Calcium binding proteins: optical stopped-flow and proton nuclear magnetic resonance studies of the binding of the lanthanide series of metal ions to parvalbumin

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Abstract
Optical stopped-flow techniques were used to determine the dissociation rate constants (koff) for the lanthanide(III) ions from carp (pI [isoelectric point] 4.25) parvalbumin. For most of the 13 different lanthanides studied, the release kinetics were diphasic, composed of both a fast phase (whose rate varied across the series, La3+ .fwdarw. Lu3+, between the limits -1.2 .ltoreq. log kFAST .ltoreq. -0.7) and a slower phase (whose rate varied across the series, La3+ .fwdarw. Lu3+, between the limits -1.2 .gtoreq. log kSLOW .gtoreq. -2.9). In addition, the La3+, and Lu3+-induced changes in the 270-MHz 1H NMR spectrum of parvalbumin were used to calculate the dissociation constants for these specific lanthanides from the 2 high-affinity Ca2+ binding sites. The KD for one site appears to remain constant across the lanthanide series, determined to be 4.8 .times. 10-11 M for both La3+ and Lu3+. The other site, however, is evidently quite sensitive to the nature of the bound Ln3+ ion and shows a strong preference for La3+ (KD,La = 2.0 .times. 10-11 M; KD,Lu = 3.6 .times. 10-10 M). Reports of nearly indistinguishable CD/EF binding site affinities for parvalbumin complexes of the middle-weight lanthanides (i.e., Eu3+, Gd3+ and Tb3+) are quite reasonable in view of the crossover in relative CD/EF site affinities across the lanthanide series.