BODIPY-alpha-casein, a pH-independent protein substrate for protease assays using fluorescence polarization

BODIPY-alpha-casein is a new fluorescent protein substrate designed for fluorescence polarization studies to measure proteolytic activity at any pH over the range from pH 2 to 11. Kinetic protease assays in real-time were performed in 1 to 5 min using an FPM-1 fluorescence polarization instrument. A purified enzyme or bacterial culture was mixed with the BODIPY-alpha-casein in a buffer of an appropriate pH and the decrease 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 degrees 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 K at pH 7.4, and 2 mU for Streptomyces griseus alkaline protease at pH 11. Only 1-10 microliters of a growing culture was necessary to assay the protease activity of Porphyromonas gingivalis or Treponema denticola, oral bacteria that possess certain proteases on their surfaces. These assays have clinical applications, since certain pathogens use proteolytic activity as a virulence mechanism and differ from their nonpathogenic counterparts in this characteristic. Fluorescence polarization assays are simple, rapid, and reproducible.