STRUCTURE-BASED DESIGN OF A SULFONAMIDE PROBE FOR FLUORESCENCE ANISOTROPY DETECTION OF ZINC WITH A CARBONIC ANHYDRASE-BASED BIOSENSOR

Given the avid and selective metal binding properties of naturally-occ urring metalloproteins, it is possible to exploit these systems in the development of novel sensors, i.e., ''biosensors'', for the detection of trace quantities of metal ions. Here, we exploit the high affinity of human carbonic anhydrase II (CATI) for zinc in the detection of na nomolar concentrations of this metal ion by fluorescence anisotropy us ing a fluorescein-derivatized arylsulfonamide probe, 1-(4-N-(5-fluores ceinylthioureido)butyl)]benzamide (3). This probe was designed through an iterative, structure-based approach and was demonstrated to bind t ightly only to the zinc-bound holoenzyme (K-d = 2.3 nM) and not the me tal-free apoenzyme. Furthermore, the probe exhibits anisotropy that is proportional to the concentration of bound zinc, and this behavior ca n be exploited in the detection of zinc in the 10-1000 nM range. Strat egies for the structure-based design of improved CAII-based metal ion biosensors are considered in view of these results.