A spin labeling study of the effects of inorganic ions and pH on the conformation of spectrin.

The structure of spectrin from human erythrocytes has been investigated by the EPR technique measuring the mobility of the protein spin label, 4-maleimido-2,2,6,6-tetramethylpiperidinooxyl. Conformational changes in the protein induced by variation of the concentrations of NaCl, Na2SO4, KCl, CaCl2 and MgCl2 and of pH have been studied. It could be demonstrated that both Ca2+ and Mg2+ give rise to structural changes by binding to specific sites, whereas the monovalent cations (K+, Na+) seem to act via ionic strength. A model is used to correlate the spin label mobility with the radius of the protein. In the Ca2+- and Mg2+-binding experiments, the decrease in the spin label mobility has been interpreted on the basis of the theory of multiple chemical equilibria. These experiments have been compared with EPR spectra measured at different pH values. The results support the model in that binding of H+, Ca2+ or Mg2+ reduces the charges located on the protein surface: the 'discharging' reduces the repulsive forces on the surface of the molecule and consequently, the protein contracts in discrete steps.