INTERACTION OF A FLUORESCENT DERIVATIVE OF PACLITAXEL (TAXOL) WITH MICROTUBULES AND TUBULIN-COLCHICINE

A fluorescent derivative of paclitaxel, 2-debenzoyl-2-(m-aminobenzoyl) paclitaxel (2-AB-PT), has been prepared. 2-AB-PT induces microtubule a ssembly in vitro, but is about 3-fold less potent than paclitaxel itse lf. The absorption and emission characteristics of 2-AB-PT were analyz ed as a function of solvent. It was found that both spectra were pertu rbed by specific solvent effects when the solvent contained a hydrogen bond donor. The absorption and fluorescence spectra of 2-AB-PT bound to microtubules could not be mimicked by a single solvent, but the abs orption and emission maxima of the tubulin-bound species could be dupl icated by a solvent mixture of DMSO and water. These results indicate that the fluorophore binding site on the microtubule is in an environm ent of intermediate polarity that is accessible to a hydrogen bond don or in the vicinity of the m-amino group, In addition, tubulin fluoresc ence is quenched in the 2-AB-PT/microtubule complex, and energy transf er from tubulin to 2-AB-PT is apparent. These results indicate that su bstituents on the C-2 position of paclitaxel associate with tubulin wh en bound to the microtubule. 2-AB-PT binding to microtubules was quant itatively analyzed by fluorescence titrations, Two classes of binding sites for 2-AB-PT on microtubules were found. The high affinity site h as an apparent association constant (K-1app) Of 2.0 (+/-0.9) x 10(7) M (-1) and an apparent binding stoichiometry (n(lapp)) of 0.8 (+/-0.1) s ites/tubulin dimer in the microtubule, The apparent association consta nt for the lower affinity site is about 100-fold less than that of the higher affinity site (K-2app= 2.1 (+/-0.7) x 10(5) M(-1)), and the st oichiometry of the lower affinity site or class of sites (n(2app)) was found to be 1.3 +/- 0.1. Paclitaxel blocked 2-AB-PT binding to the hi gh affinity site, No binding of 2-AB-PT to unassembled tubulin was obs erved, but the emission spectrum of 2-AB-PT in the presence of the tub ulin-colchicine complex resembled the emission spectrum of the ligand bound to microtubules. It was previously shown that paclitaxel can ind uce GTPase activity in the tubulin-colchicine complex, indicating that paclitaxel can bind to unassembled tubulin in its complex with colchi cine [Carlier, M.-F., & Pantaloni, D. (1983) Biochemistry 22, 4814-482 2]. Rigorous characterization of the aggregation state of the protein under these conditions demonstrates that 2-AB-PT is also capable of bi nding to the tubulin-colchicine complex.