Fluorescent oligopeptide substrates for kinetic characterization of the specificity of Astacus protease (original) (raw)
Related papers
Analytical Biochemistry, 2007
Fluorescence protease assays were investigated with peptide substrates containing a 2,3-diazabicyclo[2.2.2]oct-2-ene-labeled asparagine (Dbo) as a fluorescent amino acid. The special characteristic of the fluorophore Dbo is its exceedingly long fluorescence lifetime (ca. 300 ns in water under air), which allows the use of nanosecond time-resolved fluorescence (Nano-TRF) detection to efficiently suppress shorter-lived background emission. In addition, the natural amino acids tryptophan and tyrosine can be employed as intramolecular fluorescence quenchers, which facilitates substrate design. Fourteen synthetic peptide substrates (composed of 2-19 amino acids) and five enzymes (trypsin, pepsin, carboxypeptidase A, leucine aminopeptidase, and chymotrypsin) were investigated and, in all 28 examined combinations, enzymatic activity was detected by monitoring the increase in steady state fluorescence with time and determining the reaction rates as k cat /K m values, which ranged from 0.2 to 80 · 10 6 M À1 min À1 . The results suggest an excellent compatibility of the very small and hydrophilic fluorescent probe Dbo with solid-phase peptide synthesis and the investigated proteases. For all 14 peptides the fluorescence lifetimes before and after enzymatic cleavage were measured and Nano-TRF measurements were performed in 384-well microplates. The fluorescence lifetimes of the different peptides provide the basis for the rational design of Dbo-based fluorescent substrates for protease assays. Measurements in Nano-TRF mode revealed, in addition to efficient suppression of background fluorescence, an increased differentiation between cleaved and uncleaved substrate. The Dbo-based assays can be adapted for high-throughput screening.
Characterization of bacterial proteases with a panel of fluorescent peptide substrates
Analytical Biochemistry, 2009
PS-SCL, posi tional scan ning syn thetic com bi na to rial library; FRET, fluo res cence res o nance energy trans fer; AMC, 7-amino-4-meth yl coum a rin; AE BSF, 4-(2-ami no ethyl)ben zene sul fo nyl fluo ride hydro chlo ride; E-64, N-(transep ox ysucci nyl)-l-leu cine 4-gua nid in obu tyla mide; OPT, 1,10-phe nan thro line; PIC, pro te ase inhib i tor cock tail; DMSO, dimethyl sulf ox ide; PBS, phos phate-buf fered saline. Characterization of bacterial proteases with a panel of fluorescent peptide substrates
Scientific Reports, 2017
Internally quenched fluorescent (IQF) peptide substrates originating from FRET (Förster Resonance Energy Transfer) are powerful tool for examining the activity and specificity of proteases, and a variety of donor/acceptor pairs are extensively used to design individual substrates and combinatorial libraries. We developed a highly sensitive and adaptable donor/acceptor pair that can be used to investigate the substrate specificity of cysteine proteases, serine proteases and metalloproteinases. This novel pair comprises 7-amino-4-carbamoylmethylcoumarin (ACC) as the fluorophore and 2,4-dinitrophenyl-lysine (Lys(DNP)) as the quencher. Using caspase-3, caspase-7, caspase-8, neutrophil elastase, legumain, and two matrix metalloproteinases (MMP2 and MMP9), we demonstrated that substrates containing ACC/ Lys(DNP) exhibit 7 to 10 times higher sensitivity than conventional 7-methoxy-coumarin-4-yl acetic acid (MCA)/Lys(DNP) substrates; thus, substantially lower amounts of substrate and enzyme can be used for each assay. We therefore propose that the ACC/Lys(DNP) pair can be considered a novel and sensitive scaffold for designing substrates for any group of endopeptidases. We further demonstrate that IQF substrates containing unnatural amino acids can be used to investigate protease activities/ specificities for peptides containing post-translationally modified amino acids. Finally, we used IQF substrates to re-investigate the P1-Asp characteristic of caspases, thus demonstrating that some human caspases can also hydrolyze substrates after glutamic acid. The irreversible peptide bond hydrolysis of proteins and polypeptides is the most conserved post-translational modification occurring in biochemical pathways in all living organisms 1,2. This reaction is catalyzed by proteases, which specifically recognize protein targets to control numerous significant biological processes, including cell survival and cell death and the immune response to various pathogens 3. The selectivity of proteases for binding and subsequently hydrolyzing a selected group of peptides or proteins is termed substrate specificity 4,5. The increasing number of chemical tools for substrate specificity profiling allows the development of new, more efficient and more selective small molecule substrates 6,7 , inhibitors 8 , and chemical probes 9 , which are useful for the determination of protease activity and the dissection of their physiological functions. Internally quenched fluorescent (IQF) peptide substrates constitute a convenient tool for examining the specificity of the largest group of proteases-endopeptidases 10. These substrates contain a paired fluorophore (donor) and quencher (acceptor), which are located on opposite sides of the scissile peptide bond 11,12. If the fluorophore
