SPECTROPHOTOMETRIC IDENTITY OF THE ENERGY TRANSFER CHROMOPHORES IN RENILLA AND AEQUOREA GREEN‐FLUORESCENT PROTEINS

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 6 M 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 (λ max = 430 nm, uncorrected; φ f1 = 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.