BODIPY-ALPHA-CASEIN, A PH-INDEPENDENT PROTEIN SUBSTRATE FOR PROTEASE ASSAYS USING FLUORESCENCE POLARIZATION

BODIPY-alpha-casein is a new fluorescent protein substrate designed fo r fluorescence polarization studies to measure proteolytic activity at any pH over the range from pH 2 to 11. Kinetic protease assays in rea l-time were performed in 1 to 5 min using an FPM-1 fluorescence polari zation instrument. A purified enzyme or bacterial culture was mixed wi th the BODIPY-alpha-casein in a buffer of an appropriate pH and the de crease in fluorescence polarization was automatically recorded at 0.5- min intervals. The initial decrease in fluorescence polarization with time was dependent on protease concentration. In 3-min assays at 37 de grees C, the sensitivity of detection was 8 mU for pepsin at pH 2.0, 1 mU for papain at pH 6.0, 0.6 mU for proteinase It at pH 1.4, and 2 mU for Streptomyces griseus alkaline protease at pH 11. Only 1-10 mu l o f a growing culture was necessary to assay the protease activity of Po rphyromonas gingivalis or Treponema denticola, oral bacteria that poss ess certain proteases on their surfaces. These assays have clinical ap plications, since certain pathogens use proteolytic activity as a viru lence mechanism and differ hom their nonpathogenic counterparts in thi s characteristic. Fluorescence polarization assays are simple, rapid, and reproducible. (C) 1996 Academic Press Inc.