Number and distribution of chromophore types in native phycobiliproteins
TL;DR: Fluorescence lifetimes and absolute quantum yields of a number of chromatographically pure phycobiliproteins have been determined using absorption, fluorescence emission and polarization spectra to calculate the number of different types of chromophore, sensitizing and fluorescing, per chromoprotein.
Abstract: — Fluorescence lifetimes and absolute quantum yields of a number of chromatographically pure phycobiliproteins have been determined. In conjunction with absorption, fluorescence emission and polarization spectra, these data were used to calculate the number of different types of chromophore, sensitizing and fluorescing, per chromoprotein.
To characterize the spatial distributions of the chromophores, the observed emission anisotropies were compared with those calculated from models, using the Forster transfer mechanism and the Jablonski ‘active sphere’ approximation. The experimental values are more consistent with surface locations for the fluorescing chromophores rather than with their distribution throughout the volume.
Theoretical efficiencies of transfer between sensitizing and fluorescing chromophores on the same macromolecule are consistent with those observed. The transfer efficiency from phycoerythrin prosthetic groups to chlorophyll a compared with that for transfer via phycocyanin indicates that the latter process is probably the favoured migration route.
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TL;DR: It is shown that phycobiliprotein conjugates can be applied to fluorescence-activated cell sorting and analysis, fluorescence microscopy, and fluorescence immunoassay and are well-suited for two-color flow cytofluorimetric analyses employing a single excitation line.
Abstract: The synthesis of a novel class of reagents for fluorescence analyses of molecules and cells is reported. These compounds consist of a highly fluorescent phycobiliprotein conjugated to a molecule having biological specificity. Phycoerythrin-immunoglobulin, phycoerythrin-protein A, and phycoerythrin-avidin conjugates were prepared. These conjugates bind specifically to beads containing a covalently attached target molecule and render them highly fluorescent. Femtomole (10(-15) mole) quantities of phycoerythrin conjugates can be detected because of the high extinction coefficient (epsilon M = 2.4 x 10(6) cm-1 M-1 for 2.4 x 10(5) daltons) and high fluorescence quantum yield (Q = 0.8) of the phycobiliprotein moiety. An important feature of these conjugates is that they emit in the orange-red spectral region, where background fluorescence is less than at shorter wavelengths. Phycoerythrin conjugates are well-suited for two-color flow cytofluorimetric analyses employing a single excitation line. The distributions of Leu antigens (also called OKT antigens) on the surface of T-lymphocytes were analyzed using fluoresceinated antibody as the green-fluorescent stain and biotinylated antibody counter-stained with phycoerythrin-avidin as the red one. This one-laser two-color analysis showed that cells express Leu-3a and Leu-3b or neither antigen. In contrast, the distributions of Leu-2a (a marker of suppressor and cytotoxic T-cells) and Leu-3a (a marker of helper and inducer T-cells) are mutually exclusive. These studies show that phycobiliprotein conjugates can be applied to fluorescence-activated cell sorting and analysis, fluorescence microscopy, and fluorescence immunoassay.
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TL;DR: Electronic excitation energy transfer as function of distance measured, noting energy transfer process use as spectroscopic ruler as well as the use of spectroscopy ruler for measuring distance.
Abstract: Electronic excitation energy transfer as function of distance measured, noting energy transfer process use as spectroscopic ruler
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TL;DR: In this paper, the theoretical relation between the radiative lifetime and the absorption intensity has been investigated and the effect of a change in nuclear configuration, which is particularly marked in the higher diphenylpolyenes, is shown to increase the radii lifetime and to distort the spectral intensity distribution.
Abstract: Measurements have been made of the fluorescence spectra, lifetimes and quantum efficiencies and of the absorption spectra of several organic molecules in dilute solution. These have been used to test the validity of the theoretical relation between the radiative lifetime and the absorption intensity, and of the mirror-image relation between the fluorescence and absorption spectra. The former is found to be valid provided there is no major change in the nuclear configuration between the ground and excited states, but the latter is usually more sensitive to such changes. The effect of a change in nuclear configuration, which is particularly marked in the higher diphenylpolyenes, is to increase the radiative lifetime and to distort the spectral intensity distribution.
300 citations
TL;DR: In this paper, the Mesvorgang is vereinfacht und einige Fehlerquellen werden eliminiert, die in der Fluorescenzspektroskopie im Vergleich zur Absorptionsspektriscale zusatzlich auftreten.
Abstract: Die Banden in den Fluorescenzspektren von Losungen organischer Verbindungen sind meist schwach und breit und gelegentlich eine Funktion der Bestrahlungsdauer durch das Erregungslicht. Zur Messung von Fluorescenzspektren sind daher rasch registrierende, hochempfindliche lichtelektrische Spektrometer besonders geeignet. Die Gesichtspunkte werden dargelegt, die bei der Verwendung handelsublicher UV-Spektralphotometer in der Fluorescenzspektroskopie zu beachten sind. Es werden fluorescierende Losungen ausgewahlt, die als sekundare Strahlungsstandards dienen und deren Fluorescenzspektren gemeinsam mit denen der Proben gemessen werden. Auf diese Weise wird der Mesvorgang vereinfacht und einige Fehlerquellen werden eliminiert, die in der Fluorescenzspektroskopie im Vergleich zur Absorptionsspektroskopie zusatzlich auftreten.
197 citations
TL;DR: The theoretical determination of the natural radiative lifetime from the absorption spectrum is shown to have inherent ambiguities, so that only calorimetric methods provide a reliable, independent check for photometric yield measurements.
Abstract: — The fluorescence yields and lifetimes of fluorescein and nine brominated derivatives in basic ethanol are reported. Calorimetric. photometric, and lifetime methods are used independently to measure the yields. A new and simple calorimetric method is presented for this purpose, and the accuracy of the techniques is assessed. There is good agreement between the calorimetric and photometric results. The importance of parameters such as purity, pH, and fluoresence reabsorption is illustrated. The theoretical determination of the natural radiative lifetime from the absorption spectrum is shown to have inherent ambiguities, so that only calorimetric methods provide a reliable, independent check for photometric yield measurements.
180 citations