The reaction rate of ATP-limited firefly luciferase-catalysed reactions, is affected by the presence o f detergents. Anionic detergents inhibit luciferase activity without causing significant enzyme inactivation during the reaction. Cationic detergents in- crease reaction rate several-fold w i t h a sharply defined optimum concentration o f detergent for the effect. However, cationic detergents inactivate firefly luciferase during the reaction, resulting in a continuously decreasing reaction rate. Under such conditions, peak light intensity must be used as an indication o f initial reaction rate. The inactivation rate increases with increasing detergent concentration. Non-ionic and zwitterionic detergents increase reaction rate over a broad range o f detergent concentrations. Enzyme stability during the reaction is not affected by non-ionic detergents and only affected by zwitterionic detergents at high detergent concentration. Cyclodextrins, which can increase reaction rates o f some chemiluminescent reactions, have little effect on firefly luciferase activity.
Assays for ATP using firefly luciferase must be internally standardized by the constant addition technique in which a known amount o f ATP is added t o the test sample, since external calibration o f such assays, by reference to a previously prepared standard curve, can lead t o imprecision when detergents are present.