Development of a fluorescent substrate to measure hyaluronidase activity

Abstract

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.

A new affinity method for purification of bovine testicular hyaluronidase enzyme and an investigation of the effects of some compounds on this enzyme

“…K M and V Max values for BTH were calculated from a Lineweaver-Burk plot using HA (sodium salt) as substrate and found to be 2.23 mM and 19.85 U/mL, respectively. These values, using HA (sodium salt), are close to other values reported for BTH from bovine testis 24,26 . The literature shows that PIX, b-NOA and gibberellic acid have various effects, in different ways and at different levels on many enzymes and physiological systems.…”

Dynamic Changes in Cervical Glycosaminoglycan Composition during Normal Pregnancy and Preterm Birth

“…Hyaluronidase enzyme activity was measured using a modified FRET-HA assay (35). The substrate, FRET-HA, was made by covalently conjugating fluorescent donor and acceptor molecules to the carboxylic acid functional groups of HA (sodium hyaluronate, Thermo Fisher Scientific, Pittsburgh, PA).…”

Hyaluronan Endocytosis: Mechanisms of Uptake and Biological Functions

“…An activatable HA molecular probe, called FRET-HA, designed to detect hyaluronidase activity was recently reported [71]. Briefly, the HA co-polymer was chemically labeled with donor and acceptor fluorescent probes.…”