Binding of hydrophobic ligands by Pseudomonas aeruginosa PA-I lectin

Abstract The ability of Pseudomonas aeruginosa PA-I lectin to bind the fluorescent hydrophobic probe, 2-( p -toluidinyl) naphthalene sulfonic acid (TNS), and adenine was examined by spectrofluorametry and equilibrium dialysis. Interaction of TNS with PA-I caused significant enhancement of TNS fluorescence. The Hill coefficient (3.8±0.3) and the dissociation constant (8.7±0.16 μM) showed that TNS probably bound to four high affinity hydrophobic sites per PA-I tetramer. Interactions between PA-I and adenine were examined by equilibrium dialysis using [ 3 H] adenine. The results indicated the presence of at least two classes of binding sites—one high and four lower affinity sites per tetramer with dissociation constants of 3.7±1.5 and 42.6±1.2 μM, respectively. These were distinct from the TNS sites as titration of TNS-equilibrated PA-I with adenine caused TNS fluorescence enhancement. The titration curve confirmed the existence of two classes of adenine-binding sites. Conversely, when PA-I was first equilibrated with adenine and then titrated with TNS, no TNS-binding was registered. This may indicate that conformational rearrangements of the lectin molecule caused by adenine prevent allosterically TNS binding.