Evaluation of ß-Galactosidase Activity in Tissue in the Presence of Blood

The reporter gene for ß-galactosidase is frequently used to determine the efficiency of gene transfer in arteries. However, blood is often present in arterial explants and may compromise the results by the presence of hemoglobin. The light absorption of hemoglobin is similar to the absorption of several colorimetric products of the commonly used ß-galactosidase substrates, including onitrophenyl-ß-D-galactopyranoside (ONPG) and chlorophenol red galactopyranoside (CPRG). This may result in false-positive measurements of ß-galactosidase enzyme activity. The aim of this investigation was to determine the most appropriate method for quantification of ß-galactosidase activity in the presence of blood. Colorimetric substrates (ONPG, CPRG) or the chemiluminescent Galacton-Plus substrate were used, and light absorption was measured at different concentrations of erythrocyte extract. Among the ß-galactosidase substrates tested, CPRG was the most appropriate, allowing detection of enzyme activity at concentrations as low as 0.05 mU, independent of blood contamination. Addition of reducer stabilized enzyme activity for at least 5 h. Endogenous ß-galactosidase activity was evaluated and used to correct results. CPRG substrate, in combination with the reducer agent mercaptoethanol, was found to be the optimal reagent for quantifying ß-galactosidase activity in the presence of blood after nonviral in vivo reporter gene transfection, even with a relatively low transfer effi-