A novel methodology for the investigation of intracellular proteolytic processing in intact cells.

Taking advantage of the unique spectral properties of the fluorescent probe FL-Bodipy, we have developed a new methodology to study processing of exogenous proteins in intact cells. FL-Bodipy was conjugated to bovine serum albumin (BSA) at a molar ratio of 29 probe molecules to 1 albumin equivalent. The resulting conjugate was 98% self-quenched due to fluorescence resonance energy transfer (homotransfer) between neighboring Bodipy molecules. In vitro proteolytic cleavage of the conjugate led to relaxation of self-quenching and to a significant increase in fluorescence. Flow cytometry and fluorescence microscopy indicated that Bodipy-labeled BSA was readily internalized by J774 macrophages and accumulated in intracellular compartments. The kinetics of intracellular degradation of Bodipy-BSA was linear for up to 2 hours and was completely inhibited by a combination of protease inhibitors. Future applications of the methodology reported here may comprise studies of antigen processing and presentation, as well as the investigation of cellular events related to processing and disassembly of intracellular pathogens such as parasites, bacteria and viruses.