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 calo rimetry (PAC) and fluorescence study is presented on two widely used c ovalent protein probes, fluorescein-5-isothiocyanate (FITC) and 6-acry loyl-2-dimethylaminonaphtalene (acrylodan). Three proteins that contai n a single free thiol, namely carbonic anhydrase, bovine serum albumin (BSA) and papain, have been selectively labelled with FITC and acrylo dan, and their fluorescence emission was quenched with KI. Nonradiativ e decays of the excited states of FITC are used to complement the info rmation usually obtained by monitoring the quenching of fluorescence e mission. Data analysis evidences the dependence of the nonradiative qu enching constants on the exposure of the dye to the solvent, and shows the involvement of a triplet state of FITC in the non radiative deexc itation. The shielding of the binding sites from the solvent is demons trated also by the fluorescence emission of acrylodan and by the Stern -Volmer analysis of fluorescence quenching by KI. From photoacoustic d ata, an estimate of the fluorescent quantum yield of bound FITC is obt ained. This work demonstrates the complete equivalence of quenching da ta obtained by fluorescence and photoacoustics measurements and shows that this combined approach allows a better control of the photophysic s of the dyes involved in the quenching process.