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 (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 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 (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.
- Publication:
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Journal of The American Society for Mass Spectrometry
- Pub Date:
- January 2013
- DOI:
- 10.1007/s13361-012-0533-5
- Bibcode:
- 2013JASMS..24...49D
- Keywords:
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- Electrospray ionization mass spectrometry;
- Rate constants;
- Protein-ligand complexes