acetyl-biotin lotin ta vidin


Figure 9 Schematic of a FP-serine-threonine kinase assay. A fluorescein labeled peptide is thiophosphorylated (step 2) by incubation with ATPyS and kinase. Biotin is attached to the sulfur by incubation with iodoacetyl-LC-biotin (step 3), and the binding of streptavidin to thio-biotinyl peptide (step 4) produces a FP signal.

RNA of the RNA-DNA hybrid is hydrolyzed. The fl-RNA will no longer be attached to the avidin bound biotin-DNA strand and hence the FP signal will decrease with increasing enzyme activity in this assay. The FP signal decreased to 110 mP from 306 mP. There was no enzyme activity in the absence of reaction mixture (Mg2+ and Mn2+).

The time dependence of the FP signal with HIV-RNase H showed that 60 min incubation is optimal (Fig. 10A). RNA hydrolysis was measured as a function of RNase H concentration; the FP signal showed that 2 units of RNase H was

Figure 10 FP RNase H assay. (A) Time-course of RNase H reaction. (B) RNase H activity as a function of RNase H enzyme concentration. (C) Validation with random compounds (10 ||M). The distribution of the activity was normal with > 95% of compounds falling between 90 and 110%.

optimal (Fig. 10B). Evaluation of FP RNase H assay with a plate of random compounds at 10 |M (Fig. 10C) showed that the distribution of activity was very tight, with most of the activity between 90 and 110%. This suggests that the assay does not give spurious results and that synthetics can be tested at 10 |M or higher concentrations. The results with FP RNase H assay suggest that the signal is robust and reproducible and can be used for HTS.

Protease Assay. Standard FP protease assay kits using fluorescein labeled a-casein as substrate are available (Panvera and Molecular Probes). The FP signal of the relatively large fluorescein substrate is high and when cleaved by protease action produces smaller labeled fragments that have low FP signal. Fl-casein substrate, however, is not stable at lower pHs below 7. To assay various proteo-lytic enzymes by FP, a new pH-independent substrate BODIPY-a-casein was used between pH 2 to 11 [21]. A peptide substrate derivatized by biotinylation of a Y-aminobutyric acid modified amino terminus and labeled with 5-(4,6-dichlorotriazinyl)aminofluorescein at the carboxy terminus was used for cytomegalovirus protease [22]. The substrate binds to avidin and increases FP signal. Enzyme action cleaves the substrate generating a fluorescein containing peptide separated from biotin that will not be able to interact with avidin thereby resulting in the loss of FP signal.

FP Receptor Binding Assay. The bulk of HTS targets are receptors such as G-protein coupled receptors (GPCRs) and other membrane receptors, and nuclear receptors. For successful application of the FP method, a membrane receptor has to be expressed in a high copy number (~ 100,000) in each cell. The ligand has to have very high affinity for the receptor, and a substantial amount of fluorescent ligand has to bind to the receptor for FP assay. Previously, FP membrane receptor binding assays were limited to analytical methods using large amounts of membranes [19]. Recently, FP assay has been used for both peptide (vasopressin Vja and S-opioid) and nonpeptide (Pi-receptor and 5-HT3) receptors in 96-and 384-well formats [20].

The FP method has been successfully used for nuclear receptors [18]. FP assays amenable for HTS using purified nuclear receptor protein and a fluorescent ligand have been developed for estrogen receptors ERa and ERP [17] and other nuclear receptors. ERa and ERP FP assays have been developed using full length receptors and Fluormone™ ES1 (a natural fluorescent ligand) and ES2 (a fluores-cein labeled estradiol). The ligand is incubated with the receptor protein for 1 hr, and the FP signal is measured in a plate reader. The assay produced a robust signal of 200 mP. The FP ligand binding assay can be extended to all other nuclear receptors. The FP nuclear receptor and membrane receptor assays are discussed in greater detail elsewhere in this volume (Chap. 7).


FP is a simple and reasonably predictive technology and can be used for a variety of different assays. FP is a robust homogeneous assay that can be applied to higher density 384- and 1536-plates. Only one component of the assay is required to be labeled with a fluorophore and is suitable for small ligands (< 15 kDa). FP is insensitive to inner-filter effects. Since the FP signal is a ratiometric measurement it is less susceptible to quenching from colored compounds. However, autofluorescence compounds can interfere with a FP assay. Sometimes, propeller effects on the fluorescent tag may restrict the use of the FP assay. FP plate readers from several manufacturers are available in different price ranges.

C. FRET Assays

Fluorescence resonance energy transfer (FRET) is a phenomenon wherein excitation energy is transferred from a donor molecule to an acceptor molecule without emission of a photon. FRET has been used for measuring the distances between interacting molecules under physiological conditions with near angstrom resolution [25]. For FRET to occur, donor and acceptor molecules have to be in close proximity (10-100 A), excitation of the acceptor must overlap with the emission of donor, and donor and acceptor transition dipole orientations must be parallel.

