven-coordinate three-dimensional metal complexes are considered to be quite unstable and kinetically labile species, and their solution chemistry is largely undefined. Over the last few years it was shown that these species exhibit extremely interesting chemical properties and catalytic activity. They can catalyze disproportionation of deleterious superoxide radicals, even faster than natural enzymes, and therefore this became a challenging research area. Recently, Rudi van Eldik reported a detailed rapid-scan stopped-flow kinetic study of the substitution behavior of the seven-coordinate [Fe(dapsox)(L)2]C104 complex with thiocyanate as a function of the thiocyanate concentration, temperature, and pressure in protic and aprotic organic solvents. 

Kinetic experiments are performed in two different ways. In one an initial disequilibrinm exists between two or more reactants, which after being rapidly mixed, combine to react toward equilibrium see Rapid Scan, Stopped-Flow Kinetics). Ideally, the mixing time is short with respect to the timescale of the reaction or actually with respect to the formation of intermediates. In contrast, in the relaxation experiment, the reactants are together and in equilibrium, and the whole system is instantaneously displaced from equilibrium. Subsequently, the system relaxes to the same or a new equilibrium state. Table 1 suimnarizes the approximate time resolution of various commonly applied mixing and relaxation techniques. The table indicates the superiority of the relaxation methods with respect to time resolution, mainly due to the development of ultrafast lasers. Mixing liquids on the (sub)microsecond time scale appears to present an important experimental barrier. 

A major breakthrongh in time resolution ( 1 ms), dynamic range ( unlimited ) and reduction of sample amount came with the development by Britton Chance of the stopped-flow techniqne see Rapid Scan, Stopped-Flow Kinetics), which is stUl widely used today. The stopped-flow techniques finds a major application in the... 

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... 

Applications of Rapid-Scanning, Stopped-Flow (RSSF) UV-Visible Spectroscopy to the Study of Biological Systems... 

Rapid-Mixing, Rapid-Scanning Stopped-Flow Systems... 

APPLICATIONS OF RAPID-SCANNING, STOPPED-FLOW (RSSF) UV-VISIBLE SPECTROSCOPY TO THE STUDY OF BIOLOGICAL SYSTEMS... 

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