Proton nuclear magnetic resonance determination of the sequential ytterbium replacement of calcium in carp parvalbumin

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Abstract
The substitution of the paramagnetic lanthanide ion Yb3+ for the Ca2+ bound to the CD and EF sites of carp parvalbumin results in a series of 1H NMR resonances which are shifted far outside the envelope of the 1H NMR spectrum of the diamagnetic form of the protein. Titrations of Ca2+-saturated parvalbumin with Yb3+ demonstrate that Yb3+ sequentially replaces the 2 bound Ca2+ of the protein. Analysis of the 1H NMR data yields the relative affinities of the 2 sites (CD and EF for Yb3+ with respect to Ca2+. The dissociation constants for Yb3+ are then calculated to be KYb1+ CD = (4-7) .times. 10-10 M and KYb3+EF = (2-6) .times. 10-10 M from the known dissociation constants for Ca2+. The approximate equality of these constants is verified by Yb3+ titrations of apoparvalbumin.