title: Dissociation kinetics of the streptavidin-biotin interaction measured using direct electrospray ionization mass spectrometry analysis.
abstract: Dissociation rate constants (k (off)) for the model high affinity interaction between biotin (B) and the homotetramer of natural core streptavidin (S(4)) 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 (S(4) + 4B) complex, the other involving nonlinear fitting of the time-dependent relative abundances of the (S(4) + iB) species. The two methods were found to yield k (off) 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 k (off) 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 k (off), the results of the ESI-MS measurements revealed that the apparent cooperative distribution of (S(4) + iB) species observed at short reaction times is of kinetic origin and that sequential binding of B to S(4) occurs in a noncooperative fashion with the four ligand binding sites being kinetically and thermodynamically equivalent and independent.
authors.affiliation: Department of Chemistry and Alberta Glycomics Centre, University of Alberta, Edmonton, Alberta T6G 2G2, Canada.
authors.forename: Lu
authors.initials: L
authors.lastname: Deng
authors.forename: Elena N
authors.initials: EN
authors.lastname: Kitova
authors.forename: John S
authors.initials: JS
authors.lastname: Klassen
mesh_terms: D001710:Biotin / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D011485:Protein Binding; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D019809:Streptavidin / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D001710:Biotin; D019809:Streptavidin