MULTIPARAMETER FLOW CYTOMETRIC ANALYSIS OF A PH SENSITIVE FORMYL PEPTIDE WITH APPLICATION TO RECEPTOR STRUCTURE AND PROCESSING KINETICS

Environmentally sensitive molecules have many potential cellular appli cations. We have investigated the utility of a pH sensitive ligand for the formyl peptide receptor, CHO-Met-Leu-Phe-Phe-Lys (SNAFL)-OH (SNAF L-seminaphthonuorescein), because in previous studies (Fay et al.: Bio chemistry 30:5066-5075, 1991) protonation has been used to explain the quenching when the fluoresceinated formyl pentapeptide ligand binds t o this receptor. Moreover, acidification in intracellular compartments is a general mechanism occurring in cells during processing of ligand -receptor complexes. Because the protonated form of SNAFL is excited a t 488 nm with emission at 530 nm and the unprotonated form is excited at 568 nm with emission at 650 nm, the ratio of protonated and unproto nated forms can be examined by multiparameter flow cytometry. We found that the receptor-bound Ligand is sensitive to both the extracellular and intracellular pH. There is a small increase in the pK(a) of the l igand upon binding to the receptor consistent with protonation in the binding pocket. Once internalized, spectral changes in the probe consi stent with acidification and ligand dissociation from the receptor are observed. (C) 1994 Wiley-Liss, Inc.