The rate at which the energy is transferred from donor to acceptor is governed by the Forster equation [26]

The rate of Forster energy transfer kT is dependent on td, the fluorescence lifetime of donor (d) in the absence of acceptor molecules. R is the distance between donor and acceptor, and Ro is the Forster radius, the distance at which energy transfer is 50% efficient (typically between 10 and 50 A). Thus kT = 1/td when R0 = R. This equation applies to a single configuration of donor and acceptor, and distributions of donor and acceptor have to be averaged appropriately over distance and orientations. The Forster distance is dependent on k2, the dipole orientation factor between donor and acceptor; is the quantum yield of the donor in the absence of acceptor; n, the refractive index of the medium (which is 1.4 in aqueous solution), and J, the overlap integral between the donor and acceptor. J = JFd(X) ■ Ea(X) -X4 • dX where Fd(X) is the peak-normalized fluorescence intensity of donor and eA is the molar absorption coefficient of the acceptor at wavelength X [3,25-27].

In most cases the donor and acceptor dyes are different, and FRET can be detected as the quenching of donor fluorescence by the acceptor or appearance of the sensitized fluorescence of the acceptor. Typical R0 for the donor-acceptor pair of fluorescein and tetramethylrhodamine is 55 A, 5-(2-aminoethylamino) naphthalene-1-sulfonic acid (EDANS) and 4-(4-dimethylaminophenylazo)ben-zoic acid (DABCYL) is about 33 A, and 5-(2-iodo-acetyl aminoethylamino) naphthalene-1-sulfonic acid (IAEDANS) and fluorescein is 46 A [2,5,25,26].

1. FRET Applications

FRET applications include quench and quench relaxation assays.

Protease Assay. In a peptide containing C-terminus EDANS and N-terminus DABCYL, the fluorescence of EDNAS is quenched by the acceptor DABCYL. Cleavage of this internally quenched fluorogenic substrate leads to an enormous increase in fluorescence intensity as the donor is separated from the acceptor. The fluorescence intensity is proportional to the hydrolysis of the substrate. Thus the protease activity can be monitored by the fluorescence intensity. Several protease assays (e.g., trypsin, HIV-1 protease, renin, hepatitis C

virus protease, human cytomegalovirus protease) using FRET have been described [27].

Time-resolved fluorescence (TRF) is based on the long lifetime properties of lanthanides, europium (Eu), samarium (Sm), terbium (Tb), and dysprosium (Dy). Homogeneous time-resolved fluorescence (HTRF), as the name implies, is a homogeneous assay method that uses fluorescence resonance energy transfer from europium cryptate (EuK) donor to an acceptor fluorophore provided they are at a distance less than 10 nm from one another and that the emission energy of donor overlaps with the excitation of the acceptor. HTRF™ technology is a trademark of Packard and CIS/Bio, and this phenomenon is variously called time-resolved fluorescence resonance energy transfer (TR-FRET) or Lance™ (Lanthanide Che-late Excitation) technology, a trademark of Wallac. For the energy transfer, the donor and acceptor have to be brought into close proximity. The fluorescence lifetime of most of the fluorescent compounds is very short (typically a few nanoseconds). The interference from assay components, microtiter plates, light scatter, and biological samples and compounds is short-lived fluorescence (prompt fluorescence). To avoid this interference, lanthanide chelates with long lifetimes, 100-1000 |s (no polarization), are used as fluorescence energy donors. The common acceptor fluorophores used are modified allophycocyanine, a phycobilipro-tein from red algae (XL665, Cy 5), fluorescein, or tetramethylrhodamine. The long-lived fluorescence of lanthanide due to Forster dipole-dipole energy transfer to the acceptor results in a long-lived fluorescence of the acceptor. The energy transfer emission has a decay time directly proportional to donor decay time and inversely to the distance between acceptor and donor. Time resolution in HTRF/ Lance reduces scattering and prompt decay background interference from shortlived fluorescence due to delay in the time of measurements of fluorescence.

The rare-earth elements, Eu+3, Su3+, Tb3+, and Dy3+, are poor fluorophores by themselves, and to measure the lanthanide fluorescence, the lanthanides have to be complexed. Europium fluorescence is protected from decay by conversion to the macropolycyclic compound europium cryptate [Eu]K to enhance their fluorescence, and cryptate protects from fluorescence quenching (Fig. 11) [26,2830]. [Eu]K has convenient linker arms to complex covalently with peptides, proteins, and nucleic acids. Also, selected generic reagents, streptavidin, biotin, WGA, ConA, protein A, anti-DNP antibody labeled with XL665 and biotin, streptavidin, and antiphosphotyrosine antibody labeled with [Eu]K are available from Packard. New chelates of Eu3+ and Tb3+ are available from Advant (a joint venture between Xenova and Wallac) that are used in Lance technology (Fig. 11) [30]. The fluorescence resonance energy is transferred from Eu chelates emitting

Was this article helpful?

0 0

Post a comment