ANALYSIS OF THE INTRACELLULAR PROCESSING OF PROTEINS - APPLICATION OFFLUORESCENCE POLARIZATION AND A NOVEL FLUORESCENT-PROBE

Previous studies indicated that fluorescein derivatized bovine serum a lbumin was an ideal probe to monitor the time-dependent kinetics of an tigen processing in the murine macrophage cell line J774, Whereas prev ious work focused on fluorescence intensity measurements, the present study relied on fluorescence polarization to dissect the local environ ment of the fluorescent hapten-protein within the endocytic system of the cell. A steady increase in both fluorescence intensity and fluores cence polarization of the cell population was detected for the first 1 00 min, However, at 100 min, a plateau in both fluorescence intensity and polarization was observed and was followed by a decrease in fluore scence polarization and a corresponding increase in fluorescence inten sity. Western blot analyses revealed that the decrease in fluorescence polarization was due to proteolytic degradation of the probe within t he cell. Using a combination of in vitro experiments and an additional fluorescent probe, it was determined that the initial increase in flu orescence polarization was due to movement of the probe through a pH g radient within the cell, suggestive of transport through the endocytic system, By combining fluorescence polarization, flow cytometry, and a unique fluorescent enhancement substrate, these studies represented a novel approach for monitoring intracellular trafficking and processin g of proteins within macrophages. (C) 1997 Wiley-Liss, Inc.