Characterization of bacterial proteases with a panel of fluorescent peptide substrates (original) (raw)
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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.
Continuous Assay of Proteases Using a Microtiter Plate Fluorescence Reader
Analytical Biochemistry, 1997
. The drawback of these assays is that proteases sometimes behave differently toward small peptides or David A. Menges, Damian L. Ternullo, in hydrolysis of a bond that is not a true peptide bond. Recently, an assay that combines the uses of a natural protein, the sensitivity of fluorescence, and the ease
A Continuous Fluorimetric Assay for Tail-Specific Protease
Analytical Biochemistry, 1998
A continuous fluorimetric assay for tail-specific protease (Tsp) has been developed using a fluorescence donor/quencher system, in which 5-[(2-aminoethyl) amino]naphthalene-1-sulfonic acid (EDANS) and 4-(4dimethylaminophenylazo)benzoic acid (DABCYL) are attached to the N-terminus and the lysyl side chain of peptide AARAAK-(6-aminocaproyl) 2-ENYALAA, respectively. Tsp-mediated cleavage of the Ala-Arg peptide bond separates the quencher, DABCYL, from the donor, EDANS, and results in a large increase in the fluorescent yield of EDANS (>50-fold). Using this sensitive assay, Escherichia coli tail-specific protease was shown to exhibit typical Michaelis-Menten kinetics with a k cat of 0.086 ؎ 0.002 s ؊1 , K M of 4.0 ؎ 0.3 M, and k cat /K M of 2.2 ؋ 10 4 M ؊1 s ؊1. A control substrate, which only differs from the above substrate by having a charged residue (glutamate) at the C-terminus, showed drastically reduced activity to Tsp (k cat /K M ؍ 58 M ؊1 s ؊1). A peptide containing the C-terminal sequence of the substrate, GRGYALAA, was shown to be a competitive inhibitor of Tsp with a K I value of 31 M. These results demonstrate the utility of this assay for the rapid assessment of Tsp activity.
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.
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. #
A bioluminescent assay for the sensitive detection of proteases
BioTechniques, 2011
A bioluminescent general protease assay was developed using a combination of five luminogenic peptide substrates. The peptide-conjugated luciferin substrates were combined with luciferase to form a homogeneous, coupled-enzyme assay. This single-reagent format minimized backgrounds, gave stable signals, and reached peak sensitivity within 30 min. The bioluminescent assay was used to detect multiple proteases representing serine, cysteine, and metalloproteinase classes. The range of proteases detected was broader and the sensitivity greater, when compared with a standard fluorescent assay based on cleavage of the whole protein substrate casein. Fifteen of twenty proteases tested had signal-to-background ratios >10 with the bioluminescent method, compared with only seven proteases with the fluorescent approach. The bioluminescent assay also achieved lower detection limits (≤100 pg) than fluorescent methods. During protein purification processes, especially for therapeutic proteins, ...
Detection, quantification, and characterization of proteases in recombinant Escherichia coli
Biotechnology Techniques, 1993
Electrophoresis of crude cell exlracts on PAGE gels in the presence of SDS copolymerized with a nonspecific protease substrate has been used to detect, characterize, and quantify intracellular proteases in recombinant Escherichia coli. After electrophoresis, the gels are incubated, SDS is removed, and protease activity is revealed by clear zones on the stained gel due to proteolysis of the nonspecific protease substrate (gelatin or casein). The method differentiates proteases based on activity and molecular weight.
Molecular and Biotechnological Aspects of Microbial Proteases
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Isolation and Biochemical Characterization of Extracellular Microbial Proteases
Proteases are a broad family of hydrolytic enzymes with various applications in chemical, cosmetics, and pharmaceutical industries. Owing to their physiological necessity, proteases are found in diverse sources including microorganisms. Our objective study was to search for a high quality and inexpensive source for the production of microbial proteases under different culture and growth conditions. Also, we aimed to characterize microbial proteases. Proteases-producing bacteria were isolated from soil samples collected from a poultry waste site. Soil samples were inoculated in skimmed agar media and 48 h later, colonies producing clear zones were selected as the source of microorganisms producing enzyme. The isolates were used to inoculate liquid media and the clear supernatant was taken as a crude for enzyme preparation. The enzyme was isolated and purified with ammonium sulfate at 60-80% saturation followed by dialysis. Subsequently, characterization of the enzyme fraction with the highest activity was carried out. The results indicated that the isolated enzyme with (60-80%) fractionation of ammonium sulfate exhibited the highest specific activity. In addition, the optimal temperature for enzyme activity was determined at 70ºC at pH values of 0.05M of acetate buffer 3.6 and 0.05M of glycine-NaOH buffer 10.0. Finally, the kinetic parameters (Michaelis–Menten constant, Km and maximal reaction velocity, Vmax) were calculated as 0.11 µmole/ml and 0.5x104 nmole of tyrosine/ml/hour, respectively. In conclusion, our findings provide evidence that the isolated bacteria represent a rich source of thermostable proteases. Indeed, more studies are still required to obtain such proteases in a purified form suitable for studying their applications.
Micromethods for the spectrophotometric determination of bacterial protease activities
Journal of Microbiological Methods, 1983
Microassays for the spectrophotometric determination of bacterial proteases were developed using congo red elastin, a substrate specific for elastolytic activity, and hide powder azure, a substrate sensitive to more general proteolytic activity. The small reaction volume (0.1 ml) allows incubation, filtration and quantitation to be carried out in 96 well microassay plates. Using a simple spin filtration device constructed from microassay plates a large number (768) of microassays can be filtered simultaneously. The microassays are particularly useful for screening large numbers of bacterial colonies for proteolytic mutants since they allow the rapid and efficient handling of multiple samples. These assays also permit the qualitative estimation of enzyme levels.