54) Fluorescence Polarizationassays Foreign Patent Documents of Enzymes and Substrates

Fluorescent-labeled substrates are provided for fluorescence 21 Appl. No.: 566,390 polarization assays of enzymes. These Substrates are pro 1-1. teins labeled with derivatives of BODIPY(R), 44-difluoro 22 Filed: Dec. 1, 1995 5,7-dimethyl-4-bora-3a,4a-diaza-S-indacene. The 51) Int. Cl. ........................ G01N 33/573; G01N 33/53; BODIPYOR) fluorescent tag of the present invention is pH C12O 1/00 independent, and can be used over a pH range of from about 52 U.S. Cl. .............................. 435/7.4; 435/4; 435/7.72; 2 to about 11. Thus one can assay, in real time, enzymes with 435/15; 435/24; 548/126; 548/405 pH maxima at pH below 7 using fluorescence polarization 58 Field of Search ................................ 435/4, 7.4, 7.72, methodology, which could not be done with fluorescein 435/7.9, 15, 24; 548/126, 405 derivatives. Different enzymes can be compared using the same BODIPYOR conjugate by merely changing the buffer 56) References Cited System which changes the pH conditions.