strong and specific binding feature of this biotin -The binding of 5-end biotinylated DNA, ranging in streptavidin system not only offers many powerful size from 100 to 5000 base pairs, was studied using bioanalytical applications but also generates considstreptavidin-coated polystyrene latex particles with erable interest as a versatile model in studying macdiameters between 0.944 and 0.090 mm. The experimenromolecule -ligand interactions (1 -4).
tal binding constants and forward rate constants of
The binding kinetics of several biotin-streptavidin this solid-phase reaction were determined to be sevsystems have been well studied in solution (1). The eral orders of magnitude lower than values for the bioexperimental results from such studies conform with tin-streptavidin interaction in solution as expected classical chemical theories, i.e., the law of mass action and were shown to depend on the size of both ligand with its rigorous thermodynamic underpinnings. Howand substrate. An observed inflection in the binding ever, a few reports exist in which the focus has been constant of the biotinylated DNA appeared around the interaction between either biotin or streptavidin in 1000 base pairs, possibly indicating different surface the form of a solid-phase reagent. These studies show orientations of the macroligand above and below this that the heterogeneous phase kinetics differ substancritical size. This effect was more pronounced for the tially from the theoretical models that have been develsmaller latex particles used in this study and highoped to explain the results of biotin-streptavidin reaclighted possible differences in the surface arrangetions in solution (5-7). The initial forward reaction ment of streptavidin on the differently sized particles.
Diffusion limitation to the binding reaction was found often becomes diffusion limited at the interface, possito be significant in all cases. In this present work, an bly due to steric hindrance and the inflexibility of the exponential relationship was established between the immobilized ligand. On the other hand, the dissociation experimental binding constant and the number of base rate of the bound molecule is often faster than that pairs in the biotinylated DNA. This relationship possicharacteristic of the complex in solution as a result of bly provides a means to predict capacity and binding surface-induced conformational shifts in the binding speed in cases where adsorption, purification, and resites presented by the immobilized molecules. Thus, lease of larger DNA chains are required. แญง 1996 Academic both the forward and reverse reactions tend to reduce Press, Inc.
the magnitude of the binding constant (7).
Uniform latex particles have been used in biomedical research for more than two decades. Since the surface area per mass of particles increases with smaller diam-Streptavidin, a protein produced by Streptomyces eters, the amount of immobilized ligand relative to the avidinii, binds d-biotin with an extremely high affinity (the K A of biotin -streptavidin in solution is total mass of the particles also increases. Furthermore, around 10 15 M 01 ); the formation of this complex can in binding large biotinylated macromolecules (e.g., biobe regarded as practically irreversible, as the binding tinylated DNA) to streptavidin-coated surfaces, the energy is comparable to that of a covalent bond. The size of the biotinylated ligand is likely to affect not only the equilibrium binding level, but also the rate at which this binding occurs. Since mass transfer limitations