Protonation kinetics of GFP and FITC investigated by FCS - aspects of the use of fluorescent indicators for measuring pH

In this work, the protonation kinetics of fluorescein isothiocyanate (FITC) and a F64L S65T variant (BioST) of green fluorescent protein (GFP) has been investigated using fluorescence correlation spectroscopy (FCS). It is shown that buffer effects, in general, must be considered when using fluorescent species as pH-probes and that the pH behaviour of BioST, in contrast to that of FITC, cannot be modelled as a single-step reaction. The outer beta-barrel structure of the GFP molecule not only slows the exchange of protons within the microenvironment of the chromophore-bearing unit but also apparently prevents buffer molecules and even protons from directly reaching the fluorescently active residues in the interior of the barrel. This would mean that the active residues are only affected indirectly, where changes in fluorescence are a secondary effect mediated intramolecularly, following a proton exchange at some exterior part of the molecule. (C) 1999 Elsevier Science B.V. All rights reserved.