An internally quenched fluorescent substrate for collagenase

Saikumari, Y. K.; Balaram, Padmanabhan

doi:10.1002/bip.20952

Cited by 5 publications

(5 citation statements)

References 26 publications

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“…Barrett et al (1989) assayed a clostridial collagenase using N-(2,4-dinitrophenyl)-ProLeu-Gly-Pro-Trp-Lys substrate. Bickett et al (1993); Gould et al (1999) and Saikumari and Balaram (2008) used similar fluorescently-labeled substrates to achieve high sensitivity in collagenase activity measurements. Variation in these fluorescent techniques include: (a) labeling the products of collagenase activity rather than employing a synthetic substrate, (b) employing a reagent that forms a fluorescent complex with amino acids, (c) adding fluoresamine after incubating the enzyme and (d) using fluorescent substrate (Evans and Ridella, 1984).…”

Section: Synthetic Peptides and Fluorescent Assaymentioning

confidence: 99%

Extraction and Purification of Collagenase Enzymes: A Critical Review

Daboor¹,

Budge²,

Ghaly

et al. 2010

American Journal of Biochemistry and Biotechnology

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Problem statement: Enzymes have vital roles in several industrial processes (foods, cosmetics, nutraceuticals and pharmaceuticals) due to their highly selective nature and high activity at very low concentrations. Recent efforts to identify new sources of useful enzymes have been concentrated on the marine environment because of the potential to make use of processing wastes. About 35-50% of the mass of the fish caught is a waste that is disposed off at sea or in landfills. The extraction of enzymes from fish processing waste can reduce environment problems and improve the economics of the fish industry. Collagenases are a group of enzymes that can be extracted from fish waste. Approach: Comprehensive reviews of the literature on the extraction, purification, characterization and use of collagenases was carried out. Results: Collagenases have different molecular weights based on their types and sources. They have the ability to break down the peptide bonds in collagen at physiological pH. They are classified into two types: serine and metallocollagenase. Collagenolytic activities have been shown at a wide range of temperatures (20-40°C) and pH (6-8). Many activators can be used to achive collagenase activity including 4-Aminophenylmercuric Acetate (APMA), trypsin, potassium or sodium thiocyanate, iodoacetamide and potassium iodide. Dithiothreitol (DTT), mercaptoethanol, ethylendiaminetetracetic acid, ophenanthroline and cysteine inactivate the enzyme. Collagenases enzymes can be extracted with a variety of techniques using different buffering systems (tris-HCl, sodium bicarbonate, calcium chloride and cacodylate). All techniques involve the use of ammonium sulphate fractionation and centrifugation to precipitate the enzyme. Collagenases are normally purified using chromatographic techniques such as gel-filtration, ion-exchange and affinity column chromatography. Collagenase can be assayed with a number of methods, including: colorimetric absorbance, viscometry, radioactivity and fluorescence spectroscopy. Collagenases are partly responsible for toughness in red meats and are used as tenderizers in food industry, have application in the fur and hide tanning to ensure uniform dying of leather, used in medicine to treat burns and ulcers, eliminate scar tissues, transplantation of organs. Conclusion: Understanding of the nature of the enzymes and identifying the most suitable resources and the methods for their extraction and purification will have significant impact on the fish processing, food and medical industries.

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Section: Synthetic Peptides and Fluorescent Assaymentioning

confidence: 99%

Extraction and Purification of Collagenase Enzymes: A Critical Review

Daboor¹,

Budge²,

Ghaly

et al. 2010

American Journal of Biochemistry and Biotechnology

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“…The unique characteristic of the enzyme substrate developed is described in Table 1. Our new substrate is 5Fam-AGGPLGPPGPGGK-dabcyl, which has different donor/acceptor fluorophores group localization, which can be cleaved by thermolysin as compared to a previous study18. Complete amino-acid sequences of the ECM: collagens (I, II, III, IV, V), laminin, and fibronectin were obtained in FASTA format from UniProtKB22.…”

Section: Resultsmentioning

confidence: 99%

“…In this study, we first synthesized and characterized a novel peptide substrate that can be used to assess the collagenolytic activity of the enzymes including collagenase class I, II and thermolysin/neutral protease using Fluorescence Resonance Energy Transfer (FRET). The method is sensitive to determine the kinetic parameters of various enzymes using micromolar concentrations of substrate1718192021. The substrate contains 5-CarboxyFluorescein-Aminohexyl Amidite (5-Fam) fluorogenic groups.…”

