Luciferases, the enzymes responsible for the bioluminescence reaction, are present in multiple animal phyla and bacteria. The luciferases oxidize luciferins to produce light and the chemical nature of the luciferins can vary widely. Perhaps the best known are the ATP-dependent beetle luciferases that catalyze the oxidation of firefly luciferin in a photochemical reaction that has been widely used for the detection of low levels of ATP. In contrast, most marine luciferases do not use ATP, requiring only their luciferin and molecular oxygen as substrates with oxyluciferin, CO2 and light as products. Different species use different luciferins: coelenterazine is the cognate luciferin for Renilla, Gaussia and Metridia luciferases (RLuc, GLuc and MLuc, respectively), while cypridina luciferin is the cognate luciferin for Cypridina luciferase (CLuc) (). The two chemically related luciferins share the common chromophore, imidazopyrazinone.
Chemical structure of Coelenterazine and Cypridina luciferin.
Some of the marine luciferase proteins have been extensively studied and mutated in order to improve their yield and bioluminescent properties 
. The three dimensional structure of RLuc has been solved. It is a 37 kDa monomer and it contains a single substrate binding site 
. CLuc is a 62 kDa protein 
. GLuc and MLuc are 18.5 kDa and 22 kDa respectively with similar amino acid sequence and both contain a duplication of a proposed catalytic domain 
. RLuc and CLuc sequences are neither similar to each other nor to GLuc or MLuc.
Alignment of both the amino terminal and carboxyl terminal halves of GLuc and MLuc.
In the past twenty years, the genes of Renilla
, and more recently Gaussia
luciferases have been used as expression reporters for determining how particular genes are regulated by placing the luciferase gene downstream of particular regulatory sequences. Typical reporter assays measure the amount of luciferase protein that has been produced by measuring its activity in relative light units (RLU) from cell extracts or conditioned media by adding nearly saturating luciferin concentrations. Furthermore bioluminescent imaging of whole animals with marine luciferases has been used to identify tumors or tissue specific reporter gene expression 
. Despite extensive structural and molecular biology characterizations, no classical kinetic characterization of these enzymes has been reported.
The range of sensitivity for detection of these luciferases spans many orders of magnitude. At a fixed high substrate concentration, very low levels of luciferase can be detected. In contrast here, it was of interest to determine whether the presence of very low levels of substrate (picomolar) could be detected in the presence of a fixed amount of luciferase. In this study we compared the kinetic properties of several marine luciferases in relation to their luciferin concentration, in an effort to identify a luciferase enzyme that offers the highest sensitivity in such an assay. Similar bioluminescent assays utilizing marine luciferases have been performed with RLuc in linked reactions to measure the concentration of 3′–5′ adenosine diphosphate (PAP) 
. This assay requires extreme sensitivity, and it was anticipated that luciferases having a higher rate of turnover than RLuc 
e.g. GLuc 
would perform substantially better for the detection of PAP.