Lj. Jones et al., QUENCHED BODIPY DYE-LABELED CASEIN SUBSTRATES FOR THE ASSAY OF PROTEASE ACTIVITY BY DIRECT FLUORESCENCE MEASUREMENT, Analytical biochemistry, 251(2), 1997, pp. 144-152
We have prepared casein conjugates of two BODIPY dyes for use as fluor
ogenic protease substrates in homogeneous assays. Both conjugates are
labeled to such an extent that the dyes ave efficiently quenched in th
e protein, yielding virtually nonfluorescent substrate molecules. Thes
e fluorogenic substrates release highly fluorescent BODIPY dye-labeled
peptides upon protease digestion, with fluorescence increases proport
ional to enzyme activity. These quenched substrates are suitable for t
he continuous assay of enzymatic activity using standard fluorometers,
filter fluorometers, or fluorescence microplate readers using either
fluorescein excitation and emission wavelengths to measure BODIPY FL c
asein hydrolysis or Texas Red wavelengths to detect proteolysis of BOD
IPY TR-X casein. Most current techniques for detecting protease activi
ty, such as the fluorescein thiocarbamoyl casein (FTC-casein) protease
assay, require extensive manipulation, including separation steps, an
d are therefore labor intensive and error-prone. In comparison, we fou
nd the BODIPY dye-labeled casein protease assays to be simple and prec
ise and to have greater sensitivity and a broader dynamic range of det
ection than the FTC-casein assay. We were able to sensitively detect t
he activities of a wide variety of enzymes with these new substrates,
including serine, acid, sulfhydryl, and metalloproteases. We also foun
d the assay suitable for quantitating protease inhibitor concentration
s and for real-time analysis of proteolysis. (C) 1997 Academic Press.