title: Fluorometric detection of ca(2+) based on an induced change in the conformation of sol-gel entrapped parvalbumin.
abstract: We report the development of a fluorometric detection strategy for Ca(2+) 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 Ca(2+) 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 Ca(2+). Entrapment also caused improvements in protein stability against chemical denaturants. However, entrapment caused the apparent affinity constant for binding of Ca(2+) to decrease substantially with aging time. Even so, in optimum cases, fluorometric detection of Ca(2+) could be done over a 600 μM range with a limit of detection of 3 μM and with no interference from divalent ions such as Mg(2+), Sr(2+), or Cd(2+), indicating the viability of using sol-gel entrapped FAST proteins for the detection of Ca(2+).
authors.affiliation: Department of Chemistry, Brock University, St. Catharines, Ontario, Canada, L2S 3A1.
authors.forename: K
authors.initials: K
authors.lastname: Flora
authors.forename: J D
authors.initials: JD
authors.lastname: Brennan