Spectrophotometric titration with cobalt(III) for the determination of accurate absorption coefficients of transferrins

A rapid and sensitive technique, involving difference spectral titration with cobalt(III), to measure the a values of chicken ovotransferrin, human serum transferrin, the N-lobe of human transferrin and several single point mutants is reported. The resulting epsilon values were compared with the values calculated from the equation proposed by Pace, Vajdos, Fee, Grimsley and Gray [(1995) Protein Sci. 4, 2411-2423]. The titrations with cobalt feature sharp break-points and do not destroy the protein samples. The choice of buffer was found to be important, depending on the metal-binding avidity of the proteins. Cobalt titration should prove useful for studying the comparative metal-binding properties of transferrin and mutants of transferrin being generated by recombinant technology.