SPECTROPHOTOMETRIC IDENTITY OF THE ENERGY TRANSFER CHROMOPHORES IN RENILLA AND AEQUOREA GREEN-FLUORESCENT PROTEINS |
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Authors: | William W Ward Chris W Cody Russell C Hart Milton J Cormier |
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Institution: | **Department of Biochemistry and Microbiology, Rutgers University, Cook College, New Brunswick, NJ 08903;†Bioluminescence Laboratory, Department of Biochemistry, University of Georgia, Athens, GA 30602, U.S.A. |
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Abstract: | 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 (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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