Quantum yield phycobiliproteins

Grabowski, J., and Gantt, E. (1978) Photophysical properties of phycobiliproteins from phycobilisomes Fluorescence lifetimes, quantum yields, and polarization spectra. Photochem. Pbotobiol. 28, 39-45. 

The absorption spectmm of the monomer is essentially equal to the sum of absorptions of its components. On the other hand, the fluorescence quantum yield ofthe ap monomer is much higher than that of any ofthe separate components. These results suggest that association ofthe a- and P-subunits to form an ap monomer results in a reduction ofthe flexibility ofthe molecular skeletons of the bilins without altering their environment in any other spectroscopically significant way, as it is well known that a less flexible chromophore conformation inhibits the excited state from following alternative de-excitation pathways such as internal conversion. In the case of a denatured phycobiliprotein, the absorption is sharply decreased in the visible and it is non-fluorescent. 

Phycobiliproteins are highly fluorescent, with a quantum yield between 0.3 and 0.9, which does not vary markedly with temperature. The emission intensity is constant over a broad range of pH and it is not known to be quenched by many biomolecules. These properties have in fact made phycobiliproteins very... 

A third and relatively new application of FRET is the generation of new compound dyes with spectral characteristics that combine the best of both dyes. The idea is to attach covalently a donor and acceptor together in close proximity to one another. In the simplest case, where the absorption or emission properties of the individual dyes do not change, the absorption characteristic of the compound dye is the sum of the two individual dyes. At the same time, the emission is dominated by the acceptor since almost all of the energy absorbed by the donor is transferred to the acceptor. This results in dyes having potentially large Stokes shifts (the sum of the donor and acceptor Stokes shifts) and excellent quantum yields. So far, this work has mainly been applied to phycobiliproteins and DNA dyes. " ... 

Other dyes such as dansyl and AEDANS are popular donors in protein studies, in part because of the relatively long lifetimes (13-20 nsec), large (150 nm) Stokes shifts, and reasonable quantum yields (0.1-0.5). If steric hindrance is not a problem and a large-sized donor or acceptor can be used, the multichromophoric phycobiliproteins (molecular weight of 104,000 for B- or R-phycoerythrin 240,000 for allophycocyanin) make excellent donors or acceptors, having extinction coefficients which can exceed... 

The phycobiliproteins are hard to beat as extremely bright antibody labels for fluorescence analysis of cell surface antigens by flow cytome-try 15,16 These bacterial photosynthetic macromolecules each have up to 34 individual bilin fluorophores wrapped within the polypeptide structure. An example is R-phycoerythrin (R-PE), which has an extinction coefficient of 2 X 10 L/mol cm and a quantum yield of 0.68. Usually there is room for only one PE molecule per antibody, because the size of an R-PE label is 1.5 times that of an IgG antibody. The large size of the complex reduces the kinetics of binding to cell surface antigens, and some intracellular markers are inaccessible to the R-PE-labeled antibody. For intracellular measurements, lower molecular mass fluorophores (<1 kDa) are usually preferred. 

Phycobiliprotein Molecular weight (nm) e (cm- NT ) Max (nm) Fluorescence quantum yield... 

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