Abstract
Stopped-flow rapid mixing is a common, direct technique for the study of ligand-binding reactions. In this method, protein and ligand are mixed together at relatively high velocities directly into an observation chamber, so that time courses for reactions occurring on time scales as short as a few milliseconds can be measured. This chapter presents an introduction to this technique, including a discussion of experimental and technical issues that must be addressed when designing stopped-flow experiments. Simple experiments for measuring reaction dead time and flushing volume are also described along with the details of several common reaction schemes and methods for data analysis.
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References
Gutfreund H. (1969) Rapid mixing: continuous flow. Meth. Enzymol. 16, 229–249.
Gibson Q. (1969) Rapid mixing: stopped flow. Meth. Enzymol. 16, 187–228.
Hammes G. (1974) in Techniques of Chemistry, Vol. VI, Part II, Wiley-Interscience, New York, pp. 1–61.
Wilson M. and Torres J. (2000) In Spectrophotometry & Spectrofluorimetry (Gore G., ed.), Oxford University Press, Washington DC, pp. 209–239.
Gore G. and Bottomley S. (2000) in Spectrophotometry & Spectrofluorimetry (Gore G., ed.), Oxford University Press, Washington DC, pp. 241–264.
Rodger A. and Carey M. (2000) in Spectrophotometry & Spectrofluorimetry (Gore G., ed.), Oxford University Press, Washington DC, pp. 265–281.
Espenson J. H. (1995) Chemical Kinetics and Reaction Mechanisms. McGraw-Hill Series in Advanced Chemistry, McGraw-Hill.
Pilling M. and Seakins P. (1997) Reaction Kinetics, Oxford University Press, Washington DC.
Capellos C. and Bielski B. (1972) Kinetic Systems, John Wiley & Sons, Inc., New York.
Trent J. T., III., Hvitved A. N., and Hargrove M. S. (2001) A model for ligand binding to hexacoordinate hemoglobins, Biochemistry 40, 6155–6163.
Brancaccio A., Cutruzzolá F., Allocatelli C. T., Brunori M., Smerdon S. J., Wilkinson A. J., Dou Y., Keenan D., Ikeda-Saito M., Brantley R. E., Jr., and Olson J. S. (1994) Structural factors governing azide and cyanide binding to mammalian metmyoglobins, J. Biol. Chem. 269, 13,843–13,853.
Zimmerle C. and Frieden C. (1989) Analysis of progress curves by simulations generated by numerical integration, Biochem J. 258, 381–387.
Barshop B., Wrenn R., and Frieden C. (1983) Analysis of numerical methods for computer simulation of kinetic processes: development of KINSIM-a flexible, portable system. Anal. Biochem. 130, 134–145.
Johnson K. A. (1998) Advances in transient-state kinetics, Curr. Opin. Biotech. 9, 87–89.
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© 2005 Humana Press Inc., Totowa, NJ
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Hargrove, M.S. (2005). Ligand Binding With Stopped-Flow Rapid Mixing. In: Ulrich Nienhaus, G. (eds) Protein-Ligand Interactions. Methods in Molecular Biology, vol 305. Humana, Totowa, NJ. https://doi.org/10.1385/1-59259-912-5:323
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DOI: https://doi.org/10.1385/1-59259-912-5:323
Publisher Name: Humana, Totowa, NJ
Print ISBN: 978-1-58829-372-5
Online ISBN: 978-1-59259-912-7
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