The calcium- and magnesium-binding properties of four parvalbumins, two from each genetic lineage α and β, have been studied by equilibrium dialysis, flow dialysis, and ultraviolet differential spectroscopy. In the absence of calcium, α-parvalbumins from rabbit pI = 5.55 and from frog pi = 4.88 and 0-parvalbumin from frog pi = 4.50 bind two Mg2+ ions with high affinity (Kd = 16, 21, and 27 /iM, respectively) at two equivalent and independent sites. Magnesium binding to the ion-free proteins induces conformational changes at the level of the hydrophobic core and of the AB loop, which can be monitored by UV differential spectroscopy. Absorbance differences increase linearly with the molar ratio up to 2 mol of Mg2+/mol of parvalbumin. The affinity of parvalbumins for Ca2+ decreases in the presence of Mg2+ according to a simple competition for the same sites. In the absence of Mg2+, two Ca2+ ions are bound at equivalent and independent sites with a calculated Kd = 6.6, 7.8, and 2.2 nM for rabbit, frog pI = 4.88, and frog pI = 4.50 parvalbumins, respectively. Substitution of Mg2+ by Ca2+ ions induces structural changes that are especially visible at the level of the hydrophobic core and of the AB loop, but the overall structure is similar in proteins that bind either Ca2+ or Mg2+. Therefore, these three parvalbumins exhibit two independent and equivalent high-affinity Ca2+-Mg2+ sites. In contrast, when studied by the above techniques, β-parvalbumin from hake exhibits two nonequivalent high-affinity Ca2+-Mg2+ sites. Since no more than 1.5 Mg2+- or Ca2+-binding sites could be found, the protein may have lost part of its Ca2+-binding capacity upon removal of divalent metals. Hake parvalbumin was shown to bind 2 Ca2+ ions at independent sites with = 3-5 nM and Kd2 ≥ 17 nM, when conformational changes induced by addition of EGTA were followed by UV differential spectroscopy. Binding of EGTA to parvalbumin (K0.5 = 35 mM) was also demonstrated by this technique. β-Parvalbumins appear to bind Ca2+ ions more strongly than a-parvalbumins. The Ca2+-Mg2+ sites of parvalbumins exhibit either a lysyl residue (CD sites) or a glycyl residue (EF sites) between the Y and Z coordination sites. There is, therefore, no correlation between the nature of the residue in this position and the class of site. Parvalbumins bind 2 Mg2+ ions in resting muscle. The delay in calcium binding resulting from the dissociation of bound Mg2+ explains why Ca2+ can trigger contraction. Relaxation occurs when Ca2+ is displaced from the low-affinity sites of troponin C to the high-affinity Ca2+-Mg2+ sites of parvalbumins. More generally, low-affinity Ca2+-specific sites, which pick up Ca2+ with diffusion-limited kinetics, are “triggering sites”, whereas high-affinity Ca2+-Mg2+ sites are “relaxing sites”. © 1979, American Chemical Society. All rights reserved.
