Sistla Jyothi C, Desai Umesh R
Institute for Structural Biology, Drug Discovery, and Development, Virginia Commonwealth University, Richmond, VA 23219, USA.
Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, VA 23219, USA.
Bio Protoc. 2019 Sep 5;9(17):e3356. doi: 10.21769/BioProtoc.3356.
Heparanase, an endo-β--glucuronidase, cleaves cell surface and extracellular matrix heparan sulfate (HS) chains at distinct sites and plays important biological roles including modulation of cell growth and metastasis. Although a number of different types of heparanase assays have been reported to date, most are labor intensive, complex and/or expensive to carry out. We reasoned that a simpler heparanase assay could be developed using heparin labeled with Dabcyl and EDANS as donor and acceptor fluorophores so as to generate a FRET signal. Our results show that a more robust heparanase assay could be developed based on the principle studied herein and more homogeneous preparation of heparin. Yet, the assay in its current form could be used for routine screening of potential inhibitors in a high-throughput manner as well as for studying heparanase activity expressed in tumors as well as biological fluids like plasma.
乙酰肝素酶是一种内切-β-D-葡糖醛酸酶，可在不同位点切割细胞表面和细胞外基质硫酸乙酰肝素（HS）链，并发挥重要的生物学作用，包括调节细胞生长和转移。尽管迄今为止已报道了许多不同类型的乙酰肝素酶检测方法，但大多数方法实施起来劳动强度大、操作复杂且/或成本高昂。我们推断，可以开发一种更简单的乙酰肝素酶检测方法，使用标记有Dabcyl和EDANS的肝素作为供体和受体荧光团，从而产生荧光共振能量转移（FRET）信号。我们的结果表明，基于本文研究的原理和更均匀的肝素制剂，可以开发出更强大的乙酰肝素酶检测方法。然而，目前形式的该检测方法可用于以高通量方式对潜在抑制剂进行常规筛选，以及研究肿瘤和血浆等生物流体中表达的乙酰肝素酶活性。
