Spectrophotometric Titration of Proteins

  • Alexander P. Demchenko


Titration methods are usually applied to determine the ionization constants of the ionizable groups in proteins studying the effects of pH variation. In spectrophotometric titration, as distinct from potentiometric titration, changes in the absorption spectra are observed. This makes the method selective to certain definite ionizable groups. The method for spectrophotometric titration of proteins is described in a number of reviews (Tanford 1962; Wetlaufer 1962; Donovan 1969, 1973 a b). This method has been attracting great interest in recent years because the electrostatic interactions (Wada and Nakamura 1981) and local electrical fields (Topolev and Krishtalik 1983) in the protein molecule are decisive in the stabilization of the structure of native proteins and in mechanisms of their functioning. Of interest is the problem of accessibility of protein internal regions to protons as well as on the nature of considerable shifts of ionization constants. At the same time, spectrophotometric titration is a simple and convenient method for analyzing protein conformational changes. This chapter considers not only the theoretical principles of the method and analysis of the results, referring mainly to titration of tyrosine residues in the alkaline pH region by the difference spectroscopic method, but also the new possibilities suggested by the TPDS and derivative spectroscopic methods.


Tyrosine Residue Titration Curve Difference Spectrum Tryptophan Residue Ionization Constant 
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Copyright information

© Springer-Verlag Berlin Heidelberg 1986

Authors and Affiliations

  • Alexander P. Demchenko
    • 1
  1. 1.Palladin Institute of BiochemistryAcademy of Sciences of the UkraineKievUSSR

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