Assay of Retinol-Binding Protein–Transthyretin Interaction and Techniques to Identify Competing Ligands
The principles of fluorescence resonance energy transfer have been utilized to develop a high-throughput assay which detects compounds that interfere with interaction between retinol-binding protein (RBP) and transthyretin (TTR). In this assay, the intrinsic fluorescence from the RBP–retinol complex excites a probe molecule which is covalently coupled to TTR. Generation of an emission signal from the TTR probe indicates interaction between RBP–retinol and TTR. Importantly, the inclusion of retinol in the assay allows discrimination of test compounds which bind RBP versus those which bind to TTR. Thus, compounds which bind to RBP must compete with retinol in order to affect RBP–TTR interaction. This feature of the assay will be useful to identify test compounds which are more likely to have an effect in vivo.
Key wordsFluorescence resonance energy transfer (FRET) inner filter effect Cogan plot retinol retinol-binding protein (RBP) transthyretin (TTR) N-(4-hydroxyphenyl)retinamide (HPR)
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