title: Application of the pH-jump method to the titration of tyrosine residues in bovine alpha-lactalbumin.
abstract: A stopped-flow technique has been developed for the zero-time spectrophotometric titration of tyrosine residues in the purely native or in the purely alkaline denatured state of alpha-lactalbumin that undergoes an alkaline conformational transition in the pH region of tyrosine ionization. The progressive absorption change at 298 nm caused by a pH jump from neutral pH is shown to result from the change in ionization of the tyrosine residues brought about by a first-order process of the conformational transition. Extrapolation to zero time gives the titration curve for purely native alpha-lactalbumin. Similarly, the pH jump from highly alkaline pH gives the titration curve for the purely alkaline denatured protein. The method should be generally applicable to other proteins that contain tyrosines. Analysis of the titration curves suggests that the four tyrosines in native alpha-lactalbumin have pK values of 10.5, 11.8, 11.8, and 12.7, respectively. After the alkaline transconformation, all of them become titrated normally with a pK value of 10.3. A comparison of these results with the ionization behavior of tyrosines in hen egg white and human lysozymes is presented and discussed in terms of differences in the sequences of the proteins.
mesh_terms: D000818:Animals; D002417:Cattle; D055598:Chemical Phenomena; D002621:Chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007768:Lactalbumin; D009113:Muramidase; D009994:Osmolar Concentration; D011489:Protein Denaturation; D013056:Spectrophotometry, Ultraviolet / Q000379:methods; D014443:Tyrosine
chemical_list: D014443:Tyrosine; D007768:Lactalbumin; D009113:Muramidase