Fluorometric detection of Ca2+ based on an induced change in the conformation of sol-gel entrapped parvalbumin

We report the development of a fluorometric detection strategy for Ca2+ based on induced changes in the conformation of cod III parvalbumin entrapped within a sol-gel processed glass. The detection scheme utilizes a fluorescent allosteric signal transduction (FAST) strategy wherein conformational changes induced by Ca2+ binding result in alterations in the intrinsic fluorescence from the single tryptophan residue at position 102. Intrinsic fluorescence was also used to examine chemically induced changes in protein structure to ascertain the effects of entrapment on the conformational. motions and stability of the protein. Fluorescence analysis indicated that the behavior of the protein depended on the entrapment protocols used. The entrapped protein retained conformational flexibility similar to that observed in solution and remained accessible to analytes such as Ca2+. Entrapment also caused improvements in protein stability against chemical denaturants. However, entrapment caused the apparent affinity constant for binding of Ca2+ to decrease substantially with aging time. Even so, in optimum cases, fluorometric detection of Ca2+ could be done over a 600 mu M range with a limit of detection of 3 mu M and with no interference from divalent ions such as Mg2+, Sr2+, Or Cd2+, indicating the viability of using sol-gel entrapped FAST proteins for the detection of Ca2+.