A Method for Rapid Protease Substrate Evaluation and Optimization
Combinatorial Chemistry & High Throughput Screening, 2006
We have developed a high throughput assay for the measurement of protease activity in solution. This technology will accelerate research in functional proteomics and enable biologists to streamline protease substrate evaluation and optimization. The peptide sequences that serve as protease substrates in this assay are labeled on the carboxy terminus with a biotin moiety and a fluorescent tag is attached to the amino terminus. Protease cleavage causes the biotin containing fragment to be detached from the labeled peptide fragment. Following the protease treatment, all biotin containing species (uncleaved substrates and the cleaved carboxy terminal fragment of the substrate) are removed by incubation with streptavidin beads. The cleaved fluorescently labeled amino terminal part of the substrate remains in solution. The measured fluorescence intensity of the solution is directly proportional to the activity of the protease. This assay was validated using trypsin, chymotrypsin, caspase-3, subtilisin-A, enterokinase and tobacco etch virus protease.
Journal of Combinatorial Chemistry, 1999
To map the substrate specificity of cysteine proteases, two combinatorial peptide libraries were synthesized and screened using the archetypal protease, papain. The use of PEGA resin as the solid support for library synthesis facilitated the application of an on-resin fluorescence-quenched assay. Results from the screening of library 2 indicated a preference for Pro or Val in the S 3 subsite and hydrophobic residues in S 2 ; the most prevalent residue not being Phe but Val. The S 1 subsite exhibited a dual specificity for both small, nonpolar residues, Ala or Gly, as well as larger, Gln, and charged residues, Arg. Small residues predominated in the S 1 ′-S 4 ′ subsites. Active peptides from the libraries and variations thereof were resynthesized and their kinetics of hydrolysis by papain assessed in solution phase assays. Generally, there was a good correlation between the extent of substrate cleavage on solid phase and the k cat /K M 's obtained in solution phase assays. Several good substrates for papain were obtained, the best substrates being Y(NO 2 )PMPPLCTSMK(Abz) (k cat /K M ) 2109 (mM s) -1 ), Y(NO 2 )PYAVQSPQK(Abz) (k cat /K M ) 1524 (mM s) -1 ), and Y(NO 2 )PVLRQQRSK-(Abz) (k cat /K M ) 1450 (mM s) -1 ). These results were interpreted in structural terms by the use of molecular dynamics (MD). These MD calculations indicated two different modes for the binding of substrates in the narrow enzyme cleft. † 1X, set as standards100 times dilution of enzyme (8.34 µM); 20X, 20 times dilution of 1X enzyme solution; Abz, 2-
Measurement of Specific Protease Activity Utilizing Fluorescence Polarization
Analytical Biochemistry, 1997
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 subapproach less attractive. strate for human cytomegalovirus protease. The pep-We present an assay for site-specific proteolytic actide substrate was derivatized by biotinylation of a tivity utilizing fluorescence polarization. The substrate g-aminobutyric acid-modified amino-terminus and lais a protease-specific peptide derivatized by biotinylabeled with 5-(4,6-dichlorotriazinyl)aminofluorescein tion of the amino-terminus and coupling of a fluoroat 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 polarizaand subsequent addition of egg white avidin produced tion of the mixture of cleaved and uncleaved peptide a polarization signal that was proportional to the relaafter incubation with the protease. Since the fluorestive amounts of cleaved and uncleaved substrate. The cence polarization value is the ratio of orthogonal fluouncleaved 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, biovalue. The inhibitory activity of a 3,4-dichloroisocoumtin, and avidin in the presence of increasing concentraarin against the protease was evaluated by comparing tions of dyes that absorb where fluorescein either abthe change in polarization with a noninhibited consorbs 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-dichlorotriazinyl)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)-dimethyage sites is usually measured using substrates conlammonio]-1-propane sulfonate (Chaps), g-aminobusisting 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 4ЊC after preparation in ultramination of substrate cleavage. Substrates of this type pure water (Millipore Milli-Q) and further filtered are widely used in the characterization of many differthrough 0.2-mm filters. Recombinant human cytomegaent proteases. However, evaluation of potential inhibitors in complex mixtures such as natural products ex-1 Abbreviations used: DTAF, 5-(4,6-dichlorotriazinyl)aminofluortracts 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.
