| Abstract: | The reaction rate of ATP-limited firefly luciferase-catalysed reactions, is affected by the presence of detergents. Anionic detergents inhibit luciferase activity without causing significant enzyme inactivation during the reaction. Cationic detergents increase reaction rate several-fold with a sharply defined optimum concentration of 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 of initial reaction rate. The inactivation rate increases with increasing detergent concentration. Non-ionic and zwitterionic detergents increase reaction rate over a broad range of 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 of 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 of ATP is added to the test sample, since external calibration of such assays, by reference to a previously prepared standard curve, can lead to imprecision when detergents are present. |
