Evaluation of beta-galactosidase activity in tissue in the presence of blood

The reporter gene for beta -galactosidase is frequently used to determine t he efficiency of gene transfer in arteries. However, blood is often present in arterial explants and may compromise the results by the presence of hem oglobin. The light absorption of hemoglobin is similar to the absorption of several colorimetric products of the commonly used beta -galactosidase sub strates, including o-nitrophenyl-beta -D-galactopyranoside (ONPG) and chlor ophenol red galactopyranoside (CPRG), This may result in false-positive mea surements of beta -galactosidase enzyme activity. The aim of this investiga tion was to determine the most appropriate method for quantification of bet a -galactosidase activity in the presence of blood. Colorimetric substrates (ONPG, CPRG) or the chemiluminescent Galacton-Plus substrate were used, an d light absorption was measured at different concentrations of erythrocyte extract. Among the beta -galactosidase substrates tested, CPRG was the most appropriate, allowing detection of enzyme activity at concentrations as lo w as 0.05 mU, independent of blood contamination. Addition of reducer stabi lized enzyme activity for at least 5 h. Endogenous beta -galactosidase acti vity was evaluated and used to correct results. CPRG substrate, in combinat ion with the reducer agent mercaptoethanol, was found to be the optimal rea gent for quantifying beta -galactosidase activity in the presence of blood after nonviral in vivo reporter gene transfection, even with a relatively l ow transfer efficiency. Copyright (C) 2000 S. Karger AG, Basel.