Filter binding assay for the geldanamycin-heat shock protein 90 interaction

A filter binding assay to measure affinity of [H-3-allyl]17-allylamino geldanamycin ([H-3]AAG) for the ATP binding site of the N-terminal domain of human Hsp90alpha (hHsp90alpha9-236) was developed. Diethylaminoethyl cellulose or glass fiber filters impregnated with polyethyleneimine were used to capture the [H-3]AAG-Hsp90 complex, and conditions which washed >98% of free [H-3]AAG from the filters were developed. The complex formed at a rapid rate (k(on) = 2.5 x 10(7) L mol(-1) s(-1)) and dissociated with a half-life of 2.3 min (k(off) = 5 x 10(-3) s(-1)). hHsp90alpha9-236 bound to [H-3]AAG with a K-d value of 0.4 +/- 0.1 mum. [H-3]AAG had similar affinities for full-length hHsp90alpha and for hHsp90alpha9-236 variants containing biotinylated N-terminal biotinylation signal sequences and N- or C-terminal His(6) tags. Geldanamycin, ADP, ATP, and radicicol-all known to bind to the ATP domain of Hsp90-competed with [H-3]AAG for binding to hHsp90alpha9-236, showing K-d values in good agreement with reported values. (C) 2003 Elsevier Science (USA). All rights reserved.