Limited Proteolysis of Luciferase as a Reporter in Nanosystem Biology: A Comparative Study

Abstract

Firefly luciferase is a 62 kDa protein that produces a flash of light on the oxidation of luciferin in the presence of ATP, Oxygen and Mg2+. Luciferase has a broad range of applications for analytical purposes and in vivo imaging for nanosystem biology studies. However, the enzyme is highly susceptible to proteolytic degradation that reduces its half-life. Rate of proteolytic digestion between two members of luciferase family (Photinus pyralis and Lampyris turkestanicus) is compared. Proteolytic sensitivity of L. turkestanicus luciferase was found to be more than P. pyralis luciferase, due to higher rate of hydrolysis under identical conditions. Both luciferases showed more sensitivity to chymotrypsin than trypsin with different digestion pattern. Digestion of P. pyralis by trypsin produced some fragments which were found to be more resistant to further degradation, whereas in L. turkestanicus initial fragments subdigested by trypsin, like chymotrypsin effect on both luciferases. Furthermore, both luciferases become increasingly labile to proteolysis as the temperature increases. The rate of inactivation and the rate of degradation between luciferases were different in a specific time of incubation. Appearance of similar bands for both luciferases confirmed exposure of specific regions, in spite of structural differences.