Use of nonradiative decays of extrinsic fluorophores as structural and dynamical probes in protein environments: Fluorescence quenching

In this work a combined pulsed-laser, time-resolved photoacoustic calorimetry (PAC) and fluorescence study is presented on two widely used covalent protein probes, fluorescein-5-isothiocyanate (FITC) and 6-acryloyl-2-dimethylaminonaphtalene (acrylodan). Three proteins that contain a single free thiol, namely carbonic anhydrase, bovine serum albumin (BSA) and papain, have been selectively labelled with FITC and acrylodan, and their fluorescence emission was quenched with KI. Nonradiative decays of the excited states of FITC are used to complement the information usually obtained by monitoring the quenching of fluorescence emssion. Data analysis evidences the dependence of the nonradiative quenching constants on the exposure of the dye to the solvent, and shows the involvement of a triplet state of FITC in the non radiative deexcitation. The shielding of the binding sites from the solvent is demonstrated also by the fluorescence emission of acrylodan and by the Stern-Volmer analysis of fluorescence quenching by KI. From photoacoustic data, an estimate of the fluorescent quantum yield of bound FITC is obtained. This work demonstrates the complete equivalence of quenching data obtained by fluorescence and photoacoustics measurements and shows that this combined approach allows a better control of the photophysics of the dyes involved in the quenching process