A study of the carboxyl groups of sheep brain tubulin

Tubulin was isolated from whole sheep brain by ammonium sulphate fractionation followed by ion exchange chromatography using DEAE-Sephadex in 10 mM sodium phosphate buffer pH 7.0. It was essentially free from contaminating proteins as shown by SDS-PAGE electrophoresis and was active by the criterion of colchicine binding. Hydrogen ion titration of the sheep brain tubulin was performed under native conditions. In the present study, only that pH range has been selected for analysis where carboxyl groups (most of which are located in the C-terminal regions of both the alpha- and beta- subunits) deprotonate. The results indicated the presence of 75 carboxyl groups per dimer upto pH 5.5. Of these, 46 titrated with a pK of 3.7 and the remaining 29 with a pK of 3.5.