Fluorogenic substrates for proteases based on intramolecular fluorescence energy transfer (IFETS)
Journal of Biochemical and Biophysical Methods, 1996
A prospective class of intramolecular fluorescence energy transfer substrates (IFETS) is described. In contrast to the known chromogenic and fluorogenic substrates that are used widely in the scientific and medical research, IFETS allow to control the enzymatic cleavage at any point of the peptide chain and thus permit simultaneous studies of enzymes of different specificity. We discuss the main types of donor and acceptor, their advantages and drawbacks and the effectiveness of exited-state energy transfer between them. High sensitivity and selectivity of IFETS in the assay of proteinases was demonstrated. They prove to be a very useful and very promising instrument for enzymology and medicine.
International journal of biological macromolecules, 2018
Circular dichroism (CD) and fluorescence spectroscopy (FS) were used to monitor the pH-dependent conformational and structural stability changes induced by temperature and UV light on the protease from Aspergillus tamarii URM4634 at different pH values. The formation of photoproducts, such as N-formylkynurenine, dityrosine and kynurenine, were monitored with FS. The pH-dependent melting temperatures (T) were determined using CD and FS from 20 to 90 °C. Conformational changes were correlated with the pH-dependent biochemical activities. CD revealed that the protease is rich in α-helices. Thermal denaturation was irreversible at all pH range and displayed T values from 42.8 to 67.8 °C (CD) and from 38 to 60.3 °C (FS), which the highest T was observed at pH 6. The light and temperature induced to the formation of photoproducts was more intense at high pH value. Despite the biochemical data shows optimum pH 9, the highest stability was at pH 6, maintaining 100% of activity after 24 h. T...
Bioorganic & Medicinal Chemistry Letters, 2003
The water soluble fluorescein-based ligand 1 forms a non-fluorescent complex with Cu 2+ . This complex serves as a fluorescent sensor for amino acids in the 10 À3 M concentration range. Since the signal response is very fast, the sensor can be used to detect the hydrolytic activity of various proteases (trypsin, chymotrypsin, subtilisin) on bovine serum albumin as a whole protein substrate, and more generally to follow reactions releasing or removing free amino acids, in real time. #
Journal of Medicinal Chemistry, 1992
A general scheme for obtaining a fluorescent donor/acceptor peptide substrate via solid-phase synthesis methodology is presented. The key feature of this method is the design of a glutamic acid derivative that has been modified on the carboxyl side chain with a 5-[(2'-aminoethyl)aminolnaphthelenesulfonic acid (EDANS) to create a fluorescent donor moiety that can be incorporated near the C-terminus of the peptide substrate. The corresponding fluorescent acceptor group containing a 4 4 [4-(dimethylamino)phenyl]am] benzoic acid (DABCYL) can then be attached (16) Yamaoka, K.; Tanigawara, Y.; Nakagawa, T.; Uno, T. A. Pharmacokinetic Analy~is Program (Multi) for Microcomputer. J. P h r m .