Cleavage, fluorescence energy transfer

The recent investigation of Mb adsorption on PDMS (above) has probed the conformation and orientation of a layer of protein adsorbed on a hydrophobic surface. Our present goal is further characterization of the conformation and orientation of Mb on PDMS. Two techniques, fluorescence energy transfer and proteolytic enzyme cleavage, can be used to achieve this goal. 

A peculiar effect was observed in the decomposition of 19 a with anthracene as fluorescer when oxygen was carefully removed from the solutions an increase of the chemiluminescence decay rate and of the dioxetane cleavage resulted. It was suggested that this was due to a catalytic effect of triplet anthracene (formed by energy transfer from triplet formate) on the decomposition of the dioxetane. When oxygen is present, triplet anthracene is quenched. Whether such a catalytic effect of triplet anthracene or similar compounds on dioxetane cleavage actually exists has not yet been fully established positive effects were observed by M. M. Rauhut and coworkers 24> in oxalate chemiluminescence and by S. Mazur and C. S. Foote 80> in the chemiluminescent decomposition of tetramethoxy-dioxetane, where zinc tetraphenylporphy-rin seems to exert a catalytic effect. However, the decomposition of trimethyl dioxetane exhibits no fluorescer catalysis 78h... 

Another approach of energy transfer-based probe has been demonstrated for a-Amylase sensing/98 In this case amylose was doubly labeled, by fluorescein derivative (donor) and Procion Red MX8B (acceptor). As a-Amylase catalyzes the cleavage of the amylose into smaller units, the average distance between fluorescein and Procion Red increases, which reduces the degree of quenching. The rate of increase in fluorescence intensity is proportional to ee-amylase activity. 

In 1994 and 1995, two crystal structures of hammerhead ribozymes [31,32] and a structural analysis based on fluorescence resonance energy transfer studies [41] were published. In case of the crystal structure analyses, both ribozyme variants contained certain modifications that had been introduced to avoid self-cleavage [31,32]. In one case a DNA-analog of the substrate oligonucleotide was used [31], in the other case the all-RNA substrate contained a I -O-CR modification at the attacking 2 -OH group to avoid cleavage in the crystal [32] for reviews see [8,42,43]. 

The emission spectrum is similar to fluorescence of 9,10-diphenyl-l,4-tiinethoxy anthracene although it is not excited directly. The decomposition of the endoperoxide to (1) and (3) is the excitation producing step in wtcatalyzed cleavage and (2) is activated by energy transfer. The formation of 102 by elimination reaction is a very minor reaction step. 

FIGURE 2.5 FRET quench readout principle. In the intact peptidic substrate (amino acids symbolized by X, Y and Z) labeled with a fluorophore (dye) and a quencher (Q) at the opposite sites of the scissile bond, the fluorescence emission is quenched through an energy transfer (ET) from the fluorophore to the quencher. After cleavage of the substrate between amino acids X and Y by a protease, the energy transfer is disrupted and an increase in fluorescence emission is observed (light gray arrow). An increase of fluorescence intensity over time dependent on the enzymatic velocity is recorded. 

T.L. Chew, W.A. Wolf, P.J. Gallagher, F. Matsumura, R.L. Chisholm,A fluorescent resonant energy transfer-based biosensor reveals transient and regional myosin light chain kinase activation in lamella and cleavage furrows. J. Cell Biol. 156(3), 543-553 (2002)... 

An example of this technology is the HIV protease assay shown in Fig. 9, which was published by Wang and co-workers [67]. The peptide substrate is labeled at the amino terminus with EDANS (5-((2 -aminoethyl)amino)naphthalene-l-sulfonic acid) as a donor fluorophore and at the carboxyl terminus with DABCYL (4-((4 -(di-methylamino)phenyl)azo)benzoic acid) as the acceptor chromophore. In the intact peptide, fluorescence resonance energy transfer (FRET) from EDANS to DABCYL results in quenching of the EDANS fluorescence. On cleavage of the peptide by HIV protease, the fluorescence of EDANS is restored. 

Fio. 5. (A) Use of FRET technique for measuring HIV protease activity. The polypeptide was Arg-Glu(EDANS)-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Lys(DABCYL)-Arg. Because a simple system which could be commercialized was desired (fluorescence means HIV activity, no fluorescence means no HIV activity), the nonfluorescent acceptor was beneficial because it does not contribute signal after cleavage. This system also has the attribute that detailed information about the dyes and their energy transfer properties are not needed. (B) Good overlap between donor emission (maximum at 490 nm, excitation at 340 nm) and acceptor absorbance (maximum of 28,000 cm" ). (From Molecular Probes Catalog. )... 

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