Fluorescence dynamics of N-terminal Trp-Trp residues in polypeptide: intrinsic indicators for monitoring pH

pH plays a vital role in various cellular activities, and real-time observation of the intracellular pH through a pH indicator is very important for studying many physiological processes. In this paper, we studied the pH response of Trp-Trp dipeptide and its derivatives (NATrp2Me, NBTrp2 and Trp2Me) by steady-state and time-resolved fluorescence spectroscopy. Both the fluorescence intensities and lifetimes of Trp-Trp dipeptide as well as Trp2Me were functions of pH in the physiological range from 5.5 to 9.0. However, NATrp2Me and NBTrp2 showed no difference. The exposed amino was found to be pivotal for its pH dependence. Moreover, an artificially synthesized tetrapeptide (Trp-Trp-Ala-Ser) confirmed the pH sensitivity of N-terminal Trp-Trp residues. The pH values could be quantitatively determined from the fluorescence intensities and lifetimes of the N-terminal Trp-Trp residue. Thus, the N-terminal Trp-Trp residues may be fused into the polypeptides/proteins to serve as an intrinsic pH indicator in fluorescence spectroscopy and imaging.