Hydrophobic Chromatography of Tubulin on Immobilized Colchicine Columns with Various Spacers

Abstract

Eleven immobilized-colchicine adsorbents were synthesized from deacetylcolchicine (DAC) and Sepharose with various spacers (R) and their tubulin-adsorbing capacity was investigated. The adsorption capacity of DAC-R-Sepharose for tubulin depended on the hydrophobicity rather than the length of the spacer and was three to five times that of DAC-Sepharose without any spacer.The results of the present studies can be summarized as follows. (1) The adsorbed tubulin showed two binding modes : a reversible binding which is abrogated at weak ionic strength (such as 0.1 M NaCl), and an irreversible binding which is abrogated by denaturants such as 6 M urea and 7 M guanidine-HCl. (2) The adsorbed tubulin was not eluted by colchicine but was eluted as colchicine-binding tubulin by 0.1 M NaCl. (3) Tubulin was adsorbed on hydrophobic ligands such as the phenyl, diphenyl, trimethoxyphenyl or naphthyl group. (4) Tubulin-colchicine complex was adsorbed by immobilized colchicine.It is considered that the immobilized colchicine binds tubulin at a hydrophobic region, not necessarily at a specific colchicine-binding site.