Fluorescence properties of [Ca.sup.2+]-independent discharged obelin and its application prospects

Discharged obelin, a complex of coelenteramide and polypeptide, is a fluorescent protein produced from the photoprotein obelin, which is responsible for bioluminescence of the marine hydroid Obelia longissima. Discharged obelin is stable and nontoxic and its spectra are variable, and this is why it can be used as a fluorescent biomarker of variable color in vivo and in vitro. Here we examined light-induced fluorescence of [Ca.sup.2+]-independent discharged obelin (obtained without addition of [Ca.sup.2+]). Its emission and excitation spectra were analyzed under variation of the excitation wavelength (260-390 nm) and the emission wavelength (400-700 nm), as well as the 40°C exposure time. The emission spectra obtained with excitation at 260-300 nm (tryptophan absorption region) included three peaks with maxima at 355, 498, and 660 nm, corresponding to fluorescence of tryptophan, polypeptide-bound coelentera-mide, and a hypothetical indole-coelenteramide exciplex, respectively. The emission spectra obtained with excitation at 310-380 nm (coelenteramide absorption region) did not include the 660-nm maximum. The peak in the red spectral region ([λ.sub.max]=660 nm) has not been previously reported. Exposure to 40°C under excitation at 310-380 nm shifted the obelin fluorescence spectra to the blue, whereas excitation at 260-300 nm shifted them to the red. Hence, red emission and variation of the excitation wavelength form a basis for development of new medical techniques involving obelin as a colored biomarker. The addition of red color to the battery of known (violet to yellow) colors increases the potential of application of obelin. Keywords Fluorescent protein * Discharged photoprotein obelin * Fluorescence color * Thermoinactivation * Excitation wavelength * Proton transfer