[1] Purification and properties of firefly luciferase

This chapter discusses purification and properties of firefly luciferase. Firefly luciferase in the presence of luciferin (d-LH2), adenosine triphosphate (ATP)-Mg, and molecular oxygen catalyzes the production of light. The color of the emitted light shows a peak at around 560 nm. This emission can be affected by temperature, pH, and metal ions. At low pH or in the presence of lead, mercury, or other heavy metals, the emission peak is shifted to red, showing an emission around 615 nm. Thus, when this system is used for the assay of ATP, it is critical to run internal controls to make sure that the light color has not been shifted. Because of the greater sensitivity of phototubes in blue in contrast to the red, a shift to red will give an apparent lower level of ATP. A dark-adapted eye can readily discern the difference in color between the control and one emitting at longer wavelengths. In crude firefly extracts and some luciferin preparations, dehydroluciferin is usually present. It is a potent inhibitor of the light reaction. The chapter describes the method of collecting and drying fireflies in order to prepare an appropriate acetone powder, the preparations and properties of a crude and partially purified luciferase and finally the preparation of crystalline firefly luciferase.