Detailed characterization of the solution kinetics and thermodynamics of biotin, biocytin and HABA binding to avidin and streptavidin

PloS One
Roberto Fabio DelgadilloLawrence J Parkhurst

Abstract

The high affinity (KD ~ 10-15 M) of biotin for avidin and streptavidin is the essential component in a multitude of bioassays with many experiments using biotin modifications to invoke coupling. Equilibration times suggested for these assays assume that the association rate constant (kon) is approximately diffusion limited (109 M-1s-1) but recent single molecule and surface binding studies indicate that they are slower than expected (105 to 107 M-1s-1). In this study, we asked whether these reactions in solution are diffusion controlled, which reaction model and thermodynamic cycle describes the complex formation, and if there are any functional differences between avidin and streptavidin. We have studied the biotin association by two stopped-flow methodologies using labeled and unlabeled probes: I) fluorescent probes attached to biotin and biocytin; and II) unlabeled biotin and HABA, 2-(4'-hydroxyazobenzene)-benzoic acid. Both native avidin and streptavidin are homo-tetrameric and the association data show no cooperativity between the binding sites. The kon values of streptavidin are faster than avidin but slower than expected for a diffusion limited reaction in both complexes. Moreover, the Arrhenius plots of the kon values r...Continue Reading

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Citations

Oct 1, 2019·Biomedical Optics Express·Enxiao LuanLukas Chrostowski
Apr 2, 2020·International Journal of Molecular Sciences·Denis V VoroninDaniil N Bratashov
Apr 5, 2020·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Marek Trojanowicz
Oct 23, 2020·Biotechnology and Bioengineering·Liem Bui-LeJason P Hallett
Oct 15, 2021·Organic Letters·Mahmoud ElkhalifaGary A Molander

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Methods Mentioned

BETA
Fluorescence
glycosylation
fluorescence assay
FRET
chip
affinity
pull-off
Scanning Force Microscopy
biosensors

Software Mentioned

Fluofit
Accelrys DS visualizer
LabVIEW
PicoQuant

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