Biotin derivatives of folate compounds: Synthesis and utilization for visualization and affinity purification of folate transport proteins

Abstract

A single-step, affinity purification procedure, utilizing biotin derivatives of folate/methotrexate (MTX) and folate, has been devised to isolate both folate transporters in homogeneous form and in high yield from L1210 cells. These biotin–folate compounds are also useful probes for visualizing the folate transporters in individual cells. Fluorescein derivatives of MTX and folate have been synthesized by linking the γ-carboxyl group of the latter compounds to the fluorophore via a diaminopentane (DAP) spacer. These probes, after activation of the open α-carboxyl by N -hydroxysuccinimide (NHS), were used to label covalently the folate transport proteins. In the procedures presented in this chapter, the folate compounds are derivatized with biotin, and the two functional units are joined either with a stable linking group (DAP) or a group that can be cleaved by reduction [DAP plus -CO-(CH 2 ) 2 -S-S-(CH 2 ) 2 -NH-]. After the activation with N -hydroxysulfosuccinimide (NHSS), these probes are covalently attached to the transporters in situ . Membrane proteins are extracted with detergent, and the labeled transporters are purified from the mixture by adsorption and elution from streptavidin beads. Other procedures allow the labeled transporters to be visualized on individual cells by light, fluorescence, or electron microscopy.