Michael P. Valley, Erika M. Hawkins, Mike A. Scurria, James Unch, Troy Good, Dave Good, Laurent Bernad, Dieter H. Klaubert and Keith V. Wood
Promega Corporation, 2800 Woods Hollow Rd., Madison, WI 53711-5399 USA
Luciferase assay reagents containing luciferin are inherently limited by the conflict between a substrate that is activated with increasing pH and reagent components that are more stable with decreasing pH. Addition of a fluorine at the 5’-position of luciferin allows for activation of the substrate at a lower pH, and this provides a number of advantages in a luciferase reagent. The oxidation and/or degradation of luciferase reagent components is less at lower pH, so 5’- fluoroluciferin-containing reagents are more stable than those containing luciferin. Luciferase is also less affected by enzymatic inhibitors in a low pH reagent and the color quenching of phenol red, a dye commonly found in cell culture media, is reduced. Furthermore, low pH leads to a greatly reduced thiol odor; luciferin-containing reagents typically require high concentrations of thiols to control the rate of enzymatic turnover and generate a steady signal, but the low pH of reagents containing 5’-fluoroluciferin can reduce the rate of turnover using 100-fold less thiol. Thus, the use of 5’-fluoroluciferin allows for a luciferase assay reagent that is easier to use, more robust to reaction environment, and less aromatic than reagents containing unmodified luciferin.