Quantitation of macromolecular binding using size exclusion filters: application to a fucosyltransferase assay

A method to quantify the covalent attachment of small radiolabeled substrates to macromolecules on the basis of molecular weight using size exclusion filters in an Amicon Centrifree micropartition system is described. GDP-[14C]-L-fucose was covalently attached to asialofetuin in a fucosyltransferase reaction catalyzed by mouse spermatogenic cell extracts. Radiolabeled product was separated from unreacted substrate by centrifuging 200-400 microliter of cell extract through a 10-kDa size exclusion filter at 1000 g for 10 to 20 min. After 10 washes with an appropriate buffer, no detectable radioactivity was found in the eluant and the membrane-bound radiolabeled product was counted in a scintillation vial. Using this method the fucosyltransferase activity of mouse spermatogenic cells was approximately 17 pmol/mg protein/min which is essentially identical to values obtained using size exclusion chromatography. This technique provides a rapid, efficient, and inexpensive alternative for the isolation and detection of acceptor-substrate complexes.