Flourescence studies of platelet tubulin

Temperature-related changes in tubulin conformation were investigated by several flourospectroscopic methods utilizing steady-state flourescence techniques. Tubulin was isolated from human platelets by three cycles of temperature-dependent polymerization-depolymerization. 8-Anilino-1-naphthalenesulfonate (ANS) was used as a probe of polarity of tryptophanyl fluorescence in tubulin. The microtubule protein displayed definite differences at 4 and 37 degrees C. While the total number of ligand sites was the same, differences in the binding affinity of ANS were noted. Acryamide, a discriminating and efficient quencher of intrinsic protein fluorescence, also revealed temperature-dependent changes in the exposure of trypthophanyl residues of tubulin. ANS-induced quenching was associated with a transfer of energy from tryptophanyl residues to this fluorophor which could be used to calculate a critical distance between the acceptor-donor pair. The temperature-related changes were not found to be contingent upon the presence of polymerized tubulin.