Stopped-flow spectrophotometry

Chance B 1951 Rapid and sensitive spectrophotometry. I. The accelerated and stopped-flow methods for the measurement of the reaction kinetics and spectra of unstable compounds in the visible region of the spectrum Rev. Sci. Instrum 22 619-27... 

Basically the kinetic results are consistent with the first (rapid) reaction being the addition of a hydroxide ion to the diazonium ion followed by the very fast deprotonation of the (Z)-diazohydroxide to give the (Z)-diazoate (steps 1 and 2 in Scheme 5-14). In addition, however, the stopped-flow experiments showed that the diazonium ion also reacts with the water molecule, initially forming the conjugate acid of the (Z)-diazohydroxide (ArN2OH2), which is then very rapidly deprotonated (reaction 1 in Scheme 5-14). The rate of the relatively slow (Z/E)-isomerization (reaction 5 in Scheme 5-14) can in general be measured by conventional spectrophotometry. 

Temperature jump method. " Stopped flow method. Direct spectrophotometry. 

The fast interaction of O2 with Fe(II)-cysteine complexes to give an oxygen adduct which rapidly undergoes one-electron breakdown to an Fe(III)-cysteine complex and -OJ has been examined by stopped-flow spectrophotometry at 570 nm . Subsequent decomposition of the Fe(IlI) complex to yield Fe(II) and the disulphide, cystine, was much slower. Both mono- and bis-complexes of Fe(Il) are involved and the reaction is first-order in both Fe(II) complex and O2 k (mono) = (5 +1) x 10 l.mole ksec" and k (bis) = (2 0.5) x lO l.mole . sec at 25 °C, corresponding to factors of 10 and 10 times faster than the analogous reactions with sulphosalicylic acid complexes of Fe(II), a feature attributed to Fe(ll)-S bonding in the cysteine complexes. ... 

Pausch and Margerum [656] have described a differential kinetic method using stopped-flow spectrophotometry. 

Atienza et al. [657] reviewed the applications of flow injection analysis coupled to spectrophotometry in the analysis of seawater. The method is based on the differing reaction rates of the metal complexes with 1,2-diaminocycl-ohexane-N, N, N, A/Metra-acetate at 25 °C. A slight excess of EDTA is added to the sample solution, the pH is adjusted to ensure complete formation of the complexes, and a large excess of 0.3 mM to 6 mM-Pb2+ in 0.5 M sodium acetate is then added. The rate of appearance of the Pbn-EDTA complex is followed spectrophotometrically, 3 to 6 stopped-flow reactions being run in succession. Because each of the alkaline-earth-metal complexes reacts at a different rate, variations of the time-scan indicates which ions are present. 

In order to use the stopped-flow technique, the reaction under study must have a convenient absorbance or fluorescence that can be measured spectrophotometri-cally. Another method, called rapid quench or quench-flow, operates for enzymatic systems having no component (reactant or product) that can be spectrally monitored in real time. The quench-flow is a very finely tuned, computer-controlled machine that is designed to mix enzyme and reactants very rapidly to start the enzymatic reaction, and then quench it after a defined time. The time course of the reaction can then be analyzed by electrophoretic methods. The reaction time currently ranges from about 5 ms to several seconds. 

As might be expected, the problem of obtaining spectra of a reacting system increases as the time resolution involved decreases. The spectral changes associated with a reaction may be constructed by wavelength point-by-point measurements. The method, although tedious and costly on materials, is still used. However rapid-scan spectrophotometry, linked to stopped-flow, is now more readily available and reliable. Two systems are used, shown schematically in (3.29) and (3.30). An example of its use is shown in Fig. 3.9. Rapid scan... 

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... 

The latter two processes are slow as a result of which the detection of the protonated carbonato complex, and determination of its protonation constant by stopped-flow, rapid scan spectrophotometry were possible in some cases, van Eldik et al. (83) interpreted the initial spectral changes (shift of absorption maxima and the isosbestic points) of ( -cis-[Co(edda)C03l and [Co(nta)COa] at [H+]=0.05 M due to the formation of the protonated carbonato complexes, p-cis-[Co(edda)C03H] and [Co(nta)C03H], Repetitive rapid scan spectral measurements at [H + ] = 2.0 M, however, led them to the identification... 

Wang, T.-T. Bishop, S.H. Himoe, A. Detection of carbamate as a product of the carbamate kinase-catalyzed reaction by stopped flow spectrophotometry. J. Biol. Chem., 247, 4437-4440 (1972)... 

The most spectacular effect resulting from the highly rigid and compact structure of Cu2(K-84)p+ is undoubtedly its extraordinary kinetic inertness in the cyanide demetalation process. Measurement of the absorbance decay of its MLCT band in the visible region (A = 524 nm) could be performed by classical absorption spectrophotometry (whereas stopped-flow techniques were required for the methylene-bridged knots) and allowed to demonstrate that its demetalation implies two rate-limiting steps, well resolved in time, as schematically represented in Figure 27. 

B. Chymotrypsin Detection of intermediates by stopped-flow spectrophotometry, steady state kinetics, and product partitioning... 

The strategy is to measure the rate constants k2 and k3 of the acylenzyme mechanism (equation 7.1) and to show that each of these is either greater than or equal to the value of kCM for the overall reaction in the steady state (i.e., apply rules 2 and 3 of section Al). This requires (1) choosing a substrate (e.g., an ester of phenylalanine, tyrosine, or tryptophan) that leads to accumulation of the acylenzyme, (2) choosing reaction conditions under which the acylation and deacylation steps may be studied separately, and (3) finding an assay that is convenient for use in pre-steady state kinetics. The experiments chosen here illustrate stopped-flow spectrophotometry and chromopboric procedures. 

The kinetic behavior of solvated electrons has been followed directly using flash radiolysis (44, 45, 58) or flash photolysis technique (62, 94, 107). The former method is more universally applicable owing to the high absorption coefficient of e soiv in a spectral region where most reactants contribute little to the overall optical density. Stopped-flow spectrophotometry has also been applied in the specific case of the eaq + H20 reaction (43), but it is not applicable to reactions where the e soiv half-life is below 0.1 msec. 

Rush JD, Koppenol WH (1987) The reaction between ferrous polyaminocarboxylate complexes and hydrogen peroxide an investigation of the reaction intermediates by stopped flow spectrophotometry. J Inorg Biochem 29 199-215... 

Alcohol oxidation requires release of a proton, which formally comes from the alcohol. In other dehydrogenases such as lactate dehydrogenase, proton release occurs simultaneously with hydride transfer. In liver ADH proton release can be demonstrated, by reaction of the proton with an indicator such as thymol blue or phenol red in stopped-flow spectrophotometry, to be faster than hydride transfer, 270 vs. 150 s and unaffected by use of deuterated substrate, so it occurs before hydride transfer. Binding of the NAD+ nicotinamide ring is accompanied by a conformational change of ADH bringing the catalytic zinc about 0.1 nm closer to the... 

The formation of 2(NO) was too rapid to monitor by standard stopped-flow spectrophotometry, but the kinetics could be accessed using a low-temperature stopped-flow accessory. Second-order rate constants varied little in the range of —40 to —70 °C, but a significantly negative (—118 J/(mol K)) value of the entropy of activation was obtained. The reverse reaction, dissociation of NO, could be characterized kinetically, but some doubt was expressed about the accuracy of the parameters. Laser flash photolysis could be applied in the ambient temperature range and the value of AS for the forward reaction of Equation 7.48 was confirmed at... 

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