Luminometric quantitation of photinus pyralis firefly luciferase and Escherichia coli beta-galactosidase in blood-contaminated organ lysates.
Firefly luciferase and Escherichia coli beta-galactosidase chemiluminescent reporter gene assays are rapid and sensitive means of detecting reporter enzyme activities in cell lysates of both eukaryotic and prokaryotic systems. In these assays, expression vectors containing the luciferase or beta-galactosidase genes are transferred to cells in culture or animal tissues in vivo. Crude cell or organ lysates are then prepared and submitted to enzyme assays. The level of enzyme activity is proportional to the efficiency of gene delivery and expression. When used with modified substrates that emit light when cleaved by the appropriate enzyme, luciferase and beta-galactosidase activity can be detected luminometrically. Attempts to apply these assays to cell lysates contaminated with blood, as from any whole organ lysate, have had questionable results thus far because of light absorption by hemoglobin in the ranges of light emission by both of these assays. We have made several adjustments to standard chemiluminescent reporter gene assay protocols to minimize errors in quantitation contributed by hemoglobin. To this end, we have developed a method for quantitating the protein due to blood and due to the organ itself in a blood-contaminated organ lysate. We have also found that the use of a colorimetric protein assay that is unaffected by hemoglobin absorbance is preferred for protein quantitation. In conclusion, luciferase and beta-galactosidase assays can be applied to blood-contaminated organ lysates; however, the luciferase assay proved to be superior due to minimal endogenous activity and lower absorption by hemoglobin of light emitted by the enzyme product.
Smith
A D
AD
Department of Biochemistry and Molecular Biology, Medical College of Ohio, Toledo, Ohio 43614, USA.
Trempe
J P
JP
D000818:Animals; D002417:Cattle; D002624:Chemistry, Clinical / Q000379:methods*; D001517:Coleoptera; D004926:Escherichia coli / Q000201:enzymology; D017930:Genes, Reporter; D006454:Hemoglobins / Q000378:metabolism; D008099:Liver / Q000201:enzymology; D008156:Luciferases / Q000097:blood* / Q000378:metabolism; D008163:Luminescent Measurements; D008168:Lung / Q000201:enzymology; D051379:Mice; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012709:Serum Albumin / Q000378:metabolism; D001616:beta-Galactosidase / Q000097:blood* / Q000378:metabolism
D006454:Hemoglobins; D012709:Serum Albumin; D008156:Luciferases; D001616:beta-Galactosidase