Fluorescence properties of Ca2+-independent discharged obelin and its application prospects

Abstract

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(2+)-independent discharged obelin (obtained without addition of Ca(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 coelenteramide, 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(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.

Ultraviolet fluorescence of coelenteramide and coelenteramide-containing fluorescent proteins. Experimental and theoretical study

“…3A. In our previous studies [24][25][26][27][28][29][30], the visible spectra of discharged photoproteins had been approximated by a set of Gauss components. The components were found in "violet" (400-423 nm) and "blue-green" (506-566 nm) regions, and attributed to neutral and ionized forms of protein-bound CLM, respectively, Fig.…”

“…1). As mentioned before, the neutral form of discharged photoproteins was found in "violet" (400-423 nm) region of their fluorescence spectra [24][25][26][27][28][29][30].…”

“…Time-resolved fluorescence experiments were conducted in [23] to determine fluorescence characteristics of neutral and ionized forms of discharged obelin. Components of complex photoluminescent spectra of discharged obelin and aequorin were studied and attributed to the different forms of CLM [24]; changes of spectral composition were analyzed under variation of calcium concentration [25], exposure to higher temperature [26,27] and exogenous compounds [28][29][30].…”

“…In [26,30], fluorescence spectra of discharged obelin measured under different excitation energies were analyzed. It was found that the spectra depend on the excitation wavelengths: higher energy excitation (260-300 nm) initiated fluorescence not only in the visible spectral region, but in the near UV region, too.…”

Low-intensity Exposures via Luminescent Bioassays of Different Complexity: Cells, Enzyme Reactions and Fluorescent Proteins

“…The reactions are Ca-dependent, and this is a basis for their biomedical application [34][35]. Biochemical and photophysical mechanisms of the bioluminescence reactions [36][37][38][39][40][41] and spectral characteristics of their products -coelenteramide-containing proteins [42][43][44][45][46][47][48][49] are under intensive investigations now.…”