Rapid scanning stopped-flow studies

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 ... 

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. 

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... 

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

Measurements of absorption as a function of time can provide a wide range of useful information. Brzovic and Dunn [8] describe instrumentation for measuring the time dependence of absorption spectra after rapid mixing of reactants. Several rapid-scanning stopped-flow instruments are commercially available reactions that take place in a millisecond or longer can be studied. Enzyme-catalyzed reactions with natural chromophores, such as NADH, are discussed, and the substitution of a colored metal center [Co(II)] for a colorless one [Zn(II)] are also described. Detailed mechanistic conclusions for horse liver alcohol dehydrogenase (LADH) are given. 

If the investigation of the chemical mechanisms of enzyme-catalyzed reactions is to be successful, then detection and identification of transient intermediates along the reaction path are essential components. The application of rapid-scanning stopped-flow (RSSF) UV/visible spectroscopy to the study of enzyme catalysis may be of considerable significance in certain instances as a tool for both the direct detection and the identification of transient chemical intermediates. 

A newly designed, high-resolution rapid-scan-stopped-flow spectrophotometer has been used to study the sequential aquation reactions shown in equation (34), where ki = 86.6 s" k2 = 5.2 s and k = 0.145 s at 298.2 K in... 

Optical Spectroscopy General principles and overview, 246, 13 absorption and circular dichroism spectroscopy of nucleic acid duplexes and triplexes, 246, 19 circular dichroism, 246, 34 bioinorganic spectroscopy, 246, 71 magnetic circular dichroism, 246, 110 low-temperature spectroscopy, 246, 131 rapid-scanning ultraviolet/visible spectroscopy applied in stopped-flow studies, 246, 168 transient absorption spectroscopy in the study of processes and dynamics in biology, 246, 201 hole burning spectroscopy and physics of proteins, 246, 226 ultraviolet/visible spectroelectrochemistry of redox proteins, 246, 701 diode array detection in liquid chromatography, 246, 749. 

Fig. 1. Rapid-scanning stoppcd-flow (RSSF) study of the reaction ofN-furylacryloylr-tryptophan methyl ester (FATME) with a-chymotrypsin (a-Ct) at pH 5.0 in the absence and presence of proflavin. (A) RSSF difference spectra for the reaction of 19 pM a-Ctwith 7.5 pM FATME in 0.1 M pH 5.0 sodium acetate buffer at 25°. Spectrum 0 is 7.5 pM free FATME, spectra 1 -5 are difference spectra measured during reaction wherein the spectrum of a-Ct has been subtracted from the set. Spectrum 6 is the spectrum of the hydrolysis product furylacryloyl i-tryptophan with the spectrum of a-Ct removed by subtraction. Spectra were measured at the following time intervals after flow had stopped (1) 8.54, (2) 162.3, (3) 341.6 (4) 1409.1 and (5) 3074.4 ms. Spectrum 6, t = oo.

The formation and decay of the oxyferrous forms of the cytochromes P-450 isolated from Rhizobium japonicum. Rapid spectral scan and stopped flow studies. Biochim. Biophys. Acta 828, 144-150. 

Rapid-Scanning Ultraviolet/Visible Spectroscopy Applied in Stopped-Flow Studies... 

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 ... 

There is information from other studies which probe the question of delineation of the electronic spectra and kinetic stability of organic radical intermediates produced by Ce(IV) oxidations. For example, Kemp et al. (1980) determined such spectra for the radical-cation and neutral radical produced in the first two kinetic steps when phenothiazine and phenoxazine are the reductants. The combination of stopped-flow and rapid-scan spectrophotometry were used to evaluate kinetic parameters for these steps. 

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