The Effect of the Crocus Sativus L. Carotenoid, Crocin, on the Polymerization of Microtubules, in Vitro

Abstract

Objective(s): Crocin, as the main carotenoid of saffron, has shown anti-tumor activity both in vitro and in vivo. Crocin might interact with cellular proteins and modulate their functions, but the exact target of this carotenoid and the other compounds of the saffron have not been discovered yet. Microtubular proteins, as one of the most important proteins inside the cells, have several functions in nearly all kinds of cellular processes. The aim of this study was to investigate whether crocin affects microtubule polymerization and tubulin structure. Materials and Methods: Microtubules were extracted from sheep brains after two cycles of temperature-dependant assembly-disassembly in the polymerization buffer (PMG). Then phosphocellulose P11 column was used to prepare MAP-free tubulin. Turbidimetric assay of microtubules was performed by incubation of tubulins at 37 ºC in PIPES buffer. To investigate the intrinsic fluorescence spectra of tubulins, the emission spectra of tryptophans was monitored. To test the interaction of crocin with tubulin in more details, ANS has been used. Results: Crocin extremely affected the tubulin polymerization and structure. Ultraviolet spectroscopy indicated that crocin increased polymerization of microtubules by nearly a factor of two. Fluorescence spectroscopic data also pointed to significant conformational changes of tubulin. Conclusion: We showed that crocin increased tubulin polymerization and microtubule nucleation rate and this effect was concentration dependant. After entering cell, crocin can modulate cellular proteins and their functions. Concerning the results of this study, crocin would be able to affect several cell processes through interaction with tubulin proteins or microtubules.

Keywords

• Carotenoid
• Fluorescence spectroscopy
• Microtubules
• Tubulin