150] Cypridina and firefly Luciferase

Abstract

This chapter discusses the determination of cypridina and firefly luciferase. Luciferase is the enzyme which catalyzes the oxidation of luciferin (LH 2 ) in the presence of molecular oxygen to yield visible radiation and unknown products. Luciferin and luciferase differ, depending on the organism used. It is necessary, therefore, to specify the source of both substances. Since the intensity of the light emitted depends on both enzyme and substrate concentration, the quantitative determination of these substances can be made by measuring the light intensity by a suitable photocell arrangement. The Farrand photofluorometer is satisfactory. Qualitative estimations can be made with the naked eye. With the purified enzymes described below, light can be obtained when these are diluted l: 1000, which is still visible to the dark-adapted eye. A crude active luciferase can be obtained from air-dried Cypridina by grinding the organisms first in a mortar and then in a Ten Brock tissue grinder. A crude Cypridina luciferin prepared by making a fresh hot-water extract can be used in assaying for the enzyme. Since luciferin is autoxidizable, it is necessary to make up a fresh solution each time. Cypridina luciferase is known to catalyze only the luminescent oxidation of Cypridina luciferin. It does not catalyze the oxidation of luciferins from other sources. In the process of purification of firefly luciferase, five grams of the dried lanterns of Photinus pyralis is ground with sand and extracted three times with a total volume of 100 ml. of H 2 O. The pH of the extract is adjusted to 8 with NaOH, and the solution is placed in the deepfreeze. The only known function of firefly luciferase is that of catalyzing the luminescent oxidation of firefly luciferin.