Stopped-flow
Stopped-flow is an experimental technique for studying chemical reactions with a half time of the order of 1 ms, introduced by Britton Chance[1][2] and extended by Quentin Gibson [3] (Other techniques, such as the temperature-jump method,[4] are available for much faster processes.)
Description of the method
Summary
Stopped-flow spectrometry allows chemical kinetics of fast reactions (with half times of the order of milliseconds) to be used to study the fast reactions in solution. It is applied especially to enzyme-catalyzed reactions. The solutions are first forced from syringes into a mixing chamber that results in complete mixing. After a short dead time (see below) the observation cell is filled by a piston linked to a sensing switch that triggers the measuring device and the flow is stopped immediately.
Reactant syringes
Two syringes are filled with solutions that do not undergo a chemical reaction until mixed together. These have pistons that are driven by a single drive piston, so that they are coupled together and their contents are forced out simultaneously into a mixing device.
Mixing chamber
Once the two solutions are forced out of their syringes they enter a mixing system that has baffles to ensure complete mixing, with turbulent flow rather than laminar flow, which would allow the two solutions to flow side by side with incomplete mixing.
Dead time
It is impossible for the mixing of reactants to be achieved instantaneously. As a result there is a dead time before which measurements cannot be made. In older instruments this could be of the order of 1 ms, but improvements now allow a dead time of about 0.3 ms.[5]
Observation cell
The mixed reactants pass an observation cell that allows the reaction to be followed spectrophotometrically, typically by ultraviolet spectroscopy, fluorescence spectroscopy, circular dichroism or light scattering, and it is now common to combine several of these.[6]
Stopping
Once through the observation cell the mixture enters a third syringe that contains a piston that is driven by the flow to activate a switch to stop the flow and activate the observation.
Continuous flow
The stopped-flow method is a development of the continuous-flow method used by Hamilton Hartridge and Francis Roughton[7] to study the binding of O2 to hemoglobin. In the absence of any stopping system the reaction mixture passed to a long tube past an observation system (consisting in 1923 of a simple colorimeter) to waste. By moving the colorimeter along the tube, and knowing the flow rate, Hartridge and Roughton could measure the process after a known time.
In its time this was a revolutionary advance showing an apparently intractable problem (studying a process taking milliseconds with equipment that required seconds for each measurement) could be solved with simple equipment. However in practice it was limited to reactants available in large quantities: for proteins this effectively limited it to reactions of hemoglobin. For practical purposes this approach is obsolete.
Quenched flow
The stopped-flow method depends on the existence spectroscopic properties that can be used for following the reaction. When that is not the case quenched flow provides an alternative that uses conventional chemical methods for analysis.[8] Instead of a mechanical stopping system the reaction is stopped by quenching, the products being delivered to a recipient that stops the reaction immediately, either by instantaneous freezing or by denaturing the enzyme with a chemical denaturant or exposing the sample to a denaturing light source. As in the continuous-flow method, the time between mixing and quenching can be varied by varying the length of the tube.
The pulsed quenched flow method introduced by Alan Fersht and Ross Jakes[9] overcomes the need for a long tube. The reaction is initiated exactly as in a stopped-flow experiment, but there is a third syringe that brings about quenching a definite and preset time after the initiation.
Quenched flow has both advantages and disadvantages with respect to stopped flow. On the one hand, chemical analysis makes it clear what process is being measured, whereas it may not always be obvious what process a spectroscopic signal represents. On the other hand, quenched flow is much more laborious, as each point along the time course must be determined separately. The image at left for catalysis by nitrogenase from Klebsiella pneumoniae[10] illustrates both of these points: the agreement in half times indicates that the absorbance at 420 nm measured the release of Pi, but the quenched-flow experiment required 11 data points.
References
- ^ Chance, Britton (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". Review of Scientific Instruments. 22 (8): 619–627. doi:10.1063/1.1746019.
- ^ Chance, Britton; Legallais, Victor (1951). "Rapid and Sensitive Spectrophotometry. II. A Stopped‐Flow Attachment for a Stabilized Quartz Spectrophotometer". Review of Scientific Instruments. 22 (8): 627–634. doi:10.1063/1.1746020.
- ^ Gibson, Q. H. (1954). "Stopped-flow apparatus for the study of rapid reactions". Discussions of the Faraday Society. 17: 137. doi:10.1039/df9541700137.
- ^ Eigen, M. (1954). "Methods for investigation of ionic reactions in aqueous solutions with half-times as short as 10–9sec. Application to neutralization and hydrolysis reactions". Discuss. Faraday Soc. 17: 194–205. doi:10.1039/df9541700194.
- ^ Clark, Charles R. (1997). "A Stopped-Flow Kinetics Experiment for Advanced Undergraduate Laboratories: Formation of Iron(III) Thiocyanate". Journal of Chemical Education. 74 (10): 1214. Bibcode:1997JChEd..74.1214C. doi:10.1021/ed074p1214.
- ^ Guillerm, Jessica; Frère, Jean-Marie; Meersman, Filip; Matagne, André (2021). "The right-handed parallel β-helix topology of Erwinia chrysanthemi pectin methylesterase is intimately associated with both sequential folding and resistance to high pressure". Biomolecules. 11: 1083. doi:10.3390/biom11081083.
{{cite journal}}: CS1 maint: unflagged free DOI (link) - ^ Hartridge, H.; Roughton, F. J. W. (1923). "A method for measuring the velocity of very rapid chemical reactions". Proceedings of the Royal Society A. 104 (726): 376–394. Bibcode:1923RSPSA.104..376H. doi:10.1098/rspa.1923.0116.
- ^ Pinsent, B R W (1954). "A quenching method for studying rapid reactions". Discussions of the Faraday Society. 17: 140–141. doi:10.1039/df9541700140.
- ^ Fersht, A R; Jakes, R (1975). "Demonstration of two reaction pathways for aminoacylation of transfer-RNA: application of pulsed quenched flow technique". Biochemistry. 14 (15): 3350–3356. doi:10.1021/bi00686a010.
- ^ Thorneley, R N F; Cornish-Bowden, A (1977). "Kinetics of nitrogenase of Klebsiella-pneumoniae: heterotropic interactions between magnesium-adenosine 5'-diphosphate and magnesium-adenosine 5'-triphosphate". Biochem. J. 165 (2): 255–262. doi:10.1042/bj1650255.
Further reading
- Alan Fersht (1998). Structure and Mechanism in Protein Science: Guide to Enzyme Catalysis and Protein Folding. New York: W. H. Freeman. pp. 132–168. ISBN 9780716732686.
- Athel Cornish-Bowden (2012). Fundamentals of Enzyme Kinetics (4th ed.). Weinheim: Wiley-Blackwell. pp. 391–396. ISBN 978-3527330744.