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Dissociation kinetics of the streptavidin-biotin interaction measured using direct electrospray ionization mass spectrometry analysis Open Access
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Kitova, Elena N.
Klassen, John S.
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Electrospray ionization mass spectrometry
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Dissociation rate constants (koff) for the model high affinity interaction between biotin (B) and the homotetramer of natural core streptavidin (S4) were measured at pH 7 and temperatures ranging from 15 to 45 °C using electrospray ionization mass spectrometry (ESI-MS). Two different approaches to data analysis were employed, one based on the initial rate of dissociation of the (S4 + 4B) complex, the other involving nonlinear fitting of the time-dependent relative abundances of the (S4 + iB) species. The two methods were found to yield koff values that are in good agreement, within a factor of two. The Arrhenius parameters for the dissociation of the biotin–streptavidin interaction in solution were established from the koff values determined by ESI-MS and compared with values measured using a radiolabeled biotin assay. Importantly, the dissociation activation energies determined by ESI-MS agree, within 1 kcal mol–1, with the reported value. In addition to providing a quantitative measure of koff, the results of the ESI-MS measurements revealed that the apparent cooperative distribution of (S4 + iB) species observed at short reaction times is of kinetic origin and that sequential binding of B to S4 occurs in a noncooperative fashion with the four ligand binding sites being kinetically and thermodynamically equivalent and independent.
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- © 2013 Deng, L., Kitova, E. N., & Klassen, J. S. This version of this article is open access and can be downloaded and shared. The original author(s) and source must be cited.
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Deng, L., Kitova, E. N., & Klassen, J. S. (2013). Dissociation kinetics of the streptavidin-biotin interaction measured using direct electrospray ionization mass spectrometry analysis. Journal of the American Society for Mass Spectrometry, 24(1), 49-56. http://doi.org/10.1007/s13361-012-0533-5
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File title: Comparison of the Dissociation Kinetics of the Streptavidin-Biotin Interaction in Solution and in the Gas Phase
File author: John Klassen
Page count: 30
File language: en-US