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confidence: 99%

Fluorogenic Peptide Substrate for Quantification of Bacterial Enzyme Activities

Al-Abdullah

Bagramyan

Bilbao

et al. 2017

Sci Rep

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A novel peptide substrate (A G G P L G P P G P G G) was developed for quantifying the activities of bacterial enzymes using a highly sensitive Fluorescence Resonance Energy Transfer (FRET) based assay. The peptide substrate was cleaved by collagenase class I, II, Liberase MTF C/T, collagenase NB1, and thermolysin/neutral protease, which was significantly enhanced in the presence of CaCl2. However, the activities of these enzymes were significantly decreased in the presence of ZnSO4 or ZnCl2. Collagenase I, II, Liberase MTF C/T, thermolysin/neutral protease share similar cleavage sites, L↓G and P↓G. However, collagenase NB1 cleaves the peptide substrate at G↓P and P↓L, in addition to P↓G. The enzyme activity is pH dependent, within a range of 6.8 to 7.5, but was significantly diminished at pH 8.0. Interestingly, the peptide substrate was not cleaved by endogenous pancreatic protease such as trypsin, chymotrypsin, and elastase. In conclusion, the novel peptide substrate is collagenase, thermolysin/neutral protease specific and can be applied to quantify enzyme activities from different microbes. Furthermore, the assay can be used for fine-tuning reaction mixtures of various agents to enhance the overall activity of a cocktail of multiple enzymes and achieve optimal organ/tissue digestion, while protecting the integrity of the target cells.

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“…To remove trypsin activity, the Lys from substrate 1 was replaced by Pro in substrate 2 . Pro in the P 3 ' subsite is favored by bacterial collagenases [6, 11]. The Gly-Pro-Hyp tripeptides closest to the N - and C -terminus of the cleavage site were replaced with Gly-Pro-Flp tripeptides to improve the stability of the triple-helix [31].…”

Section: Resultsmentioning

confidence: 99%

“…These assays utilize collagen (gels or fluorescein-labeled fibrils), denatured collagen [dye-bound gelatin (azocoll)], or synthetic substrates based on collagen sequences [ N -[3-(2-furyl)acryloyl)]-Leu~Gly-Pro-Ala (FALGPA), (4-phenylazo-carbobenzoxy)-Pro-Leu-Gly-Pro-Arg (PZ peptide), or Dabcyl-γAbu-Lys-Gly-Gly-Pro-Leu~Gly-Pro-Pro-Gly-Pro-Gly-Gly-Cys(Aedans)-Lys-NH 2 (where Dabcyl is 4-((4-(dimethylamino)phenyl)azo)-benzoyl and Aedans is 5-[2-(acetamido)ethylamino]naphthalene-1-sulfonic acid)] [5, 11–13]. Due to the instability of Col G resulting in decreased collagenolytic activity, it is desirable to utilize an assay that monitors the triple-helical peptidase activity of the enzyme in order to evaluate its integrity.…”

Section: Introductionmentioning

confidence: 99%

Development of a Förster resonance energy transfer assay for monitoring bacterial collagenase triple-helical peptidase activity

Tokmina-Roszyk

Tokmina‐Roszyk

Bhowmick

et al. 2014

Analytical Biochemistry

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Due to their efficiency in the hydrolysis of the collagen triple-helix, Clostridial histolyticum collagenases are utilized for isolation of cells from various tissues, including isolation of the human pancreatic islets. However, the instability of Clostridial collagenase I (Col G) results in a degraded Col G that has weak collagenolytic activity and an adverse effect on islet isolation and viability. A Föster resonance energy transfer (FRET) triple-helical peptide (fTHP) substrate has been developed for selective evaluation of bacterial collagenase activity. The fTHP [sequence Gly-mep-Flp-(Gly-Pro-Hyp)4-Gly-Lys(Mca)-Thr-Gly-Pro-Leu-Gly-Pro-Pro-Gly-Lys(Dnp)-Ser-(Gly-Pro-Hyp)4-NH2] had a melting temperature (Tm) of 36.2 °C and was hydrolyzed efficiently by bacterial collagenase (kcat/KM = 25,000 sec−1M−1), but not by clostripain, trypsin, neutral protease, thermolysin, or elastase. The fTHP bacterial collagenase assay allows for rapid and specific assessment of enzyme activity towards triple-helices and thus potential application for evaluating the efficiency of cell isolation by collagenases.

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An internally quenched fluorescent substrate for collagenase

Cited by 5 publications

References 26 publications

Extraction and Purification of Collagenase Enzymes: A Critical Review

Extraction and Purification of Collagenase Enzymes: A Critical Review

Fluorogenic Peptide Substrate for Quantification of Bacterial Enzyme Activities

Development of a Förster resonance energy transfer assay for monitoring bacterial collagenase triple-helical peptidase activity

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