SPECTROPHOTOMETRIC TITRATION WITH COBALT(III) FOR THE DETERMINATION OF ACCURATE ABSORPTION-COEFFICIENTS OF TRANSFERRINS

A rapid and sensitive technique, involving difference spectral titrati on 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 c ompared with the values calculated from the equation proposed by Pace, Vajdos, Fee, Grimsley and Gray [(1995) Protein Sci. 4, 2411-2423]. Th e 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 titrati on should prove useful for studying the comparative metal-binding prop erties of transferrin and mutants of transferrin being generated by re combinant technology.