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.