Biospecific binding of carbonic anhydrase to mixed SAMs presenting benzenesulfonamide ligands: A model system for studying lateral steric effects

This work describes the binding of carbonic anhydrase (CA) to mixed self-as sembled monolayers (SAMs) presenting benzenesulfonamide ligands at a surfac e consisting primarily of tri(ethylene glycol) [(EG)(3)OH] groups. Surface plasmon resonance (SPR) quantified the binding of CB to the benzenesulfonam ide groups. Two factors influenced the binding of CA: (a) the density of be nzenesulfonamide groups at the surface, and (b) the coverage of the surface with molecules of CA adsorbed to these benzenesulfonamide groups. At low m ole-fractions of benzenesulfonamide groups in the mixed SAM where the bindi ng of CA is highly (>90%) reversible, we observe. (a an approximately 10-fo ld decrease in the observed bimolecular rate constant for association, k(on ,obs), during the binding of CA(as the fraction of the surface covered by a dsorbed CA increases from similar to 0.15 to similar to 0.35, the value of k(on,obs) decreases from -40 x 10(3) M-1 s(-1) to -4 x 10(3) M-1 s(-1)); ib ) almost no corresponding changes in the observed unimolecular rate constan t for dissociation (k(off,obs) similar to 0.005 s(-1)) during the dissociat ion of CA from the surface. These observations establish that k(on,obs) is influenced by the extent of coverage of the surface with CA, but that k(off ,obs) is not. At low surface densities of arylsulfonamide groups, one hypot hesis that rationalizes these data is that the decrease in k(on,obs) reflec ts repulsive steric interactions between molecules of CA. near the surface and those already adsorbed. Each molecule of biospecifically adsorbed CA sh ields proximal benzenesulfonamide ligands from binding to incoming molecule s of CA, and decreases the surface density of these ligands that are access ible to CA, at a rate that increases nonlinearly with the quantity of CA al ready adsorbed.