Hyaluronidases (HA-ase) are enzymes that cleave the polysaccharide, hyaluronic (HA), which is a glycosaminoglycan expressed in extracellular and pericellular matrices. In humans, five hyaluronidase genes and one hyaluronidase pseudogene has been described [1
]. The hyaluronidases are endoglycosidases that predominantly catalyze hyaluronan depolymerization via
cleavage of the β-N-acetyl-D-glucosaminidic bonds. In mammalian normal tissue, they are present in low concentrations; 60ng/ml in human serum [2
]. It is well established that an over expression of the hyaluronidase enzymes is observed in many different cancers including prostate cancer and malignant melanomas [3
]. The increased activity of the hyaluronidases has been correlated with several carcinogenic cell behaviors including tissue invasion [5
], resistance to apoptosis [6
] and the potentiation of angiogenesis [4
]. However HA-ases are also used as anticancer chemotherapeutic agents—the addition of HA-ase reduces a tumor’s resistance to chemotherapy [7
]. HA-se can have different biological activities depending on the cancer cell type. In contrast to prostate cancer and malignant melanoma, HA-se suppresses tumorigenicity in a model of colon cancer [9
HA, the substrate for HA-se, is a high molecular weight, linear, non-sulfated glycosaminoglycan composed of multiple subunits of D-glucuronic acid (GlcA) and N-acetylglucosamine (GlcNAc) and has the primary structure [β1→4GlcA β1→3GlcNAc]n. HA is known to exhibit diverse biological functions including: a) maintenance of tissue structural integrity, b) formation of highly hydrated matrices around individual cells, c) promotion of cellular migration including metastasis, and d) mediation of intercellular signaling. HA contributes to tumor cell behavior by: a) modulating the biomechanical properties of extracellular and pericellular matrices in which cells reside, b) forming a repetitive template for interactions with other macromolecules in the pericellular and extracellular environment and thus, contributing to the assembly, structural integrity and physiological properties of these matrices, and c) interacting with cell surface receptors through concomitant signal transduction. The digestion of fluorescently labeled HA (by HA-ase) can be used for detection of HA-ase enzyme activity. Several methods have been proposed. A simple assay for HA-ase activity using fluorescence polarization has been proposed by Murai and Kawashima [10
]. However, the observed changes in polarization do not exceed 0.01 (10mP). Although polarization measurements are very precise, these changes are too small for reliable detection. Another approach involves dually labeled HA with fluorophores suitable for Forster resonance energy transfer (FRET). The cleavage of HA results in the release of FRET and change of the relative intensities of the fluorophores involved. These ratio-metric measurements offer larger signal responses in the presence of HA-ase than polarization changes but involve the dual HA labeling [11
In this manuscript we propose a simpler approach for the detection of HA-ase activity. We observed that fluorescein-labeled HA (HA-Fl) shows a very short fluorescence lifetime due to the self-quenching of fluorescein, a phenomenon known for many years. Self-quenching of fluorescein and other xantene-type dyes is one of the oldest observations in fluorescence spectroscopy and is due to resonance energy transfer between fluorescein molecules (homo FRET). This process was frequently connected to decreases in the quantum yield, lifetime and polarization of viscous solutions with high probe concentrations [13
]. In the case of fluorescein, the Förster distance (50% probability of excitation energy transfer) for homo FRET is about 42Å [19
]. Since this distance is comparable to or larger than the size of many proteins, FRET is expected to occur when a macromolecule contains more than a single fluorophore.
The digestion of HA-Fl by HA-ase enzyme releases fluorescein self-quenching, thereby increasing fluorescence brightness and lifetime. We describe here the strategy for the HA-ase detection using changes in observed lifetimes (lifetime-based sensing, LBS).
There are many advantages of LBS over intensity-based sensing methods [20
]. Fluorescence lifetime measurements yield absolute quantity values that are independent of the measurement platform. LBS does not depend on the excitation intensity and optical misalignments which simplifies the calibration of the sensing device. Mentioned above, LBS properties and its robustness make this approach an ideal tool for measurements of difficult-to-control “real world” samples such as physiological fluids or tissue.