Effects of alcohols on fluorescence intensity and color of a discharged-obelin-based biomarker.

Photoproteins are responsible for bioluminescence of marine coelenterates; bioluminescent and fluorescent biomarkers based on photoproteins are useful for monitoring of calcium-dependent processes in medical investigations. Here, we present the analysis of intensity and color of light-induced fluorescence of Ca(2+)-discharged photoprotein obelin in the presence of alcohols (ethanol and glycerol). Complex obelin spectra obtained at different concentrations of the alcohols at 350- and 280-nm excitation (corresponding to polypeptide-bound coelenteramide and tryptophan absorption regions) were deconvoluted into Gaussian components; fluorescent intensity and contributions of the components to experimental spectra were analyzed. Five Gaussian components were found in different spectral regions-ultraviolet (tryptophan emission), blue-green (coelenteramide emission), and red (hypothetical indole-coelenteramide exciplex emission). Inhibition coefficients and contributions of the components to experimental fluorescent spectra showed that presence of alcohols increased contributions of ultraviolet, violet, and red components, but decreased contributions of components in the blue-green region. The effects were related to (1) changes of proton transfer efficiency in fluorescent S*1 state of coelenteramide in the obelin active center and (2) formation of indole-coelenteramide exciplex at 280-nm photoexcitation. The data show that variation of fluorescence color and intensity in the presence of alcohols and dependence of emission spectra on excitation wavelength should be considered while applying the discharged obelin as a fluorescence biomarker.

Fluorescence properties of Ca2+-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(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.

Discharged photoprotein obelin: fluorescence peculiarities.

Photoprotein obelin, the enzyme-substrate complex of polypeptide with 2-hydroperoxycoelenterazine, is responsible for bioluminescence of marine hydroid Obelia longissima. Addition of Ca(2+) to the photoprotein triggers the bioluminescent reaction with light emission. The product of the bioluminescent reaction--enzyme-bound coelenteramide--is a fluorescent protein called 'discharged' obelin. It is stable and highly fluorescent. The paper considers dependence of its light-induced fluorescence on Ca(2+) concentration. Increase of Ca(2+) concentration enhanced the fluorescence intensity of discharged obelin; the dependence was found as linear in double logarithmic coordinates at Ca(2+) concentration range 10(-7)-10(-6) M, both in excitation and emission spectra. The spectra were divided into components; contributions of the components to experimental excitation and emission spectra depended on Ca(2+) concentration. The data suggest enzymatic conformational transition in discharged obelin at approximately 5 x 10(-7) M of Ca(2+) concentration. Spectra variations were attributed to acidity changes of discharged obelin chromophore (coelenteramide) in its fluorescent state S(1)*.

Spectral components of bioluminescence of aequorin and obelin.

Complex bioluminescence spectra of photoproteins from marine coelenterates - jellyfish Aequorea victoria and hydroid Obelia longissima, and photoluminescence spectra of the bioluminescent reaction products (Ca(2+)-discharged photoproteins) were deconvolved into components. The bioluminescence spectra of aequorin were found to include three, the bioluminescence spectra of obelin - four, and the photoluminescence spectra of the Ca(2+)-discharged photoproteins - only two components. The spectral components were assigned to one unionized and three ionized forms of coelenteramide. The changes in acidity of the excited coelenteramide molecule are discussed. The differences in bioluminescence and photoluminescence spectra are considered, with protonic environment of coelenteramide taken into account.