Hydrolysis of glucuronide-based substrates mediated by tungsten, Cu2+, Fe2+, and Zn2+

A variety of metal microprojectiles are currently used for carrying foreign DNA into living cells via particle-acceleration techniques. While developing a microprojectile-mediated protocol for transforming cells of sugarbeet (Beta vulgaris L.), formation of a blue precipitate was observed with the indigoqenic substrate 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid (X-gluc) in the absence of gusA DNA encoding beta-D-glucuronidase (GUS). Tungsten microcarriers, but not gold or silicon carbide, proved capable of catalyzing the cleavage of the glucuronide residue from three histochemical substrates evaluated: X-gluc, salmon X-gluc and magenta X-gluc. Indigo-stained sugarbeet cells were observed following bombardment with tungsten in the absence of DNA. Addition of oxidative catalysts to tungsten microcarriers during substrate incubation had no apparent effect on the metal-mediated catalysis. Treatment of microcarriers with Proteinase K and heat ruled out the presence of enzymes. Microbiological evaluation indicated the absence of contaminating microbes. Similarly, metal-catalyzed hydrolysis of the fluorogenic substrate 4-methylumbelliferyl-beta-D-glucuronic acid (4-MUG) was observed in the presence of tungsten spheres but not with gold or silicon carbide particles. With this substrate. hydrolysis also occurred with millimolar concentrations of Cu2+, Fe2+ and Zn2+ ions. Consequently, careful monitoring of DNA-minus controls and avoidance of millimolar concentrations of Cu2+, Fe2+ and Zn2+ ions are recommended in microprojectile bombardment experiments where transient assays for gusA expression are performed.