Human recombinant alpha-parvalbumin and nine mutants with individually inactivated calcium- and magnesium-binding sites: biochemical and immunological properties

AbstractHuman recombinant α-parvalbumin (PVwt) and nine mutant proteins, containing inactivating substitutions at positions essential for Ca2+ binding in the CD Ca2+-binding site (PVE62V, PVD51A, PVD51A,62V), the EF site (PVE101V, PVD90A, PVD90A,E101V) or in both (PVE62V,E101V, PVD51A,D90A, PvD51A,E62V,D90A,EIO1V), were expressed and purified. Flow dialysis revealed that PVwt binds 2 Ca2+ with equal K′Ca, of 2.3 × 107M−1 and that Mg2+ competes with a K′Mg.compet.of 4.9 × 103M−1. The three mutants with an inactivated CD site bind 1 Ca2+ with K′Ca, of 2.0 to 2.3 × 107M−1and K′Mg.compet.of 3.4 to 4.6 × 103M−1, i.e. very similar to those of PVwt. The mutants with an inactivated EF site bind 1 Ca2+ with K′Ca, values of 7.9 × 106, 4.5 × 106and 3.6 × 106M−1, for PVD91A, PVE102V and PVE101V,D91A, respectively. The K′Mg.compet. values of these mutants are about 4-times lower than in PVwt. The three mutants with both sites inactivated bind neither Ca2+ nor Mg2+. After excitation at 259 nm, human PVwt which contains neither Tyr nor Trp, shows maximal fluorescence emission at 283 nm. Binding of either Ca2+ or Mg2+ to PVwt or to mutants with an inactivated EF site lead to a 1.8-fold decrease in fluorescence intensity, whereas the mutants with an inactivated CD show only a very slight decrease upon binding of Ca2+ or Mg2+. Specific antibodies against human α-parvalbumin were raised in rabbits. Their reactivity was tested against the mutant proteins, and their potential value for location and functional studies was investigated