Abstract— Spectral properties of guanidine‐denaturated and pronase‐digested green‐fluorescent proteins (GFP) from two species of bioluminescent coelenterates have been investigated. Spectrophotometric titrations of Renilla and Aequorea GFP, following denaturation in 6M guanidine HCl at elevated temperature, revealed identical absorption peaks in acid (383–384 nm) and in alkali (447–448 nm) and a single isosbestic point in the visible region at 405 nm. Both proteins exhibited a spectrophotometric pK. of 8.1 in guanidine ‐HCl. Pronase digestion of the heat‐denaturated GFP's generated a methanol‐soluble blue‐fluorescent peptide with identical fluorescence emission spectra (λmax= 430 nm, uncorrected; φf1= 0.003) for both coelenterate species. These data suggest that the large absorption differences between native Renilla and Aequorea GFP molecules result from unique protein environments imported to a common chromophore.

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The structure of native luciferin from the bioluminescent coelenterate Renilfa reniformis is shown to be 3,7-dihydro-2p-hydroxybenzyl). p-hydroxyphenyl benzylimidazo[1,2-apyrazin-3-one by mass spectral analysis of synthetic luciferin and the luciferin derived from a protein directly involved in the bioluminescent system. A previous report of the molecular weight of luciferin is shown to be incorrect by reexamination of the spectral data and by synthesis of two derivatives. Detailed analysis of kinetic, emission, and quantum yield data for the isolated and synthetic luciferins confirms this structure. Confirmation of this structure in a number of species from different phyla suggests a common substrate for a variety of bioluminescent marine organisms.

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