A novel rhodamine-riboflavin conjugate probe exhibits distinct fluorescence resonance energy transfer that enables riboflavin trafficking and subcellular localization studies.
A novel rhodamine-riboflavin conjugate probe exhibits distinct fluorescence resonance energy transfer that enables riboflavin trafficking and subcellular localization studies.
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一种新型罗丹明-核黄素偶联探针表现出独特的荧光共振能量转移，可实现核黄素运输和亚细胞定位研究。
DOI：
10.1021/mp0499510
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发表时间：
2004
期刊：
影响因子：
--
通讯作者：
Swaan,PeterW
中科院分区：
文献类型：
--
作者：
Phelps,MitchA;Foraker,AmyB;Gao,Wenqing;Dalton,JamesT;Swaan,PeterW
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-1cm-1at 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 aninsitumechanism 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.Keywords: Confocal microscopy; fluorescence resonance energy transfer; riboflavin; trafficking; transport; two-photon; vitamin B2
