NMR-STUDIES OF PRIMARY AND SECONDARY SITES OF PARVALBUMINS USING THE 2 PARAMAGNETIC PROBES GD(III) AND MN(II)

The binding of cations by parvalbumins was studied by the proton relaxation enhancement (PRE) method using the paramagnetic probes Gd(III) and Mn(II). Gd(III) appears as a specific probe of the primary sites CD and EF with the following binding parameters : n = 2, KdGd = 0.5 × 10-11 M and εb = 2.3. The low value of εb is the result of a nearly complete dehydration of the protein bound ions. Competition experiments between Gd(III) and various diamagnetic cations show the following order of affinity for the EF and CD sites : Mg2+ < Zn2+ < Sr2+ < Ca2+ < Cd2+ < La3+ ≤ Gd3+. Mn2+ is a specific probe of a secondary site with the following binding parameters : n = 1, KdMn = 0.6 × 10-3 M and εb = 17. The high value of εb suggests that the protein bound Mn(II) has retained most of its hydration shell. Competition experiments between Mn(II) and different cations show similar affinities for this site : Ca2+ ≤ Mg2+ ≤ Cd2+ ≤ Mn2+. This secondary site is located near the EF primary site. © 1979 Masson, Paris.