BODIPY–α-Casein, a pH-Independent Protein Substrate for Protease Assays Using Fluorescence Polarization
✍ Scribed by Sylvia Z. Schade; Michael E. Jolley; Brian J. Sarauer; Lloyd G. Simonson
- Publisher
- Elsevier Science
- Year
- 1996
- Tongue
- English
- Weight
- 123 KB
- Volume
- 243
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
the molecular mass of the labeled molecule, the greater BODIPY-a-casein is a new fluorescent protein subthe motion (rotation) will be and the lower the emitted strate designed for fluorescence polarization studies fluorescence polarization value. When a relatively large to measure proteolytic activity at any pH over the fluorescent molecule, such as labeled casein, is placed range from pH 2 to 11. Kinetic protease assays in realalone in solution, its fluorescence polarization value is time were performed in 1 to 5 min using an FPM-1 relatively high and remains high. If a small quantity fluorescence polarization instrument. A purified enof protease is introduced, the fluorescence polarization zyme or bacterial culture was mixed with the BOdrops with time. As the protein is cleaved, labeled frag-DIPY-a-casein in a buffer of an appropriate pH and ments are produced which rotate more quickly and the the decrease in fluorescence polarization was autoplane polarized light is depolarized to a greater degree matically recorded at 0.5-min intervals. The initial deas it passes through the solution. Thus, the change crease in fluorescence polarization with time was de-(decrease) in fluorescence polarization value is a meapendent on protease concentration. In 3-min assays at sure of proteolytic activity.
37ЊC, the sensitivity of detection was 8 mU for pepsin
Protease assays using a fluorescein-labeled protein at pH 2.0, 1 mU for papain at pH 6.0, 0.6 mU for proteinsubstrate, FTC 3 -a-casein, were developed by Spencer ase K at pH 7.4, and 2 mU for Streptomyces griseus et al. (1) and by Maeda (2) for use in fluorescence polaralkaline protease at pH 11. Only 1-10 ml of a growing ization instrumentation. Fluorescence polarization has culture was necessary to assay the protease activity also been used for immunoassays (3) and applied to the of Porphyromonas gingivalis or Treponema denticola, determination of drug levels in human plasma (4). oral bacteria that possess certain proteases on their Early instruments were bulky and expensive but resurfaces. These assays have clinical applications, since cently more sensitive, compact, and convenient instrucertain pathogens use proteolytic activity as a virulence mechanism and differ from their nonpathogenic ments have become available. Instruments are availcounterparts in this characteristic. Fluorescence po-able which record kinetic measurements of fluorescence larization assays are simple, rapid, and reproducible. polarization at constant temperature. Using one of