Luminometric quantitation of Photinus pyralis firefly luciferase and Escherichia coli beta-galactosidase in blood-contaminated organ lysates

Firefly luciferase and Escherichia coil beta -galactosidase chemiluminescen t reporter gene assays are rapid and sensitive means of detecting reporter enzyme activities in cell lysates of both eukaryotic and prokaryotic system s. In these assays, expression vectors containing the luciferase or beta -g alactosidase genes are transferred to cells in culture or animal tissues in vivo. Crude cell or organ lysates are then prepared and submitted to enzym e 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 -galacto sidase activity can be detected luminometrically. Attempts to apply these a ssays to cell lysates contaminated with blood, as from any whole organ lysa te, have had questionable results thus far because of light absorption by h emoglobin in the ranges of light emission by both of these assays. We have made several adjustments to standard chemiluminescent reporter gene assay p rotocols to minimize errors in quantitation contributed by hemoglobin. To t his end, we have developed a method for quantitating the protein due to blo od and due to the organ itself in a blood-contaminated organ lysate. We hav e also found that the use of a colorimetric protein assay that is unaffecte d by hemoglobin absorbance is preferred for protein quantitation. In conclu sion, luciferase and P-galactosidase assays can be applied to blood-contami nated organ lysates; however, the luciferase assay proved to be superior du e to minimal endogenous activity and lower absorption by hemoglobin of ligh t emitted by the enzyme product. (C) 2000 Academic Press.