DyBOBS: A dynamic biomimetic assay for odorant-binding to odor-binding protein

摘要

An in vitro system was developed to mimic the structural and flow conditions of the human olfactory epithelium and to measure the dynamics of odorant-binding to odor-binding protein (OBP). A hydrophilic fused silica capillary, coated internally with a thin (about 1.3 μm) aqueous film of recombinant rat-OBP3 mimicked the human olfactory epithelium. Isobutylthiazole in air was introduced into the capillary, and the outflow gas was monitored in real-time using on-line mass spectrometry. Time-dependent changes in the gas phase odorant concentration gave an indication of the rate and extent of mass transfer between the gas phase and the aqueous OBP layer during the initial uptake phase, during the steady state (when the OBP was fully loaded with odorant) and then during a release phase when clean air was introduced into the capillary. Control experiments showed no significant isobutylthiazole interaction with the system and measured the contribution of water to odorant uptake. Isobutylthiazole uptake was then measured under different flow and concentration regimes, from which the stoichiometry of binding was calculated. The measured flow and structural characteristics in the model system were comparable with the situation in vivo. Uptake and release of isobutylthiazole with OBP3 during a simulated tidal flow regime showed strong uptake but little release of odorant due to the strong binding characteristics of this particular compound. The model system (dynamic biomimetic odorant-binding system) allowed monitoring of the dynamic binding and release of airborne odorants to OBP, and the resulting kinetic data provide an insight into the way OBP functions in vivo.