Complexes Dissociate, and Then Some

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Science  30 Aug 2002:
Vol. 297, Issue 5586, pp. 1449
DOI: 10.1126/science.297.5586.1449a

The accurate determination of protein-ligand dissociation constants (Kd) normally requires working with purified components, but in some cases, even estimates of Kd can be valuable. Powell et al. have used a mass spectrometric method for determining protein stability, called SUPREX (stability of unpurified proteins from rates of H/D exchange), to estimate Kd within a factor of 2 to 3. In their method, the amount of D incorporation into a protein after a given amount of time is determined from the increase in molecular weight, and this incorporation is determined in solution for different concentrations of denaturants. These changes can be related to the free energy of folding of the protein; differences in the free energy with and without ligands allow Kd to be determined, such as for unpurified Trp repressor in a bacterial lysate. — PDS

J. Am. Chem. Soc. 10.1021/ja026574g (2002).

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