Stopped flow kinetic devices

Any advanced absorbance/fluorescence spectrophotometer designed for routine acquistion of absorption or emission on the subsecond time scale. The basic goal is to obtain a series of complete UV/visible or fluorescence spectra as a function of time, usually after samples are mixed in a stopped-flow device. Such data help the investigator to infer the most likely structures of transient intermediates whose electronic spectra or fluorescence spectra can be determined by deconvoluting the spectra with appropriate reaction kinetic simulation software or by some other global analysis method (Fig. 1). 

Measurements of reactions that occur in less than a few seconds require special techniques to speed up the mixing of the enzyme and substrate. One way to achieve this is to place solutions containing the enzyme and the substrate in two separate syringes. A pneumatic device then is used to inject the contents of both syringes rapidly into a common chamber that resides in a spectrophotometer for measuring the course of the reaction (fig. 7.5). Such an apparatus is referred to as a stopped-flow device because the flow stops abruptly when the movement of the pneumatic driver is arrested. In this type of apparatus it is possible to make kinetic measurements within about 1 ms after mixing of enzyme and substrate. 

By control experiments with a standard commercial stopped-flow instrument, it was checked that true kinetic information was derived, i.e. that there are no mixing effects any more, which limit the applicability of the device [133],... 

Fig. 8 Kinetics of binding of Flutaxl to microtubules at 35 °C. In the stopped-flow device a 1 pM solution of Flutaxl was mixed with 25 pM pure tubulin assembled into microtubules (concentration of sites 20 pM) (final concentrations of 500 nM Flutax and 10 pM sites) in the absence (a) and presence (b) of 50 pM docetaxel. Curve a is fitted to an exponential decay. Inset residues between the experimental and theoretical curves. Taken from [10]...

Today a stopped-flow instrument consists of oifly the unit itself combined with a diode array detector and a computer allowing fast kinetic measurements of time-resolved UV-vis spectra under anaerobic, high pressure and/or low temperature conditions. Improvements have been made as well, for example, syringes are installed vertically instead of horizontally (to avoid problems with gas bubbles) and polyetheretherketone (PEEK) is used instead of Teflon for valves and flow tubes to improve the anaerobic capabilities of the instrument. Further, the syringe drives setup was optimized. Application of rapid-scan devices (usually, but not exclusively, diode arrays) allows complete spectra to be collected at very short time intervals during a reaction. 

Figure 4 A schematic representation of the experimentai approach for time-resoived XAS measurements. XAS provides local structural and electronic information about the nearest coordination environment surrounding the catalytic metal ion within the active site of a metalloprotein in solution. Spectral analysis of the various spectral regions yields complementary electronic and structural information, which allows the determination of the oxidation state of the X-ray absorbing metal atom and precise determination of distances between the absorbing metal atom and the protein atoms that surround it. Time-dependent XAS provides insight into the lifetimes and local atomic structures of metal-protein complexes during enzymatic reactions on millisecond to minute time scales, (a) The drawing describes a conventional stopped-flow machine that is used to rapidly mix the reaction components (e.g., enzyme and substrate) and derive kinetic traces as shown in (b). (b) The enzymatic reaction is studied by pre-steady-state kinetic analysis to dissect out the time frame of individual kinetic phases, (c) The stopped-flow apparatus is equipped with a freeze-quench device. Sample aliquots are collected after mixing and rapidly froze into X-ray sample holders by the freeze-quench device, (d) Frozen samples are subjected to X-ray data collection and analysis.

Stopped-flow mixing has been used for fundamental studies of rapid reactions and for routine kinetic determinations of analytes involved in fast reactions. The principles of fluid dynamics that make stopped-flow mixing possible and the solution-handling capabilities of this and similar devices are used in many contexts to automatically mix solutions and measure analyte concentrations in numerous industrial and clinical laboratories. 

Cartoon of a stopped flow kinetic device (Courtesy of TgK Scientific Ltd). Solutions of enzyme and substrate are pushed from two syringes into a mixing chamber, then through an observation chamber and then into a third syringe. 

Although the course of a reaction can be monitored by chemical or even visual means, most kinetic methods rely on instruments for this purpose, optical (photometric and fluorimetric) and electroanalytical devices being by far the most common choices. In this context, it is worth emphasizing the ability of stopped-flow mixing methodology to boost the performance of the chemiluminescence reaction to which it is specially suited on account of the fast transient nature of chemiluminescence reactions. 

As a rule, these determinations use the initial reaction rate method. Their prominence in recent years can be ascribed to the increasing availability of inexpensive mixing systems such as modular stopped-flow and continuous-addition-of-reagent devices, which enable kinetic monitoring. 

Enzyme reaction intermediates can be characterized, in sub-second timescale, using the so-called pulsed flow method [35]. It employs a direct on-line interface between a rapid-mixing device and a ESI-MS system. It circumvents chemical quenching. By way of this strategy, it was possible to detect the intermediate of a reaction catalyzed by 5-enolpyruvoyl-shikimate-3-phosphate synthase [35]. The time-resolved ESI-MS method was also implemented in measurements of pre-steady-state kinetics of an enzymatic reaction involving Bacillus circulans xylanase [36]. The pre-steady-state kinetic parameters for the formation of the covalent intermediate in the mutant xylanase were determined. The MS results were in agreement with those obtained by stopped-flow ultraviolet-visible spectroscopy. In a later work, hydrolysis of p-nitrophenyl acetate by chymotrypsin was used as a model system [27]. The chymotrypsin-catalyzed hydrolysis follows the mechanism [27] ... 

Pure Solvents Miscellaneous.—Solvent Exchange. Recent examples of kinetic studies of solvent exchange at metal cations, formally the simplest substitution reaction in solution, include exchange of DMF at chromium(iii) and of DMSO at aluminium(m). The latter investigation used an ingeniously modified stopped-flow device integrated with a pulse Fourier-transform n.m.r. spectrometer, an apparatus of considerable potential value in fast-reaction studies. ... 

Chemical kinetics the absolute values of kRi and kR2. The magnitude of the rate constant, kRi, will determine how much A can be converted during the time required to achieve molecular mixing. The extent of the conversion will determine the amount of R that is subject to excess B concentration and hence overreaction to S as determined by kR2. In some cases the kinetics can be determined by use of a stopped-flow reactor or similar device. For... 

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