Development of a fluorescent substrate to measure hyaluronidase activity

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 Km value for bovine testes hyaluronidase was determined using FRET–HA in a continuous fluorescent assay. Importantly, the estimated Km value for bovine testes hyaluronidase using FRET–HA as the substrate was in excellent agreement with Km 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.