A fluorescent probe for selective detection of lysosomal β-hexosaminidase in live cells

Determining the activity of lysosomal β-hexosaminidase in cells is of great importance for understanding the roles that these enzymes play in pathophysiological events. Herein, we designed the new fluorescent probe, βGalNAc-Rhod-CM(NEt₂), which consisted of a βGalNAc-linked rhodol unit serving as a β-hexosaminidase reactive fluorogenic moiety and a N,N'-diethylaminocoumarin (CM(NEt₂)) group acting as a fluorescence marker for determining the degree of cell permeabilization. Treatment of βGalNAc-Rhod-CM(NEt₂) with β-hexosaminidase promoted generation of Rhod-CM(NEt₂), thereby leading to an increase in the intensity of fluorescence of Rhod. However, this probe did not respond to the functionally related glycosidase, O-GlcNAcase. The detection limit of βGalNAc-Rhod-CM(NEt₂) for β-hexosaminidase was determined to be 0.52 nM, indicating that it has high sensitivity for this enzyme. Furthermore, the probe functioned as an excellent fluorogenic substrate for β-hexosaminidase with k_cat and K_m values of 17 sec^-1 and 22 μM, respectively. The results of cell studies using βGalNAc-Rhod-CM(NEt₂) showed that levels of β-hexosaminidase activity in cells can be determined by measuring the intensity of fluorescence arising from Rhod and that the intensity of fluorescence of CM(NEt₂) can be employed to determine the degree of cell permeabilization of the probe. Utilizing the new probe, we assessed β-hexosaminidase activities in several types of cells and evaluated the effect of glucose concentrations in culture media on the activity of this enzyme.