A simple and rapid assay for heparanase activity using homogeneous time-resolved fluorescence

Enomoto, Koji; Okamoto, Hiroyuki; Numata, Yoshito; Takemoto, Hiroshi

doi:10.1016/j.jpba.2006.01.032

Cited by 32 publications

(21 citation statements)

References 26 publications

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“…HTRF has been used to measure the activity of heparanase [48]. Heparanase functions as an enzyme cleaving heparan sulfate proteoglycan to release growth factors and plays an important role in tissue invasion by malignant tumor cells and inflammatory cells.…”

Section: Htrf Assay Applicationsmentioning

confidence: 99%

HTRF: A Technology Tailored for Drug Discovery - A Review of Theoretical Aspects and Recent Applications

Degorce

Card²,

Soh³

et al. 2009

TOCHGENJ

396

285

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HTRF (Homogeneous Time Resolved Fluorescence) is the most frequently used generic assay technology to measure analytes in a homogenous format, which is the ideal platform used for drug target studies in high-throughput screening (HTS). This technology combines fluorescence resonance energy transfer technology (FRET) with time-resolved measurement (TR). In TR-FRET assays, a signal is generated through fluorescent resonance energy transfer between a donor and an acceptor molecule when in close proximity to each other. Buffer and media interference is dramatically reduced by dual-wavelength detection, and the final signal is proportional to the extent of product formation. The HTRF assay is usually sensitive and robust that can be miniaturized into the 384 and 1536-well plate formats. This assay technology has been applied to many antibody-based assays including GPCR signaling (cAMP and IP-One), kinases, cytokines and biomarkers, bioprocess (antibody and protein production), as well as the assays for protein-protein, proteinpeptide, and protein-DNA/RNA interactions.Since its introduction to the drug-screening world over ten years ago, researchers have used HTRF to expedite the study of GPCRs, kinases, new biomarkers, protein-protein interactions, and other targets of interest. HTRF has also been utilized as an alternative method for bioprocess monitoring. The first-generation HTRF technology, which uses Europium cryptate as a fluorescence donor to monitor reactions between biomolecules, was extended in 2008 through the introduction of a second-generation donor, Terbium cryptate (Tb), enhancing screening performance. Terbium cryptate possesses different photophysical properties compared to Europium, including increased quantum yield and a higher molar extinction coefficient. In addition to being compatible with the same acceptor fluorophors used with Europium, it can serve as a donor fluorophore to green-emitting fluors because it has multiple emission peaks including one at 490 nm. Moreover, all Terbium HTRF assays can be read on the same HTRF-compatible instruments as Europium HTRF assays.Overall, HTRF is a highly sensitive, robust technology for the detection of molecular interactions in vitro and is widely used for primary and secondary screening phases of drug development. This review addresses the general principles of HTRF and its current applications in drug discovery.

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Section: Htrf Assay Applicationsmentioning

confidence: 99%

HTRF: A Technology Tailored for Drug Discovery - A Review of Theoretical Aspects and Recent Applications

Degorce

Card²,

Soh³

et al. 2009

TOCHGENJ

396

285

View full text Add to dashboard Cite

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“…During FRET, the donor fluorescence would be quenched; hyaluronidase digestion of the HA co-polymer would disrupt FRET resulting in fluorescent enhancement of the donor molecule. Although most FRET based enzymatic assays have used peptide substrates, a FRET heparin substrate has recently been reported [13]. Thus, carbohydrate FRET substrates can also be designed to study other enzymatic reactions.…”

Section: Introductionmentioning

confidence: 99%

Development of a fluorescent substrate to measure hyaluronidase activity

Zhang

Mummert

2008

Analytical Biochemistry

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A novel fluorescent substrate (termed FRET-HA) to quantitatively assess hyaluronidase activity was developed. Hyaluronan (HA), the major substrate for hyaluronidase, was dual labeled with fluorescein amine and rhodamine B amine. The fluorescein amine fluorescence signal was significantly quenched while the rhodamine B amine signal was significantly enhanced due to fluorescence resonance energy transfer (FRET). In the presence of bovine testes hyaluronidase, cleavage of HA disrupted FRET resulting in a loss of the fluorescein amine quenching that was dependent upon both enzyme concentration and time. Increase in the fluorescein amine signal could be conveniently monitored in both non-continuous and continuous fashions. The K m value for bovine testes hyaluronidase was determined using FRET-HA in a continuous fluorescent assay. Importantly, the estimated K m value for bovine testes hyaluronidase using FRET-HA as the substrate was in excellent agreement with K m values previously reported for this enzyme using native (i.e., unlabeled) HA. Therefore, FRET-HA is a reliable substrate for quantitatively assessing the HA/ hyaluronidase molecular interaction. The simplicity, sensitivity and versatility of the FRET-HA substrate suggest that it will have utility in a variety of assay platforms and should be a new tool for assessing hyaluronidase activity.

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“…On the basis of the above‐mentioned considerations, we performed a docking simulations of a putative HPSE substrate, a designed oligosaccharide with the minimum recognition backbone, with the additional 2‐ N ‐sulfate and 6‐ O ‐sulfate groups on the nonreducing GlcN and a fluorogenic tag on the reducing extremity GlcN, to use it in developing, in vitro and in vivo , a quantitative fluorescence assay. Many assays reported in the literature for HPSE activity are based on either radiolabeled substrates or separation of degraded products based on the molecular size. They present many disadvantages: cumbersome, time consuming for isolation of degradation products from uncleaved substrates and detection, HS fragments produced may inhibit HPSE activity, enzymatic assays are not comparable, use of radioisotopes, and some generate hazardous radiolabeled waste products with risk of contamination.…”

Section: Introductionmentioning

confidence: 99%

Cloning and bacterial expression systems for recombinant human heparanase production: Substrate specificity investigation by docking of a putative heparanase substrate

Pennacchio

Capo

Caira

et al. 2017

Biotech and App Biochem

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Human heparanase (HPSE) is an enzyme that degrades the extracellular matrix. It is implicated in a multiplicity of physiological and pathological processes encouraging angiogenesis and tumor metastasis. The protein is a heterodimer composed of a subunit of 8 kDa and another of 50 kDa. The two protein subunits are noncovalently associated. The cloning and expression of the two protein subunits in Escherichia coli and their subsequent purification to homogeneity under native conditions result in the production of an active HPSE enzyme. The substrate specificity of the HPSE was studied by docking of a putative substrate that is a designed oligosaccharide with the minimum recognition backbone, with the additional 2-N-sulfate and 6-O-sulfate groups at the nonreducing GlcN and a fluorogenic tag at the reducing extremity GlcN. To develop a quantitative fluorescence assay with this substrate would be extremely useful in studies on HPSE, as the HPSE cleavage of fluorogenic tag would result in a measurable response.

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A simple and rapid assay for heparanase activity using homogeneous time-resolved fluorescence

Cited by 32 publications

References 26 publications

HTRF: A Technology Tailored for Drug Discovery - A Review of Theoretical Aspects and Recent Applications

HTRF: A Technology Tailored for Drug Discovery - A Review of Theoretical Aspects and Recent Applications

Development of a fluorescent substrate to measure hyaluronidase activity

Cloning and bacterial expression systems for recombinant human heparanase production: Substrate specificity investigation by docking of a putative heparanase substrate

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