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

Abstract

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.

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.…”

Loss of FZO1 gene results in changes of cell dynamics in fission yeast

“…Studies have shown that there is a clear correlation between the intensity of a fluorescent protein and expression of the other gene in the construct, and that fluorescent intensity can indirectly reflect the expression level of the attached gene product ( 16 , 17 ). Analysis of fluorescence intensity of the spindle at prophase and metaphase showed that the fluorescence intensity of the spindle in fzo1Δ cells was notably stronger compared with in wild-type cells, whereas fluores-cence intensity of the chromosome at prophase and metaphase was notably weaker in fzo1Δ cells compared with in wild-type cells (n=20; Fig.…”