Binding and dissociation kinetics of wild-type and mutant streptavidins onmixed biotin-containing alkylthiolate monolayers

The kinetics of adsorption and competitive desorption of wild-type streptav idin (WT SA) and three genetically engineered mutants (S27A, N23E, and W120 A) was studied at gold surfaces functionalized with mixed alkylthiolates, s ome terminated with biotin headgroups and the rest with oligo(ethylene oxid e) using surface plasmon resonance (SPR). The saturation coverage of the pr otein varied strongly with surface biotin concentration (X-BAT) and was ind ependent of mutation (except at very low and very high X-BAT, where a weak dependence was seen). Initial adsorption rates were nearly diffusion-limite d except at extremely low X-BAT, where the rate varied weakly between mutan ts in accordance with their differing strengths of binding to biotin. Initi al sticking probabilities were estimated to be between similar to1-6 x 10(- 6) per collision with the surface. The adsorbed SA desorbs upon introductio n of solution-phase biotin. For X-BAT below 1%, the desorption rate constan ts of the SA variants closely follow their off-rate constants measured in h omogeneous solution (which at 25 degreesC are WT = 4 x 10(-6) sec(-1), N23E = 1.6 x 10(-3) sec(-1), S27A = 1.2 x 10(-3) sec(-1) and W120A estimated to be ca. 23 s(-1)). This proves that SA is mainly bound to the surface by a single Sk biotin link at very low XBAT Importantly, for X-BAT between 10 an d 40%, where desorption is 30- to >1000-fold slower and the saturation cove rage maximizes, the ratios of off-rate constants between mutants (W120A/N23 E and W120A/S27A) are approximately the square of their ratios for XBAT bel ow 1%. This squaring strongly suggests that the dominant species at these c overages is doubly bounded SA (i.e., immobilized via two surface biotins). The kinetics are explained with a mechanism involving only two first-order rate constants, that is, for (1) the slow dissociation of any bond between an SA site and a surface-immobilized biotin and (2) the fast reforming of t his bond in the special case that it was released from a doubly bonded SA w hose other site is still linked to one surface-immobilized biotin. The rate constant for (2) is almost independent of the SA mutant, as it is for adso rption. For X-BAT > 60%, the desorption rates again approach the singly bou nd SA values, and the ratios of rate constants for the SA variants drop to slightly less than below 1% biotin. This is due to the dominance of singly bonded SA, plus a contribution from nonspecific binding, consistent with st ructural studies of these alkylthiolate films.