Evaluation of beta-galactosidase activity in tissue in the presence of blood.
The reporter gene for beta-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 beta-galactosidase substrates, including o-nitrophenyl-beta-D-galactopyranoside (ONPG) and chlorophenol red galactopyranoside (CPRG). This may result in false-positive measurements of beta-galactosidase enzyme activity. The aim of this investigation was to determine the most appropriate method for quantification of beta-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 beta-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 beta-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 beta-galactosidase activity in the presence of blood after nonviral in vivo reporter gene transfection, even with a relatively low transfer efficiency.
Pelisek
J
J
Medical Clinic I, Klinikum Grosshadern, Munich, Germany.
Armeanu
S
S
Nikol
S
S
D000818:Animals; D001158:Arteries / Q000166:cytology; D016477:Artifacts; D001769:Blood; D002417:Cattle; D002457:Cell Extracts; D002478:Cells, Cultured; D002733:Chlorophenols / Q000032:analysis; D003124:Colorimetry / Q000379:methods*; D004912:Erythrocytes / Q000737:chemistry; D005697:Galactosides / Q000032:analysis; D017930:Genes, Reporter; D006454:Hemoglobins / Q000737:chemistry; D006801:Humans; D007763:Lac Operon; D008163:Luminescent Measurements; D008623:Mercaptoethanol / Q000494:pharmacology; D009131:Muscle, Smooth, Vascular / Q000166:cytology; D009598:Nitrophenylgalactosides / Q000032:analysis; D010783:Photometry; D012333:RNA, Messenger / Q000096:biosynthesis; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000378:metabolism; D019163:Reducing Agents / Q000494:pharmacology; D012710:Serum Albumin, Bovine / Q000494:pharmacology; D013552:Swine; D014162:Transfection; D001616:beta-Galactosidase / Q000032:analysis* / Q000378:metabolism
D002457:Cell Extracts; D002733:Chlorophenols; D005697:Galactosides; D006454:Hemoglobins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D019163:Reducing Agents; C055012:2-nitrophenylgalactoside; D012710:Serum Albumin, Bovine; D009598:Nitrophenylgalactosides; D008623:Mercaptoethanol; C074184:chlorophenol red galactopyranoside; D001616:beta-Galactosidase