Development of a fluorescent substrate to measure hyaluronidase activity
Development of a fluorescent substrate to measure hyaluronidase activity
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DOI：
10.1016/j.ab.2008.04.040
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发表时间：
2008-08-01
影响因子：
2.9
通讯作者：
Mummert, Mark E.
中科院分区：
文献类型：
--
作者：
Zhang, Li-Shu;Mummert, Mark E.
开发了一种用于定量评估透明质酸酶活性的新型荧光底物（称为 FRET-HA）。透明质酸 (HA) 是透明质酸酶的主要底物，用荧光素胺和罗丹明 B 胺双重标记。由于荧光共振能量转移 (FRET)，荧光素胺荧光信号显着猝灭，罗丹明 B 胺信号显着增强。在牛睾丸透明质酸酶存在的情况下，HA 的裂解会破坏 FRET，导致依赖于酶浓度和时间的荧光素胺猝灭能力丧失。可以以非连续和连续方式方便地监测荧光素胺信号的增加。使用 FRET-HA 在连续荧光测定中测定牛睾丸透明质酸酶的 K-m 值。重要的是，使用 FRET-HA 作为底物的牛睾丸透明质酸酶的估计 K-m 值与之前报道的使用天然（即未标记）HA 的该酶的 K-m 值非常一致。因此，FRET-HA 是定量评估 HA/透明质酸酶分子相互作用的可靠底物。 FRET-HA 底物的简单性、灵敏度和多功能性表明它将在各种检测平台中具有实用性，并且应该成为评估透明质酸酶活性的新工具。 (C) 2008 Elsevier Inc. 保留所有权利。
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 and 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 on both enzyme concentration and time. Increase in the fluorescein amine signal could be conveniently monitored in both noncontinuous 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 reported previously 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. (C) 2008 Elsevier Inc. All rights reserved.