ANALYTICAL BIOCHEMISTRY 247, 83–88 (1997) ARTICLE NO. AB972047 Measurement of Specific Protease Activity Utilizing Fluorescence Polarization Leanna M. Levine,* Marshall L. Michener,* Mihaly V. Toth,† and Barry C. Holwerda† *Monsanto Corporate Research and †Searle Research and Development, 700 Chesterfield Parkway North, St. Louis, Missouri 63198 Received October 14, 1996 substrates offers an alternative method that avoids in- A fluorescence polarization assay was designed to terference, but disposal and safety concerns make this measure proteolytic cleavage of a specific peptide sub- approach less attractive. strate for human cytomegalovirus protease. The pep- We present an assay for site-specific proteolytic ac- tide substrate was derivatized by biotinylation of a tivity utilizing fluorescence polarization. The substrate g-aminobutyric acid-modified amino-terminus and la- is a protease-specific peptide derivatized by biotinyla- beled with 5-(4,6-dichlorotriazinyl)aminofluorescein tion of the amino-terminus and coupling of a fluoro- at the carboxy-terminus. Incubation of this substrate phore at the carboxy-terminal end. Proteolytic activity with recombinant human cytomegalovirus protease was quantitated from the total fluorescence polariza- and subsequent addition of egg white avidin produced tion of the mixture of cleaved and uncleaved peptide a polarization signal that was proportional to the rela- after incubation with the protease. Since the fluores- tive amounts of cleaved and uncleaved substrate. The cence polarization value is the ratio of orthogonal fluo- uncleaved substrate produced a high polarization rescence intensities, it is not sensitive to absorptive value upon binding to avidin, whereas the cleaved, interferants. We demonstrate the assay robustness to low-molecular-weight fluorescently tagged peptide absorptive interferants by measuring the polarization that cannot bind to avidin produced a low polarization of a constant concentration of biotin – fluorescein, bio- value. The inhibitory activity of a 3,4-dichloroisocoum- tin, and avidin in the presence of increasing concentra- arin against the protease was evaluated by comparing tions of dyes that absorb where fluorescein either ab- the change in polarization with a noninhibited con- sorbs or emits. trol. The fluorescence polarization protease assay does not suffer from interference due to the presence of absorptive interferants making this a convenient, ho- MATERIALS AND METHODS mogenous assay for high throughput screening.  1997 Chemicals and Reagents Academic Press Avidin, biotin – fluorescein, and 5-(4,6-dichlorotriazi- nyl)aminofluorescein (DTAF) 1 were purchased from Molecular Probes (Eugene, OR). Bovine serum albumin The activity of proteases recognizing specific cleav- (fraction V; BSA), 3-[(3-chloramidopropyl)-dimethy- age sites is usually measured using substrates con- lammonio]-1-propane sulfonate (Chaps), g-aminobu- sisting of a specific peptide modified by the addition of tyric acid (Abu), 3,4-dichloroisocoumarin, mordant blue latently colorimetric or fluorescent moieties (1 – 6). The 3, eosin B, and biotin were purchased from Sigma (St. hydrolysis products of these synthetic substrates pos- Louis, MO). All other chemicals were analytical grade. sess spectral features that enable quantitative deter- Buffers were stored at 4°C after preparation in ultra- mination of substrate cleavage. Substrates of this type pure water (Millipore Milli-Q) and further filtered are widely used in the characterization of many differ- through 0.2-mm filters. Recombinant human cytomega- ent proteases. However, evaluation of potential inhibi- tors in complex mixtures such as natural products ex- 1 Abbreviations used: DTAF, 5-(4,6-dichlorotriazinyl)aminofluor- tracts can be severely limited because these mixtures escein; BSA, bovine serum albumin; Chaps, 3-[(3-chloramidopropyl)- often contain other components that interfere with dimethylammonio]-1-propane sulfonate; PBS, phosphate-buffered saline; HCMV, human cytomegalovirus; Abu, g-aminobutyric acid. fluorescent quantitation. The use of radioactive peptide 83 0003-2697/97 $25.00 Copyright  1997 by Academic Press All rights of reproduction in any form reserved.