Purification and properties of Renilla reniformis luciferase

Abstract

Luciferase from the anthozoan coelenterate R. reniformis (Renilla luciferin:oxygen 2-oxidoreductase (decarboxylating), EC 1.13.12.5.) catalyzes the bioluminescent oxidation of Renilla luciferin producing light (.lambda.B 480 nm, QB 5.5%), oxyluciferin, and CO2 (Hori et al, 1973). Using a combination of ion-exchange, molecular-sieve, sulfhydryl-exchange, and affinity chromatography, luciferase was purified .apprx. 12,000-fold with 24% recovery, to homogeneity as judged by analysis with disk and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel filtration, and ultracentrifugation. Renilla luciferase was active as a nearly spherical single polypeptide chain monomer of 3.5 .times. 104 daltons having a specific activity of 1.8 .times. 1015 h.nu. s-1 mg-1 and a turnover number of 111 .mu.mol min-1 .mu.mol-1 of enzyme. This enzyme had a high content of aromatic and hydrophobic amino acids such that it had an .epsilon.280nm0.1% of 2.1 and an average hydrophobicity of 1200 cal residue-1. The high average hydrophobicity of luciferase, which placed it among the more hydrophobic proteins reported, probably accounts, at least in part, for its tendency to self-associate forming inactive dimers and higher molecular weight species.