Reaction Rates at Biosurfaces

Consider a labeled molecule in equilibrium between a surface-bound state 

If the solute is fluorescent, the TIRF intensity (which is proportional to 

Using a concentration jump as the perturbation, Sutherland et a/.(113) measured the kinetics of binding of fluorescein-labeled human IgG (present as an antigen in solution) to surface-immobilized sheep anti-human IgG. Two TIRF surfaces were used a planar slide and a fiber-optic cylinder. Also using a TIRF recovery after a concentration jump, Kalb et a/,(114) measured the slow ( 10 4 s 1) unbinding kinetics of anti-trinitrophenol (TNP) antibodies in solution and a TNP-derivatized lipid in a planar bilayer. 

To increase the speed of the TIRF-based kinetic techniques, the perturbation can be optical rather than chemical. If the evanescent wave intensity is briefly flashed brightly, then some of the fluorophores associated with the surface will be photobleached. Subsequent exchange with unbleached dissolved fluorophores in equilibrium with the surface will lead to a recovery of fluorescence, excited by a continuous but much attenuated evanescent wave. The time course of this recovery is a measure of the desorption kinetic rate k2. This technique1-115) is called TIR/FRAP (or TIR/FPR) in reference to fluorescence recovery after photobleaching (or fluorescence photobleaching recovery). 

Adsorption kinetics are especially interesting when compared with surface diffusion rates of the adsorbate. This is because of the theoretical possibility that nonspecific and reversible adsorption of a ligand (say, a hormone), followed by two-dimensional diffusion on the membrane, may enhance the reaction rate with a specific binding patch (say, a hormone receptor).(1161I7) A similar effect might enhance the reaction rates between a surface-immobilized enzyme and bulk-dissolved substrate, thereby speeding some reactions in industrial chemistry. 

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