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-occurring 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 (CAII) for zinc in the detection of nanomolar concentrations of this metal ion by fluorescence anisotropy using a fluorescein-derivatized arylsulfonamide probe, 4-aminosulfonyl[1-(4-N-(5-fluoresceinylthioureido)butyl)]benzamide (3). This probe was designed through an iterative, structure-based approach and was demonstrated to bind tightly only to the zinc-bound holoenzyme (K(d) = 2.3 nM) and not the metal-free apoenzyme. Furthermore, the probe exhibits anisotropy that is proportional to the concentration of bound zinc, and this behavior can be exploited in the detection of zinc in the 10-1000 nM range. Strategies for the structure-based design of improved CAII-based metal ion biosensors are considered in view of these results.