Interaction between the C-Terminal Peptides of Tubulin and Tubulin S Detected with the Fluorescent Probe 4',6-Diamidino-2-Phenylindole

Abstract

The digestion of tubulin with subtilisin and the reassociation of the digestion products was followed by means of the fluorescent probe 4',6-diamidino-2-phenylindole (DAPI). The fluorescence spectra of DAPI bound to chicken brain tubulin and to the main products of tubulin digested with subtilisin-agarose (tubulin S and C-terminal peptides) were analyzed. The corrected emission spectrum of DAPI in the presence of tubulin showed an enhancement of fluorescence intensity with a maximum at 452 nm. The digestion reaction was followed by the diminution of the area of DAPI-tubulin emission spectra, which showed biphasic pseudo-first-order kinetics. The values for the rate constants were 1.2 x 10-2 min-1 and 3.5 x 10-2 min-1 for the α and β subunits, respectively, and were similar to those determined from the undigested subunits using polyacrylamide gel electrophoresis. Tubulin S and the C-terminal peptides were purified by means of a Bio-Gel P-60 column. The C-terminal peptides obtained from