A novel rhodamine-riboflavin conjugate probe exhibits distinct fluorescence resonance energy transfer that enables riboflavin trafficking and subcellular localization studies.
Riboflavin (vitamin B2, RF) is taken up in eukaryotic cells via specialized transport mechanisms. Although RF has fluorescence properties, direct microscopic visualization of RF uptake and trafficking has been complicated by cellular autofluorescence. We describe the synthesis, cellular uptake characteristics, and spectroscopic properties of a novel rhodamine-riboflavin conjugate (RD-RF), including absorption and emission spectra, two-photon excitation spectra, and fluorescence pH dependence. The conjugate has a molar extinction coefficient of 23 670 M(-1) cm(-1) at 545 nm (excitation wavelength) with a fluorescence quantum yield of 0.94. This compound exhibits intramolecular fluorescence resonance energy transfer (FRET). Selective quenching of the FRET signal is observed when RD-RF is bound with high affinity by the chicken riboflavin carrier protein. In addition to the typical rhodamine excitation and emission, FRET provides a secondary signal for conjugate localization and an in situ mechanism for observing riboflavin binding. Solution and in vitro stability determinations indicate that the linkage between riboflavin and rhodamine is stable for the duration of typical pulse--chase and cellular trafficking experiments. The distinct spectroscopic properties of RD-RF together with a comparable affinity for RF-binding proteins render it an excellent tool for the study of RF transport and trafficking in living cells.
Biophysics Program and Division of Pharmaceutics, The Ohio State University, Columbus, Ohio 43210-1291, USA.
Mitch A
MA
Phelps
Amy B
AB
Foraker
Wenqing
W
Gao
James T
JT
Dalton
Peter W
PW
Swaan
D002451:Cell Compartmentation; D045744:Cell Line, Tumor; D004355:Drug Stability; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D013058:Mass Spectrometry; D026901:Membrane Transport Proteins / Q000502:physiology*; D018613:Microscopy, Confocal; D036641:Microscopy, Fluorescence, Multiphoton; D015335:Molecular Probes / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D015394:Molecular Structure; D021381:Protein Transport; D012235:Rhodamines / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D012256:Riboflavin / Q000378:metabolism*; D012996:Solutions / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013347:Subcellular Fractions
D026901:Membrane Transport Proteins; D015335:Molecular Probes; D012235:Rhodamines; D012996:Solutions; C019079:riboflavin-binding protein; D012256:Riboflavin